Open Access Repository of Indian Theses

V., VANDANA

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                                                                                     Synopsis


Synopsis of the thesis submitted for the award of Ph.D. degree in Biochemistry to the University of Mysore, Mysore, India.

Title of thesis: 

STUDIES ON METAL-DNA INTERACTION INDUCED DNA CONFORMATIONS IN RELEVANCE TO NEUROLOGICAL DISORDERS

Candidate:  Mohammed Shafiul Mustak. B

Trace metal ions play a very important role in biological processes. Trace elements are sub divided into three main categories: i) Essential, like Fe, Cu, Se, Zn, Mn, Cr, Mo, Co and I;  ii) Beneficial but not currently thought to be essential including F, V, Br, and Li ; iii) Toxic metals like Pb, Cd, Hg and Al. Trace metal homeostasis is essential for normal biological functions and any change in homeostasis leads to pathophysiology of cells. 
	Metal imbalances are reported to be one of the etiological factors of neurological diseases like Bipolar Disorders (BPD), Alzheimer’s disease (AD) and Parkinson’s disease (PD). Moreover, metals like Aluminium (Al) and iron (Fe) are found to be increased in specific regions of neurodegenerative brain of AD and PD. These two metals play a pivotal role in neurotoxicity and neurodegeneration. They cause neurofibrillary tangles formation (NFTs), amyloid beta and -synuclein aggregation. Further, Al in conjunction with Fe promotes oxidative stress and apoptosis. Thus Fe, Al plays a significant role in neuronal cell death. 
DNA dynamics is an essential parameter for normal neuronal cell function. Metal ions interact with DNA, might show significant changes in structure and function. Al and Fe are reported to cause cellular toxicity through their interaction with DNA. DNA damage causes genomic instability and cell death. Moreover nuclear localization of Al and Fe has been observed in AD and PD brain.  Recent reports from our lab showed the presence of Z-DNA in hippocampus of AD brain. It was also hypothesized that environmental factors like metals (Al, Fe), oxidative stress, and trace metal homeostasis imbalance might be responsible for the DNA topological changes seen in AD brain. However, there are no studies on Al, Fe  in modulating DNA topology and stability and its relevance to neuronal cell death. 
The present study is focused to understand the Al, Fe interaction with DNA, trace metal homeostasis in serum and brain samples of bipolar disorders, genomic DNA conformation and stability of bipolar depression brain. 
The objectives of the present investigation are
1.	To study trace metal homeostasis in serum and brain samples of bipolar disorder and to develop trace metals interrelation pattern. 
2.	To study Al-Fe-DNA interactions in vitro with reference to supercoiled DNA, linearized DNA and genomic DNA.
3.	To study the conformation and stability in genomic DNA of bipolar depression brain.
	The research work is carried towards achieving these objectives makes the subject matter of the thesis.  The thesis is divided in to five chapters.  
Chapter 1.  General Introduction
	This chapter begins with a general account of metals like Al and Fe in neurological diseases like AD, PD and BPD followed by an overview of current literature. The neurotoxic role of Al and Fe in AD and PD has been discussed in detail. The role of these metals in inducing the oxidative stress and apoptosis also discussed. This chapter highlights the importance of the DNA, supercoiled DNA, metal DNA interactions and gene environmental interaction in neurological diseases. The chapter also highlights the scope of metal chelation therapy and the focus of the present study. We also provided a hypothesis on the metal toxicity in neurodegeneration. 
Chapter 2.  Trace elements levels in serum and brain of BPD and control groups: 
	Bipolar disorder (BPD) or manic-depressive illness is one of the two major psychiatric disorders characterized by recurrent depressive and manic episodes. BPD affects about 1% of the population and causes severe neuropsychological impairments. The illness is implicated in functional impairment and represents an important risk factor for suicide. Pivotal biochemical alterations underlying neuropsychiatric disorders are unknown. There are limited reports on changes in trace elemental levels in the bipolar disorder. It has shown the elevation of Vanadium (V) and Molybdenum (Mo) in serum samples of bipolar mood disorder subjects. Also an increase in Al and Br levels in the serum samples of Li-treated patients has also been reported. Moreover, most of the available information is limited to few selected elements and there is no study, which examines inter-elemental relationships with regard to three different types of BPD. 
The aim of this study is to examine the levels of eleven elements namely Na, K, S, P, Fe, Mn, Ca, Mg, Zn, Cu and Al in serum samples of bipolar patients type I, II, V and compare them with controls, and also to understand the element homeostasis through element-to-element interrelationship in bipolar disorder. The present study also analyzed few trace metals like Cu, Zn, Al and Fe in the hippocampus and frontal cortex region of BPD brain.  Further, this study proposes a hypothesis explaining the possible relevance of trace elemental homeostatic imbalance in serum of BPD to possible effects on the brain. 
	The results showed a definite pattern of variation among certain elements in three types of BPD compared to control. The comparative account of trace elements between the control group and bipolar group patients’ serum samples revealed the following trends: a) in the bipolar I patients, Na, K, P, Cu, Al and Mn are elevated significantly  b) in bipolar II hypomania-Na, Mn, and Al are increased significantly, c) in bipolar II patients suffering from depression-Na, K, Cu, and Al are increased significantly and d) in bipolar V depressive patients Na, Mg, P, Cu and Al are increased significantly. But elements namely S, Fe, and Zn levels are decreased significantly in all three bipolar types. Further, bipolar I (manic) have higher concentrations of Cu, Al, and Mn than bipolar II hypomania. The total elemental concentration (µmole/mL) is higher  in the bipolar groups serum compared to the control group indicating possible imbalance in elemental homeostasis. The data also revealed a clear imbalance in the elemental interrelationship in all three types of BPD serum compared to control as shown by the direct and inverse correlations. These results suggest that a definite disturbance in the elemental homeostasis occurs during the development of BPD. In this perspective, the present study provided a comprehensive database on concentrations of 11 elements (majority of essential and few important toxic elements) in BPD serum in comparison with control groups. And also level of Al, Zn, Cu and Fe in the hippocampus and frontal cortex region of BPD are analyzed. The result showed that Al. Fe and Cu are higher in concentration in frontal cortex but not in hippocampus where as Zn is reduced in both the brain region of BPD.  Further, we proposed a model that increased Al levels in serum of BPD is likely to alter the trace elemental homeostasis pool in the brain elemental homeostasis. We also suggested that irrespective of elements being primary risk factors or consequences of disease mechanism, a change in an individual metal ion will upset the elemental homeostasis pool resulting in a significant imbalance in elemental levels and charge distribution pattern in the biological system. 
Chapter 3A.  Aluminium-DNA interactions
Al is one of the strongly suspected etiological factors in neurodegenerative disorders like AD, PD and Guam Parkinsonism/amylotrophic lateral sclerosis. Recently, our lab for the first time, provided evidence for the presence of left-handed rigid Z-DNA in hippocampus region of severely affected AD brain. It has been hypothesized that one of the etiological factors like Al might be playing a role in right to left handed helical change associated with AD. Studies from our laboratory and others showed that Al preferentially binds to DNA and also showed the strong binding to AT*-specific oligomers and induced conformational transition from Z to A in GC*-rich oligomers. 
To investigate the above mentioned hypothesis experimentally, the present study is undertaken to examine the interaction of Al with supercoiled plasmid DNA (scDNA) and genomic calf thymus DNA (ctDNA). Plasmid scDNA as model system makes an interesting study, in view of the observation that vast arrays of small scDNA packets have been found in animal and human cells and known to be involved in gene expression and these superhelical packets are analogous to plasmid DNA supercoiling.  Hence the results can be correlated to human brain genomic DNA to provide an insight into possible role of Al in the progression of AD pathology with reference to DNA topology. 
	We demonstrated in the present study, the toxic role of Al. Firstly, We showed that Al not only binds to DNA but also able to alter the conformation of the DNA. Secondly, Al also caused DNA nicking by breaking the double strand breaks and altered the stability of the DNA. It is observed that Al induced helical transitions from (B to A, B to C or BCA mixed) in scDNA. BCA mixed DNA conformation is energetically weak and is likely to go into Z-DNA conformation. Further, Al induced helical transition from B to Z in case of genomic ctDNA. This may be biological important as Z-DNA was observed in AD brain. The present study also showed that only Al is capable of inducing the DNA conformation, while other elements (group 13/IIIA) such as Ga, In and Tl are able to destabilize the DNA but could not induce any conformational change. 
Chapter 3B. Iron-DNA interactions 
Iron plays a specific role in the central nervous system (CNS). It is involved in myelin formation as well as in the synthesis of several neurotransmitters and in generation of GABAergic activity. Iron is needed as a cofactor for many enzymes that are involved in the normal function of neuronal tissue such as the non-haem iron enzyme ‘tyrosine hydroxylase’ which is essential for dopamine synthesis and as a cofactor for the production of norepinephrine and serotonin. However, Fe overload has been implicated as one of the causes of neuronal death and can lead to vast range of disorders of the CNS.  Abnormally high levels of Fe in specific brain regions related to PD and AD where neurons undergo degeneration have been reported. 
	Iron is a potentially toxic molecule because it contributes to the enhanced generation of Reactive Oxygen Species (ROS), and causes oxidative damage to biomolecules like DNA, proteins and lipids and thus suspected to play a major role in AD. The present study detected the high level of Fe in the frontal cortex of BPD depression brain. Further, a recent report has shown that Fe is localized in the chromatin region of the neurons in AD brain. We also hypothesized that metals like Al and Fe2+ might play important role in right-to-left DNA handedness change associated with AD
In this perspective, we tried to understand the effects of iron cations (Fe2+, Fe3+ ions) on the conformation, stability and strand breaks on DNA by in vitro studies.  Further, the possible effects of Fe2+ ions on linearized scDNA have been also examined.  The experiment is not based on free radical generation but via Fenton reaction. 
	From our study, we observed the differential effect of Fe2+ and Fe3+on the DNA conformation and its stability. The present finding showed that Fe2+ not only binds to scDNA but is also able to alter the conformation of DNA from normal B to C. Importantly the study showed that for the conformation of DNA to occur, Fe in the reduced state (II) is essential since Fe in the oxidized state (III) lacks such ability to alter the transition. Hence Fe3+ could not induce any structural change in DNA. A previous study from our lab showed that spermine could convert C-DNA to Ψ-DNA. It is known that Ψ-DNA, an ordered, twisted, tight-packing arrangement of the double helix is structurally and immunologically closely related to the left handed Z-DNA family. This indicates that DNA topological changes induced by Fe2+ ions could contribute to the DNA conformational changes as seen in AD brain.
The present study also analyzed the effect of Fe2+ ions on the linearized scDNA with a cohesive end. Interestingly, we observed that Fe2+ can circularize the sticky ends of the restricted fragments of the scDNA. This study therefore showed for the first time the role played by Fe2+ in the circularization of linearized scDNA. The chapter also discussed the possible mechanism of Fe induced DNA conformation and damage. 	
Chapter 4. DNA stability and damage in BPD brain
More recently remarkable progress has been made in identifying changes in the brain related to pathophysiology of BPD. One of the consequences of oxidative stress in BPD  is apoptosis and/or necrosis and recent preliminary study showed that apoptosis is associated with etiology of BPD.
Moreover no studies to date have presented quantitative evidence for the presence of neurodegeneration in major psychiatric disorders. However, recent study evidenced decreased DNA fragmentation in BPD anterior cingulated cortex brain region.  Further, DNA fragmentations have been shown to associate with neurodegenerative disorder like PD. More recently, reported the presence of DNA fragmentation in some region of PD brain.  Further, it is shown that presence of an altered DNA conformation in hippocampus of AD brain. However such information on topology and stability of DNA is lacking in BPD depression brain and no study has been done on DNA fragmentation with respect to different region of BPD brains compared to control brains. 
In this perspective, we studied the stability, integrity and topology of DNA isolated from ten clinically and postmortem psychiatrically confirmed BPD depressive cases and ten age-matched controls. Genomic DNA is isolated from ten regions in the human brain, namely parietal lobe, temporal lobe, occipital lobe, hippocampus, thalamus, cerebellum, hypothalamus, medulla, pons, and frontal cortex collected at post-mortem from cases of BPD and controls and are analyzed for single and double strand breaks in DNA, and their conformations and topology. 
The results showed that DNA from thalamus, pons, medulla, temporal lobe, cerebellum and frontal cortex are damaged accumulating significantly higher number of single and double strand breaks compared to control subjects. However, the complex brain region hippocampus did not show any DNA fragmentation in both control and BPD brains. 	We have also showed classical apoptotic DNA laddering pattern in some regions of BPD brains like thalamus, pons, medulla and frontal cortex evidencing the presence of apoptotic cell death in BPD depressive patients in these brain regions.	Circular Dichroism studies showed that DNA conformation is altered in frontal cortex, cerebellum, pons and parietal lobe of BPD. However, both control and BPD hippocampus DNA showed normal right handed helical B-DNA conformation. To our knowledge to date, this is the first study that showed the presence of apoptotic DNA fragmentation and genomic instability and conformation in region specific BPD brains compared to control. 
	 The thesis ends with a comprehensive Summary and Conclusions of the present study.
In a nut shell the study provides the following significant contributions
1.	The present study has generated a comprehensive data on the levels of 11 elements (both essential and toxic) in serum samples of three different types of BPD affected patients and showed that there is a definite disturbance in inter-element homeostasis pattern in three types of BPD serum. We also analyzed the four trace metal level in two human brain region of BPD and showed high concentrations of Al, Fe and Cu in frontal cortex and no change in Zn level. (Chapter 2).  
2.	We have provided evidence for the role of Aluminium and iron in the alteration of DNA topology, stability and DNA damage.  Al induces a BCA –mixed and Z-DNA conformations in case of scDNA and ctDNA respectively; however, other group 13 metals could not induce any conformational change.  It is interesting to observe that Al has property of nicking the DNA by double strand breaks and damages the DNA. (Chapter 3A).
3.	Iron in the form of Fe2+ could induce the conformational change from B-DNA to C-DNA in supercoiled and calf thymus DNA. However, the Fe3+ did not bring any conformational change to the normal DNA. We also observed that Fe2+ has ability to nick the DNA by single strand breaks and not by double strand breaks.  The present study also observed that Fe2+ has ability to circularize the linearized scDNA (Chapter 3B). These have been very intriguing observations which exposed a new toxic role of Al and Fe. The observations lead to a new debate on whether direct Al and Fe induced DNA damage and conformation change in vitro has any role to play in DNA conformation observed in AD brain and also DNA fragmentation observed in BPD brain and hence open new avenues of research in this direction .
4.	The study provided a new data on DNA stability; integrity and topology of genomic DNA isolated from ten different brain regions from BPD affected human brain samples. We also evidenced the presence of apoptosis some regions of brain of BPD. 

	The present study insights that the trace metal homeostasis is altered in the BPD serum and brain compare to respective controls. Also mapped the mechanism of Al, Fe induced DNA topological changes. Further,  the DNA topology and stability is altered in BPD brain regions except hippocampus. The significance of the results is discussed in detail in the thesis.  




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English

Chemical and enzymatic methodologies for processing and modification of lipids and their derivatives

G., Rajaiah

 I 
 
 
ABSTRACT 
Research work concerns with synthesis, characterization of biodegradable polymers and 
its evaluation in particulate drug delivery systems. The work is presented in three chapters and a 
brief outline of each is given below. 
 
Chapter I: Synthesis and characterization of biodegradable polymers 
Poly P-hydroxy acid’s synthesized previously are highly crystalline and water insoluble. 
So, their application regions are limited. On the other hand, the polydepsipeptides, alternating 
copolymers of α-amino acids and α-hydroxy acids, are also known to be biodegradable polymers 
[1,2]. These polymers are expected to serve as useful drug delivery system (DDS) because they 
strongly interact with biocells to demonstrate high metabolism [3]. The copolymerization 
technique permits the synthesis of an unlimited range of polymers. It is widely used to obtain a 
better balance of properties for the commercial applications of polymeric materials. The 
objective of this work was to design and evaluate new poly[Llactic acid-(glycolic acid-leucine)] 
copolymers (PLDL) for the development of particulate drug delivery systems. 
The PLDLs were synthesized by ring opening polymerization. Various parameters in 
polymerization reaction were studied to achieve a polymer of suitable mechanical strength such 
as effect of monomer feed ratio, initiator concentration, polymerization time and polymerization 
temperature. The optimized copolymer was characterized by FTIR, NMR spectroscopy, 
molecular weight determination by GPC, glass transition temperature by DSC, and crystallinty 
by XRD studies. The weight average molecular weight of the polymers was in range of 3500-
7500 Da. The glass transition temperature of the optimized copolymer (Tg) was 36.27
0
C and 
melt temperature (Tm) was 159.62
0
C. XRD studies showed that the optimized copolymer was 
semicrystalline. In-vitro PLDL particle degradation in pH 7.0 buffer and in presence of enzyme 
was studied by molecular weight loss by gel permeation chromatography, the polymer showed 
higher degradation in presence of enzyme. Evaluation of in-vivo biological reactivity tests for 
PLDL in albino mice as per USP26 NF 21 revealed that polymer was nontoxic and can be used 
for further investigation. 
 II 
 
Chapter II: Development and evaluation of diclofenac sodium polymeric nanosuspension 
system 
Most ocular diseases are treated with a topical application of drug solutions administered 
as eye-drops; however, they often require frequent instillation of highly concentrated solutions, 
due to the rapid pre-corneal loss from the eye. A significant effort towards new DDS for 
ophthalmic administration has then been seen over the last few decades; herein hydrogels, micro- 
and nanoparticles, liposomes and collagen shields have been investigated. Among them, 
nanoparticle technologies are at present catalyzing increasing efforts in many pharmaceutical 
areas and have shown to give efficient ocular DDS [4]. The objective of this work was 
development and evaluation of diclofenac sodium (DS) polymeric nanosuspension (NS) systems 
encapsulating optimized PLDL and commercial poly(lactide-co-glycolide) (PLGA) polymers for 
achieving higher corneal residence time.  
NS were prepared by a emulsion and solvent evaporation technique. Various formulation 
and processing parameters like ultrasonication time, polymer concentration in organic phase, 
stabilizer concentration in aqueous phase, stirring rate and drug-to-polymer ratio were optimized 
to achieve nanosize polymeric nanoparticles with higher encapsulation efficiency. The optimized 
NS were characterized on the basis of physicochemical properties and drug release features. The 
nanoparticle system showed interesting size distribution suitable for ophthalmic application. In 
vitro dissolution tests showed a controlled release profile of DS from the nanoparticles. Stability 
for NS was studied at 5
0
C or at 25
0
C/60% RH, the preparations were found to be stable at 
selected storage conditions up to 6 months. To verify the absence of irritancy toward the ocular 
structures, blank NS were applied to rabbit eye and a modified Draize test was performed. 
Polymer nanoparticles appeared to be avoiding of any irritant effect on cornea, iris and 
conjunctiva up to 24 hrs after application, thus appearing to be a suitable inert carrier for 
ophthalmic drug delivery. 
 
Chapter III: Development and evaluation of valdecoxib polymeric microparticle system 
Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease characterized by 
progressive damage of the joint [5]. Inflammation that predominates the joints often exhibits a 
 III 
 
tendency of remission in 90% of the patients. Rheumatologists have identified three forms of 
RA. In the first case the patient has mild, self-limiting disease that generally resolves within a 
period of one year. The second case involves patient in which there is mild progression of 
disease that responds to conventional treatments with near normal clinical examination. The third 
category is the one in which arthritis is more aggressive and difficult to control with drug 
treatment. There is radiological deterioration accompanied with functional decline. Thus, RA has 
significant long-term morbidity and is associated with early mortality. NSAIDs are most widely 
used in clinical practice world over because of their four-fold actions comprising analgesic, 
antipyretic, anti-inflammatory and antithrombotic effect. The therapeutic regimen often involves 
the daily administration of these agents. The objective of this work was to formulate Valdecoxib 
(a drug from the NSAID class) loaded polymeric microparticulate (MP) injectable system for the 
long-term delivery of the active agent using biodegradable polymer PLDL for avoiding the 
problems associated with daily dosing and to improve patient compliance.  
Valdecoxib (VC) loaded PLDL MP’s were prepared by o/w emulsion and solvent 
evaporation technique. Various process parameters involved in microparticle formulation were 
studied. The microparticles were characterized for process yield, particle size, moisture content 
and drug encapsulation efficiency. Drug-excipients interactions were examined by FTIR, XRD 
and DSC. Solid state stability to gamma radiation was conducted by subjecting the pure drug and 
the microparticulate system to various gamma radiation doses. FTIR and DSC revealed no 
interaction between VC, PLDL and excipients. VC and the developed MP were stable at 2.5 
Mrad dose of gamma radiation. In-vitro release of the drug loaded MP’s were investigated by the 
static method at a 37
0
± 0.5
0
C, the studies revealed that all MP formulations followed near zero 
order sustained release. Stability studied for VC loaded MPs showed that the preparations were 
stable at 5
0
C and 25
0
C/60%RH up to one year. Relative bioavailability for synthetic drug and 
VC-PLDL MPs was studied in New Zealand white rabbits. A single intramuscular dose of VC-
loaded PLDL MPs resulted in sustained therapeutic drug levels in the plasma for 49 days. The 
bioavailability was increased several-fold as compared with unencapsulated drug. Therefore, 
injectable PLDL microparticles hold promise for increasing drug bioavailability and reducing 
dosing frequency for better management of rheumatoid arthritis. 
 IV 
 
References: 
1. Ouchi T., Nozaki T., Ishikawa A., Fujimoto I., and Ohaya Y. (1997) Synthesis and 
enzymatic hydrolysis of Lactic Acid-depsipeptide copolymers with functionalized pendant 
groups. Polymer Science: Part A: Polymer Chemistry, 35, 377–383. 
2. Ouchi T., Shiratani M., Jinno M., Hirao M. and Ohaya Y. (1993) Synthesis of poly[(glycolic 
acid)-alt-(L-aspartic acid)] and its biodegradation behavior in-vitro. Makromol. Chem., 
Rapid Commun., 14, 825–831 
3. Shirahama H., Tanaka A., Yasuda H. (2002) Highly biodegradable copolymers composed of 
chiral depsipeptide and L-Lactide units with favorable physical properties. J. Poly. Sci., Part 
A: Polym. Chem., 40, 302–316. 
4. Kawashima Y. (2001) Nanoparticulate systems for improved drug delivery. Adv Drug 
Delivery Rev. 47, 1–2. 
5. Lipsky P.E. (1998) Rheumatoid Arthirits, In: Harrison's Principles of Internal Medicine 
(A.S. Fausi, E. Braunwald, K.J. Isselbacher, J. D. Wilson, J.B. Martin, D.L. Kasper, S.L. 
Hauser and D.L. Longo eds.) 14th edition, McGraw Hill, Inc., pp. 1880–1887. 
 


Synthesis of biologically active compounds and development of new methodologies

A., SWAROOPA RANI


Contents


Caffeine (1,3,7-trimethyl xanthine) is found in seeds and leaves of coffee, cola, mate and tea. Along with other methylxanthines, including theobromine, paraxanthine and methyluric acids, caffeine is a member of a group of compounds known collectively as purine alkaloids. The biosynthetic pathway of theobromine and caffeine has been the subject of much study over the years. Caffeine is synthesized through three sequential steps involving methylation of xanthine derivatives at positions 7-N, 3-N and 1-N (xanthosine  7-Methylxanthosine  7-Methylxanthine  Theobromine  Caffeine), catalyzed by N -Methyltransferases that use S-Adenosyl-L-Methionine as methyl donor. 

   A survey of literature suggests that the N-methyltransferases (NMTs) that are involved in the caffeine biosynthetic pathway in coffee comprise a multigene family. Several cDNA clones for NMTs from coffee have been reported. However, there are no reports on the genomic sequences for the NMT genes. The studies on NMT gene structure and organisation are lacking. Therefore, it was envisaged that PCR based genomic cloning of the NMT genes could provide valuable information regarding the number and size of introns and exons present in NMT genes. The study of introns and exons could possibly shed light on the mechanisms for origin of NMT multigene family in coffee and possible functional divergence among different members of the gene.

   The survey of literature and patents indicated a distinct lacuna in the studies related to NMT gene regulation. The isolation of promoter for the NMT genes has not been hitherto reported and hence it became one of the major objectives of this investigation. The promoter isolation could help in understanding the NMT gene regulation and also open up the possibilities of regulating caffeine content in coffee plants. 

   There are wide variations in the caffeine contents of different coffee species, the reasons for which are unknown. Very little is known about the NMT genes in Coffea species other than C. arabica. It may be speculated that differential transcription of various NMT genes could be one of the reasons for such variations in the caffeine content. Study of possible polymorphisms for the NMT genes and their corresponding promoters in different Coffea species could be vital in understanding differential transcription of various NMT genes. 

   The production of transgenic caffeine deficient coffee plants by RNAi using specific region of the theobromine synthase gene has been reported earlier, wherein spreading of silencing to other NMT genes was observed. The use of a conserved region common to all the NMT genes should theoretically result in silencing of all the NMT genes. Moreover, other posttranscriptional gene silencing strategies were not explored for caffeine down regulation. The use of conserved region of NMT genes for different posttranscriptional gene silencing strategies could be a viable option for obtaining phenotypes with desirable purine alkaloid profiles. 

   With this background knowledge, the present research work was undertaken. The research work carried out to meet the proposed objectives is presented in five chapters in the thesis. The references are cited at the end in alphabetical order. The title and summary of the work carried out under each chapter are presented as follows: 


Chapter I
Introduction and review of literature
   This chapter provides an overview of the current literature regarding the purine alkaloid biosynthesis in coffee. The taxonomy of coffee, distribution of various purine alkaloids and their biosynthesis in different Coffea species are highlighted. The properties of N-methyltransferases that catalyze the conversion of xanthosine to caffeine and the genes encoding them have been described in detail. The comparisons of the NMT sequences in tea and coffee with related methyltransferases are also presented. The pathway for caffeine catabolism in plants is discussed. The uses of biotechnological approaches for regulating the caffeine content in plants are discussed at the end of the chapter. This chapter also highlights the unexplored areas in coffee NMT research, which forms the basis for the present investigation.




Chapter II
Molecular Cloning of the N - Methyltransferase Gene from Coffea canephora

   Primers for PCR amplification of N-methyltransferase (NMT) genes from Coffea canephora were designed based on the available cDNA sequences for NMT genes of Coffea arabica. Different sets of primers were used to obtain amplicons for partial and full-length NMT gene. Various PCR products were cloned and sequenced. Variations were observed with respect to restriction patterns among the clones for partial NMT gene. Sequence analysis of these clones revealed the presence of introns in the gene. High degree of similarity in the nucleotide sequences of exon regions and differences in the length and sequence of introns were observed, suggesting that these clones may belong to different members of the NMT multigene family. Using primers based on 5΄ and 3΄ untranslated regions specific for theobromine synthase, involved in the second methylation step, amplicons for full-length theobromine synthase gene were obtained. Sequencing of the clones for full-length gene revealed the presence of four exons interrupted by three introns. Based on the insertions and deletions present in the cDNA sequences of various NMT genes reported so far and the sizes of their open reading frames, the genomic clone corresponded to the theobromine synthase gene encoding for 378 amino acids. Variations with respect to the size and sequence of introns in two of the full-length clones for theobromine synthase were observed, though one of them contained one base nucleotide deletion in fourth exon. From this study, it appears that the coffee plants possess multiple alleles and/or multiple copies of the same gene; some of which may have undergone pseudogenisation.


Chapter III
Cloning of Promoter for an N-Methyltransferase Gene

   A PCR based genome walking method was adopted for isolation and cloning of the promoter for one of the coffee NMT genes. Inspection of the promoter sequence revealed the presence of several motifs important for the regulation of the gene expression. The isolated promoter fragment was fused to the β-glucuronidase (GUS) reporter gene and used in Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum and also for electroporation of coffee endosperms. GUS assays proved that the isolated promoter was able to direct the expression of the reporter gene in transgenic tobacco. The cellular localization of GUS activity in transgenic tobacco confirms that coffee NMT gene is a cytosolic enzyme. Transient assays demonstrated the ability of the isolated promoter fragment to drive gene expression in endosperms. The cloning of promoter for a gene involved in caffeine biosynthetic pathway opens up the possibility of studying the molecular mechanisms that regulate the production of caffeine. Based on the promoter sequence, primer was designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Sequencing of one of the genomic clones revealed the presence of four exons and three introns in NMT gene. Sequence comparisons were made between coffee NMT gene and Arabidopsis Jasmonic acid carboxyl methyltransferase, which belongs to coffee N-methyltransferase related family. Though the gene structure was highly conserved in terms of number and position of introns, AtJMT revealed great variation for coding regions, introns, and the promoter region sequences, suggesting that the two genes have diverged greatly after their origin from common ancestor. 


Chapter IV
Polymorphism for Coffee NMT Genes

   Based on the promoter sequence for theobromine synthase gene-1, primers were designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Differences in the restriction pattern among the genomic clones of theobromine synthase gene-1 were studied by performing PCR-RFLP. Though differences were observed in restriction pattern for the clones, Southern blotting of the restricted PCR products from these clones with probes for promoter as well as the gene suggested that genomic clones are highly homologous and possibly belonged to different alleles/ members of NMT gene family. Sequencing of some of these clones from Coffea canephora revealed differences in intron length and sequences, but the promoter regions and the coding regions were highly conserved. It may be concluded that C. canephora possesses several alleles/ copies of theobromine synthase gene-1. NMT gene fragment from Indian wild species Psilanthus bengalensis was also cloned and partially sequenced. The conservation of NMT gene structure was studied by comparing the introns from various genomic clones. To assess relationships between coffee NMT family members within and across species, their exon-intron organization patterns (e.g. number, positions, and phases of introns) were determined. Intron phase is strictly conserved in all genomic NMT clones available. All the three introns were located at conserved positions, being inserted between two codons (phase 0). Intron positions are absolutely conserved in the NMT genes from various species. Intron sequences have the consensus dinucleotide GT at the 5΄-end and AG at the 3΄-end. Although the splicing sites are conserved, the sizes of introns varied for different NMT genes. Two of the genomic clones obtained in this study are probably pseudogenes, due to a single nucleotide deletion in fourth and third exon respectively. Gene duplication followed by sequence divergence has been proposed as a model to explain how novel biochemical functions may have arisen for different duplicated genes of Coffee NMTs. The promoter regions were PCR amplified from different Coffea species. The sequence analyses of promoter regions from different species revealed a very high degree of conservation. The recent origin of coffee could be the reason for this high level of conservation of promoter sequences within the species and across the species.  


Chapter V
Posttranscriptional Gene Silencing of Coffee N-Methyl transferases

   A highly conserved region among coffee N-Methyltransferase genes involved in caffeine biosynthetic pathway was used in making sense, antisense and RNA interference constructs. Sonication Assisted Agrobacterium tumefaciens (SAAT) mediated transformation was used to mobilize these constructs into Coffea canephora. Somatic embryos were used for the transformation experiments. The transformed coffee plantlets, selected on hygromycin containing selection media, were used for estimating the purine alkaloids content by High Pressure Liquid Chromatography (HPLC). The purine alkaloids viz: 7-methylxanthine, theobromine, caffeine and theophylline were estimated from the transgenic plantlets. The three constructs differed in their efficiencies and specificity of silencing. RNAi was found to be more efficient in reducing caffeine content in transformed coffee plantlets, when compared to sense and antisense constructs. Though the constructs were not specific for a single gene, sense and RNAi transformants were obtained that were mainly affected in one of the N-Methyltransferases. The results suggest that the use of highly homologous coding region is highly effective in down regulating caffeine biosynthesis, encoded by N-methyltransferase multigene family. Increased transcription of CaMTL1 gene, substrate specificity of which is not known, was observed indicating possible activation of parallel or bypass pathways. 


Chapter VI
 Conclusions and Future perspectives 
   The major findings of this investigation and the future prospects are briefly highlighted in the chapter. 





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STUDIES ON HIGH PERFORMANCE BIOREACTORS FOR PENICILLIN PRODUCTION

K., GOVINDARAJU

 
 
Synthesis of glycosides involving regio and stereo selectivity is an important and 
difficult area in synthetic organic chemistry, which is of prime concern to researchers 
working in this area. Glycosides are used as surfactants (Busch et al. 1994), colorants 
and flavoring agents (Sakata et al. 1998), sweeteners (Shibata et al. 1991), antioxidants, 
anti-inflammatory (Gomes et al. 2002), antibiotics (Ikeda and Umezawa 1999; Kren and 
Martinkova 2001), antifungal (Tapavicza et al. 2000), antimicrobial (Zhou 2000) and 
cardiac related drugs (Ooi et al. 1985). Alkyl and phenolic glycosides are even useful as 
detergents, cosmetics and food additives. For the past decade or two a variety of 
glycosidases or transglycosidases have been employed for the glycosylation of mono and 
disaccharides with a variety of acceptor molecules (Katusumi et al. 2004; Vic and Crout 
1995; Sato et al. 2003). However, reports involving amyloglucosidase for the synthesis 
of alkyl/phenolic glycosides are practically nil. Amyloglucosidase from Rhizopus sp. and 
β-glucosidase isolated from sweet almonds were found to catalyze the synthesis of alkyl, 
guaiacol, eugenol, curcumin and α-tocopherol glycosides and the results from this 
investigation is presented in this work in detail.  
Chapter ONE deals with literature reports on enzymatic transformations, 
glycosidases and their sources, structural features of glucoamylase and β-glucosidase, 
glycosylation and their methods, glycosylation mechanism and applications of various 
enzymatically synthesized glycosides. A detailed report on investigations on some 
important factors that influence the glycosidase catalyzed reactions in organic solvents 
like nature of substrate, nature of solvent, thermal stability, role of water, kinetic studies 
of glycosidase catalyzed reactions and immobilization are presented. Besides, the 
advantages of carrying out glycosylation using reverse micelles, super critical carbon di-
oxide, microwave assisted glycosylation reactions and response surface methodology 
163 
 
 
  Summary 
 
 
(RSM) in glycosylation have been discussed. This chapter ends with a brief scope of the 
present investigation. 
Chapter TWO describes materials and methods involved in the present work. 
Enzyme and chemicals employed and their sources are shown. Glycosylation procedure 
by shake flask and reflux methods and the related analytical and assay procedures are 
described in detail.   
Chapter THREE describes synthesis of n-alkyl glucosides of alcohols of carbon 
chain length C1-C18 using amyloglucosidase by both shake flask and reflux methods 
besides investigating the synthesis of n-octyl-D-glucoside 13a-c in detail. Effects of 
incubation period, pH, buffer concentration and enzyme concentration were studied by 
both shake flask and reflux methods in the synthesis of n-octyl-D-glucoside. The highest 
conversion yield of 28% (w/w D-glucose) by shake flask method and 46% (w/w D-
glucose) by reflux method were obtained. Optimum reaction conditions for the shake 
flask experiments were found to be D-glucose and n-octanol in 1: 50 equivalent ratio, 
30% (w/w D-glucose) amyloglucosidase concentration and 0.8 mM (0.4 mL of 10 mM 
buffer), pH 6.0 phosphate buffer for an incubation period of 72 h. For the reflux method 
D-glucose and n-octanol in 1: 50 equivalent ratio, 30% (w/w D-glucose) 
amyloglucosidase concentration and 0.04 mM (0.4 mL of 10 mM buffer in 100 mL 
solvent), pH 6.0 phosphate buffer for an incubation period of 72 h was found to be the 
optimum. The specific alcohols, employed for the preparation of n-alkyl glucosides are: 
methyl alcohol, ethyl alcohol, n-propyl alcohol, n-butyl alcohol, n-amyl alcohol, n-hexyl 
alcohol, n-heptyl alcohol, n-octyl alcohol, n-nonyl alcohol, n-decyl alcohol, lauryl 
alcohol, cetyl alcohol and stearyl alcohol. The reaction mixtures were analyzed by 
HPLC. Glucoside yields obtained from the reflux method (5-44%) were better than those 
from the shake flask method (3-28%). While the shake flask method favored 
164 
 
 
  Summary 
 
 
glucosylation of medium chain length alcohols, the reflux method at pH 5.0, favored 
glucosylation of all the chain lengths.  
n-Octanol 1, glycosylation with carbohydrates (D-glucose 2, D-galactose 3, D-
mannose 4, D-fructose 5, D-arabinose 6, D-ribose 7, maltose 8, sucrose 9, lactose 10, D-
mannitol 11 and D-sorbitol 12) using amyloglucosidase was attempted. Only n-octyl 
maltoside 14 (conversion yield 15%) and n-octyl sucrose 16a and b (conversion yield 
13%) were the glycosides formed besides n-octyl-D-glucoside 13a-c. However, HPLC 
indicated glycosylation of D-galactose 3, D-mannose 4 and D-mannitol 11 with 
conversion yields less than 5%. n-Octyl-β-D-glucoside 14 synthesized by the reflux 
method using β-glucosidase isolated from sweet almonds showed an extent of 
glycosylation of 23%. Spectral characterization of the isolated glycosides by UV, IR, 2D 
NMR, optical rotation and MS studies confirmed that mono glycosylated and/or mono 
C6-O-alkylated products were formed.  
Amyloglucosidase catalyzed synthesis of n-octyl-D-glucoside 13a-c by shake 
flask level was optimized using RSM. A Central Composite Rotatable Design (CCRD) 
involving 32 experiments of five variables at five levels was employed to study the 
glucosylation reaction. The variables employed were n-octanol (15-75 molar equivalents 
to D-glucose), enzyme (20 –100 mg, 20 to 100% w/w D-glucose), pH (4.0-8.0), 0.3-4.3 
mM (0.2-1.0 mL) of 10 mM buffer and temperature (30-70 °C). Amyloglucosidase 
concentration, pH and temperature were found to be significant. Experimental data fitted 
the second-order polynomial equation well, as indicated by R2 value of 0.89. Validation 
experiments carried out under predicted conditions showed good correspondence 
between experimental and predicted yields. Various surface plots were generated to 
describe the relationship between operating variables and the conversion yields. The 
165 
 
 
  Summary 
 
 
highest yield of 53.5% predicted at optimum conditions of 75 equivalents n-octanol, 20 
mg (20% w/w D-glucose) amyloglucosidase, 0.3 mM (0.2 mL), pH 7.8 buffer at 50 °C 
showed good correspondence to the experimental yield of 53.8% under these conditions. 
Chapter FOUR describes amyloglucosidase catalyzed synthesis of phenolic 
glycosides: guaiacyl-α-D-glucoside 21a and b, eugenyl maltoside 27a-c, curcuminyl-bis-
α-D-glucoside 30a and b and α-tocopheryl-α-D-glucoside 36 by reflux method in di-
isopropyl ether solvent at 68 °C. The parameters optimized were incubation period, 
enzyme concentration, pH and buffer concentration and substrate concentration. 
Maximum conversion yields obtained were guaiacyl-α-D-glucoside 52% for 21a and b, 
39% for eugenyl maltoside 27a-c, 48% for curcuminyl-bis-α-D-glucoside 30a and b and 
52% for α-tocopheryl-α-D-glucoside 36. Optimum conditions for guaiacyl-α-D-
glucoside 21a and b synthesis were: D-glucose 2 and guaiacol 17 in 1: 50 equivalent 
ratio, 50% (w/w D-glucose) amyloglucosidase, 0.06 mM (0.6 mL in 100 mL solvent) pH 
7.0 phosphate buffer and 72 h incubation period. Those for eugenyl maltoside 27a-c 
were 1: 10 equivalent ratio of maltose 8 and eugenol 18, 40% (w/w maltose) 
amyloglucosidase, 0.1 mM (1.0 mL), pH 5.0 acetate buffer and 72 h incubation period. 
For curcuminyl-bis-α-D-glucoside 30a and b the conditions were: curcumin 19 and D-
glucose 2 in 2:1 molar ratio, 50% (w/w D-glucose) amyloglucosidase, 0.1 mM (1.0 mL), 
pH 4.0 acetate buffer and 72 h incubation period. For α-tocopheryl-α-D-glucoside 36 
optimized conditions were D-glucose 2 and α-tocopherol 20 in 1:1 molar ratio, 40% 
(w/w D-glucose) amyloglucosidase, 0.2 mM (2.0 mL) pH 7.0 phosphate buffer and 72 h 
incubation period.  
Under the optimized conditions, glycosides of guaiacol 17, eugenol 18, curcumin 
19 and α-tocopherol 20 synthesis with various carbohydrates 2-12 were attempted. The 
conversion yields in the range 17-52% for guaiacyl glycosides, 7-32% for eugenyl 
166 
 
 
  Summary 
 
 
glycosides, 9-48% for curcuminyl glycosides and 52% for α-tocopheryl glucoside were 
obtained. Guaiacol 17 reacted with D-glucose 2 and D-galactose 3, eugenol 18 and 
curcumin 19 reacted with D-glucose 2, D-mannose 4, maltose 8, sucrose 9 and D-
mannitol 11 and α-tocopherol 20 reacted only with D-glucose 2. The glucosylation yields 
obtained using β-glucosidase from sweet almonds were 22% for guaiacyl-β-D-glucoside 
22, 19% for eugenyl-β-D-glucoside 25, 11% for curcuminyl-β-D-glucoside 31 and 24% 
for α-tocopheryl-β-D-glucoside 37. Of the 33 glycosides synthesized, the following 
glycosides are reported for the first time from this work: n-octyl-sucrose 16a and b, 
guaiacyl-α-D-glucoside 21a and b, guaiacyl-β-D-glucoside 22, guaiacyl-α-D-galactoside 
23a and b, eugenyl-α-D-mannoside 26, eugenyl maltoside 27a-c, eugenyl sucrose 28a-c, 
eugenyl-D-mannitol 29, curcuminyl-bis-α-D-mannoside 32, curcuminyl bis maltoside 
33a-c, curcuminyl bis sucrose 34a-c and curcuminyl-bis-D-mannitol 35. Both 2D-
HSQCT and MS studies confirmed formation of the mono glycosylated and/or mono C6-
O-arylated products for guaiacol 17, eugenol 18 and α-tocopherol 20 and bis 
glycosylated and/or bis C6-O-arylated products in case of curcumin 19. No other 
secondary hydroxyl groups of the carbohydrates were found to undergo arylation.  
Curcuminyl-bis-α-D-glucoside 30a and b exhibited water solubility at 14 g/L and 
a total colour of 10.8 in DMSO. Antioxidant activities of guaiacol 17 and curcumin 19 
and their glucosides, guaiacyl-α-D-glucoside 21a and b and curcuminyl-bis-α-D-
glucoside 30a and b showed 62% activity for guaiacyl-α-D-glucoside 21a and b (89% 
for free guaiacol) and 80% activity for curcuminyl-bis-α-D-glucoside 30a and b (79% 
for free curcumin).  
Response surface methodology (RSM) was employed to optimize 
amyloglucosidase-catalyzed synthesis of curcuminyl-bis-α-D-glucoside 30a and b by 
167 
 
 
  Summary 
 
 
reflux method. A CCRD was employed involving five variables (enzyme concentration, 
curcumin concentration, incubation period, buffer concentration and pH) at five levels. A 
second-order polynomial equation with a R2 value 0.9 showed good correspondence 
between experimental and predicted yields. Three-dimensional surface and contour plots 
generated described the catalytic efficiency of amyloglucosidase under the reaction 
conditions employed. Experiments under optimum predicted conditions of 16.9% (w/w 
D-glucose) amyloglucosidase, 0.33 mmol curcumin, 120 h incubation period, 0.1 mM 
(1.0 mL of 0.01 M buffer in 100 mL reaction mixture) buffer concentration at pH 7.5 
gave a conversion yield of 56%. Validation experiments carried out under selected 
random conditions also showed good correspondence between experimental and 
predicted yields. 
Kinetic studies of the glucosylation reaction catalyzed by amyloglucosidase from 
Rhizopus sp. leading to the synthesis of curcuminyl-bis-α-D-glucoside 30a and b from D-
glucose 2 and curcumin 19 was investigated in detail. Initial reaction rates (v) were 
determined from kinetic runs involving different concentrations of D-glucose and 
curcumin (0.005 to 0.1 M). Graphical double reciprocal plots showed that the kinetics of 
the amyloglucosidase catalyzed reaction followed Ping-Pong Bi-Bi mechanism where 
competitive substrate inhibition by curcumin led to dead end amyloglucosidase-curcumin 
complex at higher concentrations of curcumin. An attempt to obtain the best fit of this 
kinetic model through computer simulation yielded in good approximation, the values of 
four important kinetic parameters, kcat = 6.07±0.58 10
-5 Mh-1.mg, Ki = 3.0±0.28 mM, Km 
D-glucose = 10.0±0.9 mM and Km curcumin  = 4.6±0.5 mM. 
Chapter FIVE describes the Angiotensin Converting Enzyme (ACE) inhibition 
activity of the synthesized glycosides. Out of all the synthesized glycosides n-octyl-D-
glucoside 13a-c, n-octyl maltoside 15, n-octyl sucrose16a and b, guaiacyl-α-D-glucoside 
168 
 
 
  Summary 
 
 
21a and b, guaiacyl-α-D-galactoside 23a and b, eugenyl-α-D-glucoside 24a and b, 
eugenyl-α-D-mannoside 26, eugenyl maltoside 27a-c, eugenyl sucrose 28a-c, eugenyl-D-
mannitol 29, curcuminyl-bis-α-D-glucoside 30a and b, curcuminyl-bis-α-D-mannoside 
32, curcuminyl bis maltoside 33a-c, curcuminyl bis sucrose 34a-c, curcuminyl-bis-D-
mannitol 35 and tocopheryl-α-D-glucoside 36 were found to be ACE inhibitors. ACE 
inhibition activity (IC50 values) in the range 0.5±0.04 to 5.3±0.51 were obtained for the 
glycosides tested. Of these eugenyl-α-D-glucoside 24a and b exhibited the best ACE 
inhibition activity (IC50 value 0.5±0.04 mM). 
Thus the present investigation has brought out clearly the glycosylation 
potentialities of amyloglucosidase from Rhizopus sp. and β-glucosidase from sweet 
almonds of n-alkanols and phenols like guaiacol, eugenol, curcumin and α-tocopherol, 
explored with diverse carbohydrate molecules. 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
169 
 
 
  Summary 


STUDIES ON THE PREPARATION AND CHARACTERISATION OF PROTEIN HYDROLYSATES FROM GROUNDNUT AND SOYBEAN ISOLATES A

BASHEER, SHABANA

Coffee is an important plantation crop belonging to the family Rubiaceae, subfamily Cinchonoideae and tribe Coffeae (Clifford et al 1989).  The Rubiaceae members are largely tropical or subtropical with nearly 400 genera and 4800-5000 species.  ‘Quahweh’ is the Arabic word from which originated the name ‘coffee’. The word was a poetic term used for wine, which is forbidden for strict muslims. Therefore, the name was transferred to coffee, and through its Turkish equivalent quahweh became cafe (French), caffe (Italian), Kaffee (German), koffie (Dutch) and coffee (English) and Latin Coffea for the botanical genus (Smith 1985).  The stimulatory effects of roasted coffee beans were well known to the natives of Africa when the Arabs brought Coffea arabica seeds from Ethiopia to Yemen (Arabian Peninsula) during the 13th century, and established the first plantations (Monaco et al 1997). 
Classification:
  Kingdom	Plantae (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Plantae|u|140|+63​) 
       Subkingdom	Tracheobionta (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Tracheobionta|u|140|+63​) 
          Superdivision	Spermatophyta (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Spermatophyta|u|140|+63​) 
             Division	Magnoliophyta (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Magnoliophyta|u|140|+63​) 
                Class	Magnoliopsida (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Magnoliopsida|u|140|+63​) 
                   Subclass	Asteridae (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Asteridae|u|140|+63​) 
                      Order	Rubiales (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Rubiales|u|140|+63​) 
                         Family	Rubiaceae (​http:​/​​/​plants.usda.gov​/​classification​/​output_report.cgi?3|S|Rubiaceae|u|140|+63​) 
                            Genus	Coffea 


The success of transgenic research in Coffee has opened up new avenues for quality improvements. A number of transformation systems have been reported for arabica and robusta coffee, however only a few reports demonstrated the recovery of whole plant with stable integration of DNA. Rapid in vitro regeneration protocol is a basic need for the success of transgenic research in any plant species. A major hindrance for genetic transformations in coffee is slow rate of in vitro regeneration. 
Transformation has been reported using the biolistic methods (Van Boxtel et al, 1995), DNA electroporation using protoplasts (Barton et al 1991) and various Agrobacterium systems (Ocampo and Manzanera 1991, Grezes 1993, Spiral et al 1993, Leroy et al 1997, Leroy et al 2000, Fernandez-Da Silva 2003). The first report of stable transformation of coffee plant used a protoplast system for electroporation (Barton et al 1991). Barton et al (1991) reported the recovery of Coffea arabica plantlets after selection of transformed protoplasts on kanamycin and regeneration. However the root system of the regenerated transgenic plants was not well developed and plants capable of flowering were not produced. 
Although many transformation systems have been investigated, most of the current work utilized the standard Agrobacterium tumefaciens mediated gene delivery system, despite the relatively low rate of transgenic plant production. Transgenic coffee plants containing the cry1A(c) gene were produced using both the Agrobacterium rhizogenes and Agrobacterium tumefaciens systems (Leory et al 1997, Leroy et al 2000). The cry1A(c) encodes for an insecticidal protein that is toxic to certain insects including the coffee minor, Perileucoptera coffeela (Oliveiro et al, 1998). Sugiyama and co workers (1995) reported Agrobacterium rhizogenes mediated transformation and regeneration plants from transgenic roots of Coffea arabica cv typica. However, All the plantlets reported to be with typical rol gene phenotype, short internodes and stunted growth. Spiral and co-workers (1999) found that the npt II kanamycin resistance was not an effective selection marker in Coffea somatic embryos. Transgenic selection with chlorosulfuron resistance was reported to be effective (Spiral et al 1999). In Coffea sp. the frequency of transgenic plants produced is considerably below that of many other transformation systems (Leroy et al 1997). In Agrobacterium tumefaciens system, 0.4% of the somatic embryos infected gave transgenic plants (Leroy et al 1997). However improvements in the selection systems have recently been reported. Leroy and co workers reported GUS gene expression after 10-12 months of selection under chlorosulfuron (Leroy et al 2000). Ocampo and Manzanera (1991) demonstrated that C. arabica tissues could be infected by wild strains of Agrobacterium tumefaciens. Hatanaka and co-workers (1999) reported stable transformation and regeneration of transgenic Coffea canephora. The authors adopted Agrobacterium tumefaciens strain EHA101 harbouring pIG121-Hm consisting of β glucournidase (uid A), hygromycin phosphotransferase (hpt II) marker gene for transformation. Grezes and co-workers (1993) reported A. tumefaciens mediated genetic transformation. Van Boxtel (1994) transformed coffee using biolistic gene delivery method. Studies on five selection markers i.e., chlorsulfuron, glufosinate, glyphosate, hygromycin and kanamycin indicated the potential of glufosinate for the detection of stably transformed coffee tissue (Van Boxtel 1994). Kanamycin resistant plantlets were obtained from coffee leaf segments through Agrobacterium mediated transformation (Naidu et al 1998). De Pena (1995) reported optimization of conditions for direct DNA uptake using the poly ethylene glycol (PEG) method. Fernandez-Da Silva (2003) established an electroporation mediated genetic transformation method of coffee (Coffea arabica cv. Catimor) and incorporation of BAR gene for ammonium glufosinate resistance. Fernandez-Da Silva and Menendez (2003) reported optimization of gene delivery to C. arabica by electroporation mediated transformation


The physiological effect of caffeine has been dealt in detail in the following references (Anonymous 1987, www.ico.org, Tavani and Vecchia 2004). Soon after drinking a cup of coffee, or tea or cola, caffeine is distributed throughout the body. However, caffeine does not accumulate in the body, so its effects are short-lived and transitory. The caffeine half-life ranges from 0.7-1.2 h in rats, 3-5 h in monkeys, 2.5-4.5 h in humans and about 80 ± 23 h. in newborn infants (Reviewed by Nehling 1999). Effect of caffeine varies depending on a number of factors. Every individual reacts differently to caffeine. For example, caffeine may stay in the body of pregnant women for up to 3 times as long as is usual in adults, whereas smokers eliminate caffeine twice as quickly as non-smokers. Some of the effects of caffeine, such as those on the heart and blood vessels, are contradictory and have no net effect - others may only be noticeable when regular consumers suddenly cut out caffeine. The body can become habituated to caffeine so that regular users are less sensitive to the stimulant effects than others (Reviewed by Nehling 1999).
Of all the physiological effects of coffee, the best known is that it is a stimulant to the nervous system. One or two cups of coffee can make one feel more awake, alert and able to concentrate. However, in sensitive individuals, caffeine may delay the onset of sleep, decrease sleep time and even lower the subjective quality of the sleep. Caffeine has various effects on mood, ranging from pleasant stimulation and mood elevation to anxiety, nervousness and irritability, but these are transient and dose-related. Effects of caffeine have given rise to the development of several methods for removal of caffeine from beverages (Reviewed by Nehling 1999).



A programme of genetic breeding was initiated in 1987 to obtain low caffeine lines at the Instituto Agronomico de Campinas, 3,000 coffee trees, representing 300 C. arabica accession from Ethiopia have been screened. Silverolla et al (2004) discovered three caffeine free plants obtained from Ethiopia and designated as AC1, AC2 and AC3. These plants found to possess theobromine but not caffeine indicating that the plants might be deficient in enzyme caffeine synthase. The results of radiolabel study demonstrated that the low caffeine trait of these plants is not because of rapid degradation, but due to deficiency of caffeine synthase activity.


Biotechnology has come a long way to play its role in the well being of human. The progress in the transgenic research led to the development of genetic manipulation strategies for specific traits. The compiled information provides an idea about the latest developments in Coffee biotechnology area. Several workers have reported somatic embryogenesis, formation of embryogenic and non-embryogenic callus in coffee. However, information is lacking on what are the factors, which determine embryogenic nature in coffee. Development of rapid regeneration protocols in coffee is a key factor for optimizing genetic transformation. So far it was not possible to transform coffee and regenerate plants devoid of rol gene phenotype by A. rhizogenes mediated transformation. Several physical and chemical factors may influence the transformation efficiency by A. rhizogenes in plants. Information on influence of sonication, calcium ions, cellulase and pectinase enzyme on A. rhizogenes mediated transformation frequency is completely lacking. The need for naturally decaffeinated coffee can be answered in a better way by molecular breeding of coffee.  Alternative gene transfer methods such as electroporation seem to be a good approach to analyze promoters to drive the expression of reporter genes in coffee. With this background a series of experiments were designed for plant regeneration through somatic embryogenesis and genetic transformation of coffee with the following objectives. The results of these studies form the substance of this thesis. 








STUDIES ON BIODEGRADATION OF NITROPHENOL ISOMERS BY MIXED BACTERIAL CULTURES

NM, SACHINDRA

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STUDIES ON SOME CRUSTACEANS OF TROPICAL WATERS WITH SPECIAL REFERENCE TO PIGMENTS

BIDLAN, RAJKUMAR




Synopsis of the Thesis

Title of the Thesis: Studies on DNA topology and DNA-Amyloid Beta (A) peptides interaction in relevance to Alzheimer’s disease.


Name of the candidate: S.Anitha 


Alzheimer's disease (AD) is a complex neurodegenerative disorder of multiple etiology.  It is a devastating dementia, correlated with a region specific neuronal cell loss. The complex neuropathology includes cortical atrophy, formation of neurofibrillary tangles, deposition of amyloid β peptide (Aβ) in senile plaques, synaptic loss, oxidative stress, metal deposition, and apoptosis and all these coupled events lead to the neuronal loss. Despite progress in uncovering many of the factors that contribute to the etiology of the disease, the cause of the nerve cell death still remains unknown. It is very well accepted that conformational changes in the proteins namely Aβ play an important role in the neurodegeneration of AD, however little is known about the DNA conformational alteration.  The changes in the genetic material of AD, so far documented are, i) mutations in genes, amyloid precursor protein (APP), presenilin-1 (PS-1) and presenilin-2 (PS-2), ii) shift in the DNA from a potentially transcribable euchromatic state to a condensed and probably heterochromatic state,  iii) oxidative damage as revealed by damaged products namely 8-OH Guanosine and 8-OH Cytosine and iv) genetic imbalance caused by the tetraploidy status of the genome by the way of unusual replication before the cell death . However there is no data on DNA topology in AD brain.  Hence the present study aimed to understand studies pertaining to DNA topology and A-DNA interactions in relevance to neurodegeneration in AD brain.




DNA Topology in AD brain

 Brain cells are very complex and have high level of gene expression, 3-4 times more than that found in liver and kidney.  In brain cells where the level of gene expression is very high, accumulated DNA damage may have serious consequences in terms of altering genomic integrity consequently affecting the transcriptional fidelity and ultimately the survival of the organism.  It was speculated that accumulated DNA damage in the brain might result in conformational changes in the DNA making it ineffective during transcription. Nevertheless the sequence and the DNA topology have been proposed to have 
a crucial role in DNA functional aspects like replication, transcription, and susceptibility of DNA for oxidative damage and mutations.  Extensive studies on handedness and various high ordered structures were studied for the oligonucleotides, but little is known about genomic DNA topology with respect to non Watson-Crick right-handed B-DNA forms, their functional ability and possible implications in the pathogenic features in the brain. 
	
Our study on genomic DNA isolated form the hippocampus region of the brain of AD affected patients revealed altered DNA conformation. Hippocampus was selected as it is one of the first brain regions affected in AD and is involved in the processing of short-term memory and spatial memory. The DNA was isolated form moderately affected (MAD), severely affected (SAD) AD brain and control aged (CA), and control young (CY) brain hippocampus. The Circular dichroism study revealed that SAD DNA showed a typical left-handed Z-DNA conformation whereas CY and CA brain DNA have the usual Watson-Crick B- DNA conformation. Interestingly, MAD DNA has modified B-DNA conformation (probable B-Z intermediate form). Immunochemical study with anti-Z-DNA IgG polyclonal antibody revealed that only the SAD DNA have the reactivity with the antibody, confirming left-handed Z-DNA conformation in SAD. The ethidium bromide binding pattern to DNA and melting temperature (Tm) profiles also revealed the conformational transition from B to Z- DNA in SAD brain. Agarose gel studies of CY and MAD DNA did not show any fragmentation. Both CA and 


SAD DNA were fragmented as evidenced by their smear migration pattern. It is interesting to mention that even though there is no difference between CA and SAD DNA as both of them have smeared pattern, EtBr staining is faint for SAD DNA. And this is due to its Z-DNA conformation. The conformational alteration has also been seemed to follow the disease progression as MAD has B-Z intermediate form and SAD has Z-DNA form.   The study was also extended to DNA isolated from other regions of the brain, superior frontal gyrus and parahippocampal gyrus, which are also shown to be implicated in the AD. However no conformational alteration in DNA was observed in these two regions. The results reveal that the conformational alteration seems to be region specific. Estimation of DNA single strand (by Nick Translation assay) and double strand breaks (by Terminal transferase assay) in the three regions of the control and AD brain revealed more breaks in hippocampus. The topological change in DNA,  particularly the B to Z-transition in the hippocampus, will have tremendous implications in the functional biology of the brain cells e.g.  transcription, replication and recombination. Based on the above results we hypothesized that AD etiological factors such as like Aβ, phosphorylated tau, metals and oxidative stress might play role in the conformational transition.  AD specific proteins like Aβ and phosphorylated tau are lysine rich and studies have shown that lysine rich proteins favor the conversion of BZ DNA transition. Tau was reported to bind to and stabilize the DNA. We investigated the A binding to DNA. 

Amyloid  (A) peptide DNA interaction

A, a hallmark feature of the senile plaques, is a proteolytic product of the transmembrane amyloid precursor protein. Evidence implicates a central role for A in the pathophysiology of AD.  According to the amyloid cascade hypothesis, the accumulation of A deposits as amyloid plaques in the patient’s brain is the primary event in the pathogenesis of AD.  Under pathogenic conditions, the transition from a random coil to a -sheet conformation in -amyloid peptide causes deposits of the amyloid fibrils. The apparent role of A, especially A (1-
42) (the number in the bracket indicates the number of amino acids of the amyloid precursor protein sequence) is now considered as a unifying pathological feature of diverse forms of AD. In vitro studies have shown that A (1-42) rapidly aggregates into fibrils and that extracellular fibrillar A peptides induce apoptosis in cultured neurons. On the other hand, recent reports have demonstrated intraneuronal accumulation of A (1-42) in AD vulnerable regions.  Intraneuronal A (1-42) accumulation has also been reported in transgenic mice expressing familial AD proteins. In the present study, we provided new evidence for A immunoreactivity in the nucleus of apoptotic hippocampal cells of AD brain.  Based on the evidence for nuclear localization of A in AD brain, we hypothesized that A might play a role in modulating DNA topology and possibly contribute to the BZ-DNA helical transition associated with AD. 

In the present study, we evidenced that the Aβ binds to supercoiled DNA (scDNA).   Further A (1-42) not only binds to scDNA but also is able to alter the superhelicity of DNA.   An initial BC transition was observed which gradually transformed into -DNA, presumably reflecting a partial DNA collapse into a -phase.   In -DNA, the DNA molecules are tightly packed in to toroidal superhelical bundle whose chiral sense is defined by the intrinsic DNA handedness.   Specifically, the right-handed secondary conformations such as the B-DNA and C-DNA motifs stabilize a left-handed tertiary conformation. The -DNA conformation induced by A (1-42) is structurally closer to Z-DNA, which was observed in SAD brain. This evidently indicates that DNA topological change induced by A is similar to the change seen in AD brain DNA.  Other A peptides like A (1-16), A(1-28) andA (1-40) caused modified DNA forms in sc DNA. Our study also provided intriguing observation of the sequence specificity of the A peptide binding. The A peptides showed preferential binding to AT rich sequences (poly d(AT) (AT)) and caused BA DNA transition. A (25-35), the hydrophobic region of A (1-42) and another potent neurotoxic A fragment has been shown to aggregate the sc DNA. Hence the results are novel interms of their DNA binding property. 




Amyloid induced DNA damage: New toxic role

The proposed mechanisms for neurotoxicity for A are diverse and there is no unifying mechanism for the toxicity and one of the mechanisms proposed was induction of apoptosis.   Under in vitro conditions A (1-42) is reported to be capable of inducing apoptosis in cultured cortical neurons. Neuronal cells treated with A exhibit morphological and biochemical characteristics of apoptosis, including membrane blebbing, compaction of nuclear chromatin and internucleosomal DNA fragmentation. Interestingly studies also suggested that 
A exert its toxic effect via activation of transcription factors. Studies also correlated between the DNA damage and amyloid deposits however the mechanism remains unclear.

             Recent debate with in the AD community has focused on whether fibrillar (amyloid) or soluble oligomers of A are active species of the peptide that ultimately cause the synaptic loss and dementia associated with AD.  Studies suggest that A-dependent toxicity can occur before significant extracellular accumulation; possibly involving soluble intracellular A forms. 

Our study provided fundamental contribution that A peptide (1-42) could cause DNA damage directly. Agarose gel studies reveal that A (1-42) caused open circular and linear form of DNA in sc DNA by inducing single strand and double strand breaks. Transmission Electron Microscopic (TEM) study also revealed formation of open circular and linear of sc DNA induced by A peptide (1-42). Time dependent DNA nicking of A (1-42)  revealed that the soluble forms of A (corresponding to low thio T binding) are more toxic than the fibrillar forms of A  (1-42) (corresponding to high thio T binding). Further metal ions Mg and Ca which solubilize the aggregated A enhance DNA nicking ability leading to the hypothesis that soluble forms of A are more toxic than the aggregated forms.  Zn metal ion which is shown to promote the aggregation of the A did not have any effect of DNA nicking of A. The DNA nicking was also shown by other A peptides having different amino acid lengths like A (1-16), A (1-28), A (1-40) and A (1-43). However there exists subtle differences in the DNA nicking caused various peptides and are differentially modulated by metals.  DNA nicking of A was inhibited by aurintricarboxylic acid (ATA), a potent nuclease inhibitor. We propose based on the enhancement/inhibition of DNA nicking by metals and ATA respectively that histidine residues are implicated in the DNA nicking activity. Both metals and ATA bind to histidine residues. ATA prevents the formation of the protein-DNA interaction thereby inhibiting the DNA nicking. Metals by binding to histidine residues might result in solubilization of the A and enhance toxicity. 
The results evidenced a new toxic role of A in terms of its direct DNA nicking activity and there by it alters the helicity of sc DNA.  Our finding of DNA nicking activity of A peptides has biological significance in terms of causing DNA damage. 


In a nut shell the study provides the following contributions.

	Evidenced topological change in genomic DNA in the hippocampus region of AD brain.
	Shown Nuclear localization of A in the nucleus in AD affected apoptotic hippocampal neurons.
	A binds to supercoiled DNA and causes helicity change.
	Provided an evidence on DNA nicking in terms of single strand and double strand breaks by A peptides.

The relevance of the above findings in understanding the neurodegeneration in AD have been discussed in the thesis. 




PAGE  



2





Studies on DDT degradation by bacterial strains

Mitbavkar, Smita S.

 
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German

Eco-biology of marine diatoms with emphasis on the influence of physico-chemical parameters

P.T., SANGEETHA

PAGE  



i

Synopsis



C
hitin, a poly  14–N-acetyl-D-glucosamine, is the most abundant and underutilized biomass resource in India.  It is estimated that fungi provide 3.2 x 104 metric tons of chitin; however, commercially it is found in the offal of marine food processing industry with an estimated 1.2 x 105 metric tons annually accessible on a world wide basis.  The processing of shellfish is of environmental concern as presently, only a small quantity of shell waste is utilized for animal feed or for chitin isolation.  Recently, the commercial importance of chitin has increased because of its beneficial properties leading to numerous industrial applications, such as, for the preparation of dietary fibers, bandages, cosmetics, and toiletries.  However, its insolubility or poor solubility in aqueous medium, buffers and other solvents precludes its large-scale application, whereas chitosan, a partially de-N-acetylated derivative of chitin, owing to its better solubility in aqueous acidic medium, biocompatibility and non-toxicity, exhibit characteristics of considerable use in health food, agriculture, medicine, biotechnology, cosmetics, pulp, textile and water-treatment.  

Pharmaceutical properties of chitosan such as drug delivery, antimicrobial, antitumor, immuno-potentiation, hypocholesterolemic, wound healing, etc. are of therapeutic significance.  Chitosan based microcapsules (spherical particles with varying size with core substance) and microspheres (spherical empty particles) are of use in controlled drug delivery formulations.  Due to its film forming property, chitosan has been used as food wraps, films and laminated films.  The potential utility of chitosan for the removal of undesirable metals such as mercury, lead, zinc, chromium, plutonium and uranium and separation of trace elements from sea water by chromatography on chitosan has been demonstrated by many.  Chitosan finds application in photography due to its resistance to abrasion and optical characteristics.  Upon neutralization with acid, chitosan is the only natural cationic gum that becomes viscous and hence of use in cosmetic creams, lotions, permanent waving lotions and nail lacquers, etc.  Ionic conductivity when dissolved in acetic acetic acid makes chitosan suitable for the preparation of solid-state batteries. 

 	Chitosan has a mean molecular mass of up to 106 Da, which corresponds to a chain length of approximately 5000 glucosamine and N-acetyl-glucosamine residues.  Due to very high molecular weight and thus high viscosity of chitosan in solution, which restricts its in vitro applications, it is advantageous and preferable too to use depolymerized products of chitosan, namely, low molecular weight chitosans (LMWC), chitooligomers and monomers, as they overcome the drawbacks of chitosan and also find additional novel applications.  Their preparation by conventional physical, chemical or enzymatic hydrolysis using chitinase/chitosanase is not practically feasible because of the high cost of the equipments required, low yield of the desired products, formation of monomers and oligomers of lower degree of polymerization (DP 2-3) and sometimes modification of the products formed, all of them affecting their biological activity and application utility.  Moreover, the enzymes chitinases/chitosanases are very expensive and not available in bulk for commercial exploitation.  Search for alternative cheap sources of enzymes has led to the identification of a few non-specific enzymes, which are inexpensive and easily available, to depolymerize chitosan and its chemically modified derivatives.

	Earlier studies from our lab have shown the pectinase isozyme from Aspergillus niger to exhibit a higher chitosanolytic activity at acidic pH.  Of the three bands in native-polyacrylamide gel electrophoresis, the fast moving one with polygalacturonase activity was shown to exhibit both pectinase and chitosanase activities.  The latter released chitooligosaccharides of DP 2-8 and LMWC of molecular weight ~20 kDa, upon chitosan depolymerization.  Following this, a few more non-specific enzymes such as, hemicellulase, lysozyme, pepsin, papain and pronase were screened for their chitosanolytic activities, which was the basis for the present investigation.  This study was carried out with the aim of determining the kinetic properties of some of these non-specific enzymes showing optimum chitosanolytic activities, standardizing the reaction conditions for obtaining the desirable products (LMWC, chitooligomers and monomers) in maximum yield, structural-functional characterization of the products formed and determining the mechanism of action of proteases towards chitosanolysis.  The results obtained have been consolidated in the form of a Ph.D. Thesis entitled “Chitosanolytic activity of non-specific enzymes – mechanism of action and potential application of the degradation products”, and having the following layout.

	Accordingly, Chapter I (General Introduction) gives a brief account of the history of chitin-chitosan, their structure-conformation, biosynthesis in crustaceans and fungi, which are the main sources, de-N-acetylation of chitin and application of chitosan in various fields such as agriculture, medicine, pharmaceutical, foods and cosmetics and finally the necessity of depolymerization of chitosan, and scope-objectives of the present investigation.  

 	Chapter II (Kinetic study of chitosanolytic activity of non-specific enzymes) begins with a brief introduction about non-specific chitosanolysis followed by the Material and Methods used during screening of enzymes for their chitosanolytic activities.  A detailed account of the preparation of chitosan and its purification-characterization (molecular weight Mr, by viscometry and gel permeation chromatography (GPC); degree of acetylation DA, by infrared (IR) and nuclear magnetic resonance (NMR) spectral data; and crystallinity index CrI, by X-ray diffractometry) are provided.  A few non-specific enzymes such as proteases (pepsin, papain, pronase and protease), lipases including carbohydrases other than chitinase and chitosanase were screened for their chitosanolytic activity.  The effects of enzyme-substrate concentration, solution viscosity, pH, temperature and reaction time are determined.  KM (substrate concentration at half maximal velocity) and Vmax (maximum velocity) are also calculated from Lineweaver-Burk (double-reciprocal) plot.  

	Pepsin, papain and pronase were used further for depolymerization of chitosan, which forms the subject matter of Chapter III (Purification of the lytic agent involved in the chitosanolysis).  In here, emphasis was given to purify non-specific enzymes and their purity was established by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary zone electrophoresis (CZE) in order to rule out the speculation of the earlier workers that the associated contaminants are responsible for the observed chitosanolytic activity of non-specific enzymes.  Fold increase in the activity after purification, the effects of various acids as chitosan solvents, DA and Mr on chitosanolysis, pH and temperature stabilities of the proteases during chitosanolysis and activation energy (Ea) calculation by Arrhenius plot have been provided in this chapter.  Probable amino acid residue(s) in the active site of these proteases involved during chitosanolysis is discussed.  Activity of proteases towards chitin-chitosan derivatives, as well as other polysaccharides such as starch, pectin, guar gum, gum arabic and carboxymethyl cellulose, which are made up of different monosaccharide units, is compared with that of chitosan.  

Chapter IV (Characterization of LMWC) encompasses a detailed account of structural characterization of LMWC, the major depolymerization product of chitosan.  GPC, HPLC and viscometric studies were carried out to determine Mr of the LMWC, whereas scanning electron microscopy (SEM) was performed to know the topographical changes.  Primary and higher order structures of LMWC were determined by employing various biophysical techniques such as IR, circular dichroism (CD) and NMR and X-ray diffraction.  The dissolution kinetics of LMWC was also studied.  

	Chitooligosaccharides (CO’s) and monomers, the other products of chitosan depolymerization are discussed in Chapter V (Characterization of Chitooligomers & Monomers).  The percent yield of CO’s-monomeric mixture as well as individual monomers and oligomers obtained were deduced by GPC and HPLC analyses.  Spectral details provided structural information of the monomers and oligomers.  Based on MALDI-TOF-MS analysis and percent increase in reducing equivalent after N-acetyl--glucosaminidase treatment, the probable ratio of the monomers and their sequence at the non-reducing terminals of the oligosaccharides were obtained.  The probable sites of action of these proteases during chitosanolysis are also described.

	Chapter VI (Value addition to chitosanolytic products) provides the biological application potential, especially as antibacterial agents, of LMWC and CO’s-monomeric mixture as well as their purified individual fractions.  Various food borne pathogens were screened for growth inhibitory actions of LMWC and CO’s-monomeric mixture, and for detailed study, Bacillus cereus and Escherichia coli (one each from Gram-positive and Gram-negative bacteria) were used.  The mechanism of bactericidal action was determined by SEM studies and analysis of the supernatants obtained after treating the test cultures with both LMWC and CO’s-monomers.  The supernatants were analyzed for total carbohydrate, protein and enzyme activities (namely maltase, lactase and protease) and later subjected to native-PAGE, HPLC and lipid analysis by gas-liquid chromatography (GLC).  One sample each from PAGE-separated protein samples of the supernatants were subjected to N-terminal amino acid analysis and compared with that of test culture proteins.  From the results, a probable mechanism of bactericidal action of LMWC and CO’s-monomers has been proposed.  In addition, DNA and RBC membrane protective actions of CO’s-monomeric mixture as well as of individual mono-/oligomers were studied.

	Chapter VII (Mechanism of chitosanolysis by Pepsin) is an extension of chapter III, in that the purity and mechanism of action of pepsin during chitosanolysis is described at length.  The commercial porcine pepsin, which showed multiple bands/peaks upon native-PAGE, CZE and HPLC, was subjected to purification by GPC.  By zymogram analysis (activity staining), it was showed that the purified enzyme was associated with dual functions, i.e., proteolysis and chitosanolysis.   Its Mr and N-terminal amino acid sequence, confirmed it to be pepsin.  Use of active site specific inhibitors as well as covalent modification of the active site residues indicated the involvement of aspartic acid during depolymerization of chitosan, which are also responsible for proteolytic action of pepsin.  A comparative study of the 3-D structures of chitinase, chitosanase and pepsin led to their possible/probable mechanism of action of chitosanolysis.  The results obtained thus unambiguously established that pepsin is indeed a good chitosanolytic agent, and this depolymerization is not caused by any contaminating enzyme, as speculated by others, before.  

	A brief account of Summary and Conclusions is given in Chapter VIII.  Finally, a collective list of references (Bibliography), which form the basis for logical interpretation of the data obtained in comparison with earlier and contemporary published results, is provided.
















































































































MICROBIAL PRODUCTION OF FRUCTOOLIGOSACCHARIDES

Kunte, Pravin D.

Enzymatic synthesis of selected amino acid esters 
of sugars 
 
 
 
Synopsis  
Submitted to 
University of Mysore 
 
 
for the degree of 
Doctor of Philosophy 
in 
 Chemistry 
 
 
 
By 
K. Lohith 
Registration No. No. Ex 9.2/Ph.D/KL/891/03-04 
  
 
Guide: Dr. S. Divakar, 
  Fermentation Technology and Bioengineering Department 
Central Food Technological Research Institute 
Mysore- 570020 
 
 
 1 
 
Introduction: 
Enzymatic synthesis is advantageous compared to chemical synthesis in many 
ways. Very few reports are available on the synthesis of amino acyl esters of 
carbohydrates, which involves mainly proteases, protected and activated amino acids and 
carbohydrates (Maruyama et al. 2002; Park et al. 1996; 1999; Riva et al. 1988).    
However, there are practically no reports on the synthesis of amino acyl esters of 
carbohydrates using unprotected and unactivated amino acids and carbohydrates with 
lipases as catalysts. The present work describes lipases catalysed synthesis of amino acyl 
esters of few carbohydrates using unprotected and unactivated amino acids and 
carbohydrates. Amino acyl esters of carbohydrates possess wide variety of applications in 
food and pharmaceutical industries as sweeteners, surfactants, microcapsules in 
pharmaceutical preparation, anti viral nucleoside amino acids, antitumor agents and 
intermediates in the synthesis of biologically active peptides (Krik 1992; Zaks and Dodds 
1997). Use of lipases in non-polar solvents for the synthesis of amino acyl esters of 
carbohydrates offers many advantages like milder conditions, low temperature, stereo and 
regio selectivity, easy work out procedures, improved product yields, less byproducts and 
no colouring of products (Klibanov 1986).  
Work done: 
 Chapter ONE deals with literature survey on mainly lipase catalysed esterification 
reactions. Applications of lipase catalysis in different food and pharmaceutical industries 
are discussed. Factors affecting esterification reaction like nature of substrates, nature of 
solvents, thermal stability of lipases, water activity and immobilization are discussed. 
Different strategies in lipase catalysis, like esterification using reverse micelles, 
 2 
 
supercritical carbon dioxide and micro oven assisted reactions are presented. Kinetics of 
lipase catalysis and optimization of reaction parameters using response surface 
methodology are also described. The chapter ends with a brief description on the scope of 
the present investigation.   
 Chapter TWO deals with materials and methods. Chemicals employed and their 
sources are listed. Methods of preparation of L-amino acyl esters of carbohydrates and the 
other related appropriate aspects of the same are discussed in detail.    
Chapter THREE describes results from investigations on the synthesis of L-
phenylalanyl-D-glucose 24a-e and L-phenylalanyl-lactose 29a-c using lipases from 
Rhizomucor miehei (RML), porcine pancreas (PPL) and Candida rugosa (CRL). 
Enzymatic esterification between L-phenylalanine 2 with D-glucose 5 and lactose 11 
using RML, PPL and CRL were investigated in terms of incubation period, solvent, 
enzyme concentrations, substrate concentrations, buffer salts (pH and concentration) and 
enzyme reusability. Under the experimental conditions employed, all the three lipases 
exhibited good esterification potentialities. Among the various solvents mixtures 
employed for the synthesis of L-phenylalanyl-D-glucose 24a-e, CH2Cl2 and DMF (90:10 
v/v) was found to be the best. Both PPL and RML showed maximum conversion yields of 
L-phenylalanyl-D-glucose 24a-e (98% and 76% of respectively) at 40% (w/w D-glucose) 
of enzyme employed and CRL showed a maximum conversion of 64% of L-phenylalanyl-
lactose 29a-c at 50% (w/w lactose) enzyme concentration. In presence of buffer salts, 
conversion yields enhanced by 10% in case of RML and PPL whereas in case of CRL 
more than 30% esterification enhancement was observed. At higher equivalents of 
substrates, decrease in conversion yields observed could be due to inhibition at higher 
 3 
 
concentrations of L-phenylalanine 2, D-glucose 5 and lactose 11. In the synthesis of L-
phenylalanyl-D-glucose 24a-e RML could be reused upto four cycles where as PPL could 
used only upto two cycles.   
 Reaction conditions for Rhizomucor miehei lipase (RML) catalysed synthesis of L-
phenylalanyl-D-glucose 24a-e using unprotected L-phenylalanine 2 and D-glucose 5 were 
optimized using Response Surface Methodology (RSM).  A Central Composite Rotatable 
Design (CCRD) was employed involving 32 experiments of five variables (L-
phenylalanine concentration in mmol, amount of RML in mg, pH, incubation period in h 
and buffer concentration in mM) at five levels. A second order polynomial equation was 
developed in terms of linear, quadratic and cross product terms to study the effects of 
variables on esterification yields. Surface and contour plots obtained, explained the 
esterification behaviour clearly. An optimum predicted yield of 1.01 mmol for L-
phenylalanyl–D-glucose at 3 mmol L-phenylalanine, 100 mg of RML, 24h incubation 
period, 0.5 mM (0.5 mL of 0.1M buffer), pH 4.8 acetate buffer was found to agree with 
0.97 mmol obtained under these experimental conditions. Validation experiments carried 
out under random conditions also exhibited good correspondence between predicted and 
experimental yields. 
 Chapter FOUR describes the syntheses and characterization of L-prolyl 1, L-
phenylalanyl 2, L-tryptophanyl 3 and L-histidyl 4 esters of carbohydrates (D-glucose 5, D-
galactose 6, D-mannose 7, D-fructose 8, D-arabinose 9, D-ribose 10, lactose 11, maltose 12, 
sucrose 13, D-mannitol 14, D-sorbitol 15). Esterification was carried out using lipase from 
Candida rugosa (50% w/w carbohydrate) in 100 mL of CH2Cl2 and DMF (90 : 10 v/v, 40 
o
C) containing 0.2 mL of 0.1M (corresponds to 0.2 mM in 100 mL solvent) pH 4.0 
 4 
 
acetate buffer for 72 h incubation. Candida rugosa lipase (CRL) showed broad substrate 
specificity towards amino acids as well as carbohydrates. Esterification yields were 
obtained in the range of 7-79%. About 29 L-prolyl (16a-c – 23a and b, L-phenylalanyl 
(24a-e – 31a and b), L-tryptophanyl (32a-e – 39a and b) and L-histidyl (40a-e – 44) esters 
of carbohydrates were prepared, of which 19 esters have not been reported before. These 
are: L-prolyl-D-glucose 16a-c, L-prolyl-D-galactose 17a-c, L-prolyl-D-mannose 18a-e, L-
prolyl-D-ribose 19a and b, L-prolyl-D-fructose 20a-c, L-prolyl-lactose 21a and b, L-
prolyl-maltose 22a-c, L-prolyl-D-sorbitol 23a and b, L-phenylalanyl-D-arabinose 28a-c, L-
tryptophanyl-D-mannose 33, L-tryptophanyl-D-galactose 34, L-tryptophanyl-D-fructose 
35a and b, L-tryptophanyl-lactose 36a and b, L-tryptophanyl-maltose 37a-d, L-histidyl-D-
glucose 40a-e, L-histidyl-D-mannose 41a and b, L-histidyl-D-fructose 42, L-histidyl-
maltose 43a-c and L-histidyl-D-mannitol 44. Aldohexoses (5-7), ketose (8) and 
disaccharides (11 and 12) showed better conversions with all the four L-amino acids 1-4. 
L-Tryptophan 3 showed lesser conversion (7-70%) to esters compared to the other three 
amino acids (20-79%). L-Tryptophanyl-D-galactose 33, L-tryptophanyl-D-mannose 34, L-
tryptophanyl-sucrose 38, L-histidyl-D-fructose 42 and L-histidyl-D-mannose 44 formed 
only one ester. D-Glucose gave five diastereomeric esters with L-phenylalanine 24a-e, L-
tryptophan 32a-e and L-histidine 40a-e and the only exception being L-proline where only 
three monoesters 16a-c are formed. Characterization of the isolated product esters (16a-c 
– 44) using UV, IR, mass and NMR spectroscopy were carried out.  Molecular ion peaks 
in mass spectrum further confirmed the formation of esters. Two-dimensional HSQCT 
NMR spectroscopy of the product esters (16a-c – 44) gave good information on the 
nature and proportion of the esters formed. Nature of the products clearly indicated that 
 5 
 
primary hydroxyl groups of the carbohydrates (1-O-, 5-O-, 6-O-, 6′-O- and 6,6′-di-O-) 
esterified predominantly over secondary hydroxyl groups (2-O-, 3-O- and 4-O-). 
Anomeric region in NMR clearly indicated that both α and β anomers reacted in case of 
D-glucose 5, D-galactose 6, D-mannose 7, D-arabinose 9, D-ribose 10, lactose 11 and 
maltose 12.  
Chapter FIVE describes kinetic study of the esterification reaction between D-
glucose 5 and L-phenylalanine 2 catalyzed by lipases from Rhizomucor miehei (RML) and 
Candida rugosa (CRL). Detailed investigation showed that both RML and CRL followed 
Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical 
double reciprocal plots and computer simulative studies showed that competitive double 
substrates inhibition at higher concentrations leading to dead-end inhibition in case of 
RML by both L-phenylalanine and D-glucose and in case of CRL, only by D-glucose at 
higher concentrations leading to dead end lipase-D-glucose complex. An attempt to obtain 
the best fit of these kinetic models through curve fitting yielded in good approximation, 
the values of important kinetic parameters, RML: kcat = 2.24±0.23 mM/h.mg protein, Km 
L-phenylalanine= 95.6±9.7 mM, Km D-glucose = 80.0±8.5 mM, Ki L-phenylalanine = 90.0±9.2 mM, Ki 
D-glucose = 13.6±1.42 mM; CRL: kcat = 0.51±0.06 mM/h.mg protein, Km L-phenylalanine = 10.0± 
0.98 mM, Km D-glucose  = 6.0 ± 0.64 mM, Ki D-glucose  = 8.5 ± 0.81 mM. 
Chapter SIX describes potentiality of L-prolyl-D-glucose 16a-c, L-prolyl-D-fructose 
19a-c, L-prolyl-D-ribose 20a and b, L-prolyl-lactose 21a and b, L-phenylalanyl-D-glucose 
24a-e, L-phenylalanyl-D-galactose 26a-e, L-phenylalanyl-D-fructose 27a and b, L-
phenylalanyl-lactose 29a-c, L-phenylalanyl-D-mannitol 31a and b, L-tryptophanyl -D-
glucose 32a-e, L-tryptophanyl -D-fructose 35a and b, L-histidyl-D-glucose 40a-e, L-
 6 
 
histidyl-D-fructose 42 and L-histidyl-D-mannitol 44 as inhibitors towards Angiotensin 
Converting Enzyme (ACE) activity. Amino acyl esters of carbohydrates tested for ACE 
inhibition activity showed IC50 values for ACE inhibition in the 0.9 mM to 13.6 mM 
range. L-phenylalanyl-D-glucose (IC50: 1.0±0.09 mM), L-tryptophanyl-D-fructose 35a and 
b (IC50: 0.9±0.09 mM) and L-histidyl-D-fructose 42 (IC50: 0.9±0.09 mM) showed the best 
ACE inhibitory activities.  
The present investigation has thus demonstrated the potentiality of RML, PPL and 
CRL to syntheses biologically and nutritionally active amino acyl esters of carbohydrates 
(16a-c – 44) using unprotected and unactivated L-amino acids and carbohydrates.  
 
 
References: 
Maruyama, T., Nagasawa, S. I., Goto,  M. (2002). Enzymatic synthesis of sugar esters in 
organic solvents.  J. Biosci. Bioeng. 94 4), 357-361. 
 
Park, O. J., Jeon, G. J., Yang, J. W. (1999). Protease catalysed synthesis of disaccharide 
amino acid esters in organic media.   Enzyme Microb. Technol. 25, 455-462. 
 
Park, O. J., Park, H. G., Yang, J. W. (1996). Enzymatic transesterification of 
monosaccharides and amino acid esters in organic solvents.  Biotechnol. Lett. 18, 
473-478. 
 
Riva, S., Chopineau, J., Kieboom, A. P. G., Klibanov, A. M. (1988). Protease catalysed 
regioselective esterification of sugars and related compounds in anhydrous 
dimethylformamide.  J.  Am.  Chem.  Soc. 110, 584-589. 
 
Kirk, O., Bjorkling, F., Godfredsen, S. E., Larsen, T. S. (1992). Fatty acid specificity in 
lipase catalysed synthesis of glucoside esters. Biocatalysis. 6, 127-134. 
 
Zaks, A., Dodds, D. R. (1997). Applications of biocatalysts and biotransformations to the 
synthesis of pharmaceuticals. Drug Dev. Today. 2, 513 –531. 
 
 7 
 
Klibanov, A.M. (1986). Enzymes that work in organic solvents. Chem. Technol. 16, 354-
359. 
 
Kunz, H., Kullumann, R. Metal ion promoted synthesis of amino acyl esters of 
carbohydrates. Tetrahedron Lett. 1992, 33(41), 6115. 
 
 
 
 
 
 
     Signature of the candidate                    Signature of the guide 
          (K.Lohith)                                   (Dr. S. Divakar) 
 


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