5,772 research outputs found
Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring
No full textThe functionally related amidohydrolases, including D-hydantoinases, dihydropyrimidinases, allantoinases and dihydro-orotases, share a similar catalytic function of acting on the cyclic amide ring. We aligned 16 amidohydrolases by taking account of the conservative substitution and found a number of highly conserved regions and invariant amino acid residues. Analyses of the secondary structure and hydropathy profile of the enzymes revealed a significant degree of similarity in the conserved regions. Among the regions, the long stretched region I is of particular interest, because it Is mainly composed of invariant amino acid residues, showing a similarity of 69 % for the enzymes. A search of the protein data bank using the sequence of the conserved region I identified a number of proteins possessing a similar catalytic property, providing a clue that this region might be linked with the catalytic function. As a particular sequence, one aspartic acid and four histidine residues are found to be rigidly conserved in the functionally related amidohydrolases. In order to investigate the significance of the conserved residues, site-directed mutagenesis was carried out typically for the D-hydantoinase gene cloned from Bacillus stearothermophilus SD1. These residues were found to be essential for metal binding as well as catalysis, strongly implying that these invariant residues play a critical role in other enzymes as well as in D-hydantoinase. On the basis of the similar catalytic function and existence of the rigidly conserved sequence, we propose a close evolutionary relationship among the functionally related amidohydrolases, including D-hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase
Enhancement of operational stability of immobilized whole cell D-hydantoinase
No full textA bacterium Pseudomonas sp. KBEL 101 isolated from soil was immobilized within polyacrylamide gel and used for the synthesis of D-p-hydroxyphenylglycine from DL-5-substituted hydantoin. The half-life of immobilized whole cell D-hydantoinase was found to be about 50 hrs. In order to increase the operational stability of immobilized whole cell D-hydantoinase, a carbon or nitrogen source was supplied with the reaction mixture in the continuous stirred tank reactor. As a sole source of carbon, glycerol was found to be most effective, and the activity of immobilized whole cell enzyme was maintained stably during 7 days when O.1%(W/V) glycerol solution was provided. In the case of nitrogen source, supplying of O.1%(W/V) yeast extract prolonged the half-life of immobilized whole cell D-hydantoinase to about 25 days
6th issue of Experimental Software and Toolkits (EST-6)
No full textScience of Computer Programming Volume 134, Pages 1-112 (1 February 2017) 6th issue of Experimental Software and Toolkits (EST-6) Edited by Mark G.J. van den Brand, Jurgen J. Vinju and Kim Men
Characterization and evaluation of a distinct fusion ability in the functionally related cyclic amidohydrolase family enzymes
No full textThe cyclic amidohydrolase family enzymes, which include allantoinase, dihydroorotase, dihydropyrimidinase and (phenyl)hydantoinase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in vivo. Each enzyme has been independently characterized, and thus well documented, but studies on the higher structural traits shared by members of this enzyme family are rare due to the lack of comparative study. Here, we report upon the expression in E. coli cells of maltose-binding protein (MBP)- and glutathione S-transferase (GST)-fused cyclic amidohydrolase family enzymes, facilitating also for both simple purification and high-level expression. Interestingly the native quaternary structure of each enzyme was maintained even when fused with MBP and GST. We also found that in fusion proteins the favorable biochemical properties of family enzymes such as, their optimal pHs, specific activities and kinetic properties were conserved compared to the native enzymes. In addition, MBP-fused enzymes showed remarkable folding ability in-vitro. Our findings, therefore, suggest that a previously unrecognized trait of this family, namely the ability to functional fusion with some other protein but yet to retain innate properties, is conserved. We described here the structural and evolutionary implications of the properties in this family enzyme
High-level expression and one-step purification of cyclic amidohydrolase family enzymes
No full textThe cyclic amidohydrolase family enzymes, including hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in prokaryotic and eukaryotic cells. With the increasing demand for the elucidation of enzyme structures and functions, along with industrial applications, the research on the family enzymes has recently been proliferating, but the related enzymes had been purified conventionally by multistep purification procedures. Here, we reported the expression in Escherichia coli cells of maltose-binding protein-fused family enzymes and their one-step purification. The expression levels of the fusion proteins account for 20-35% of the total protein in E. coli, allowing approximately 2-3 mg of the purified proteins by affinity chromatography to be obtained per 0.3 L of bacterial culture. As more promising results, their nascent biochemical properties, after the cleavage of the fusion proteins with Factor Xa, in terms of oligomeric structure, optimal pH, specific activity, and kinetic property, were also conserved as those from the native enzymes. The availability of the family enzymes to fusion strategy shows potential as a convenient procedure to recombinant protein purification and accelerates the structure-function study of the related family enzymes. (C) 2001 Academic Press
Construction and evaluation of a novel bifunctional N-carbamylase-D-hydantoinase fusion enzyme
No full textA fully enzymatic process employing two sequential enzymes, D-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity for the production of D-amino acids. To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the D-hydantoinase. Firstly, maltose-binding protein (MBP) gene of E. coli was fused with D-hydantoinase gene from Bacillus stearothermophilus SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of native D-hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics of D-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of N-carbamylase (304 amino acids [aa]) from Agrobacterim radiobacter NRRL B11291 and D-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to the D-amino acids. A related D-hydantoinase (HYD1) gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids
‐Carbamoylase
No full textWe developed a fully enzymatic process employing D-hydantoinase and N-carbamoylase for the production of D-amino aid from 5'-monosubstituted hydantoin. For the comparison of the reaction systems using two sequential enzymes, D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoyl-D-amino acid amidohydrolase (N-carbamoylase) of Agrobacterium tumefaciens NRRL B11291 were separately expressed in each host cell and coexpressed in the same host cell. A high level and constitutive expression of both enzymes in Escherichia coli in a soluble form was achieved using a promoter derived from B. stearothermophilus SD1. The expression levels of both enzymes ranged from 17% to 23% of the total soluble protein, depending on the expression system. In the case of employing separately expressed enzymes, the product yield of D-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin and productivity were 71% and 2.57 mM/g-cell/h in 15 h, respectively. The accumulation of N-carbamoyl-D-hydroxyphenylglycine was significant over the reaction time. On the other hand, use of coexpressed enzymes resulted in 98% product yield of D-hydroxyphenylglycine in 15 h, minimizing the level of intermediates in the reaction mixture. The productivity of coexpressed whole cell reaction was estimated to be 6.47 mM/g-cell/h in 15 h. The coexpressed system was tested for an elevated concentration of D,L-p-hydroxyphenylhydantoin, and efficient production can be achieved
Design of a new Faculty of Architecture - Investigation and design of Media Facades
No full textGraduation Project [G.J. Smit] for both Architecture & Building Technology. [architecture] : design of a new Faculty of Architecture [building technology] : investigation and design of Media FacadesStrategic Architectural Design DevelopmentArchitecture & Building TechnologyArchitectur
Modeling, simulation, and kinetic analysis of a heterogeneous reaction system for the enzymatic conversion of poorly soluble substrate
No full textWe developed a kinetic model that describes a heterogeneous reaction system consisting of a solid substrate suspension for the production of D-amino acid using D-hydantoinase. As a biocatalyst, mass-produced free and whole cell enzymes were used. The heterogeneous reaction system involves dissolution of a solid substrate, enzymatic conversion of the dissolved D-form substrate, spontaneous racemization of an L-form substrate to D-form, and deactivation of the enzyme. In the case of using whole cell enzymes, transfer of the dissolved substrate and product through the cell membrane was considered. The kinetic parameters were determined from experiments, literature data, and by using Marquardt's method of nonlinear regression analysis. The model was simulated using the kinetic parameters and compared with experimental data, and a good agreement was observed between the experimental results and the simulation ones. Factors affecting the kinetics of the heterogeneous reaction system were analyzed on the basis of the kinetic model, and the efficiency of the reaction systems using free and whole cell enzymes was also compared. (C) 1999 John Wiley & Sons, Inc
Directed Evolution of a Novel N-Carbamylase/D-Hydantoinase Fusion Enzyme for Functional Expression with Enhanced Stability
No full textBifunctional enzymes find a wide application as a monitoring facility and a potential biocatalyst in molecular biology and biotechnology. Recombination of natural enzymes to a bifunctional fusion offers valuable tools, but the functional and structural instability of artificial
fusion enzymes remains to be solved. Based on
structural traits of microbial D- hydantoinase, we attempted to construct a bifunctional N-carbamylase/Dhydantoinase
fusion enzyme that would be useful for the
synthesis of nonnatural D-amino acids in a concerted fashion. The bifunctional ability of D-hydantoinase, as a fusion partner, was noticeable, but the resulting fusion
enzyme was subjected to serious proteolysis in vivo, as generally encountered in the expression of large the multidomain
polypeptide in E. coli. In an effort to improve the structural instability imposed by artificial linear fusion, directed evolution of the fusion enzyme was performed
using DNA shuffling with a consensus primer to maintain a crucial domain for the enzyme activity. The evolved fusion enzyme, F11, was selected after repeated rounds, and this enzyme was found to show sixfold increased
performance in the production of D-amino acid
compared with the parent fusion enzyme, which was mainly due to the enhanced structural stability of the evolved fusion enzyme. This result is an example showing
that directed evolution of the linearly fused polypeptide may broaden the opportunity to generate a fusion enzyme with greater potential
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