Engineering Conferences International

Please click Additional Files below to see the full abstract

McGilvray, Matthew

Hermann, Tobias

Saad Ifti,, Hassan

Chakravarthy, Kharthik

Ewenz Rocher, Marc

Williams, Benjamin

Vandeperre, Luc

Hedgecock, Rowan

Hufgard, Fabian

Lӧhle, Stefan

ULTRA-HIGH TEMPERATURE CERAMICS FOR TRANSPIRATION COOLING APPLICATIONS IN 
HYPERSONIC VEHICLES 
 
Matthew McGilvray, The University of Oxford, United Kingdom 
matthew.mcgilvray@eng.ox.ac.uk 
Tobias Hermann, The University of Oxford, United Kingdom 
Marc Ewenz Rocher, The University of Oxford, United Kingdom 
Hassan Saad Ifti, The University of Oxford, United Kingdom 
Kharthik Chakravarthy, The University of Oxford, United Kingdom 
Benjamin Williams, Oxford Thermofluids Institute, The University of Oxford, United Kingdom 
Rowan Hedgecock, mperial College London, United Kingdom 
 Luc Vandeperre, mperial College London, United Kingdom 
Fabian Hufgard, Institute of Space Systems, University of Stuttgart 
Stefan Lӧhle, Institute of Space Systems, University of Stuttgart 
 
 
This paper presents an overview of the work undertaken over the past four years to explore the application of 
transpiration cooling for hypersonic vehicles using partially sintered ultra-high temperature ceramics (UHTC). 
Due to the very high speeds these vehicles travel through planetary atmospheres, they are exposed to 
extremely high heat loads (> 1 MW/m2). These heat loads need to be mitigated in order to protect the payload. 
For high flight speeds, particularly where sharp nose radii are aerodynamically desirable, these heat loads 
cannot be managed by passive systems. To date, the thermal protection system which has been most 
successfully applied is an ablative heat shield. This has extremely high heat load to mass performance. 
However, shape stability for aerodynamic applications and reusability requires a new cooling technology.  
 
Transpiration cooling provides an active cooling approach that could be employed to thermally protect such 
hypersonic vehicles, retain shape stability, and enable re-usability. The cooling technique works by passing 
coolant gas through the porous skin of the vehicle which keeps the material temperature below critical values. 
This is mainly achieved by three mechanisms: The heat exchange from solid material to coolant while the 
coolant passes through the porous wall, the creation of a protective film at the outside of the vehicle that 
reduces boundary layer temperatures, and the reduction of catalytic and oxidation reactions at the surface due 
to creating an inert film that keeps the freestream radicals and oxygen away from the surface. The last feature is 
a key requirement to enable UHTCs as a candidate material as oxidation protection unlocks the massive re-
radiation potential of these materials. Through the exclusion of oxidation, significantly higher surface 
temperatures can be achieved which enable a huge cooling potential through re-radiation. 
 
This paper will present:  an overview of the potential performance of the technology to key hypersonic vehicles 
at key trajectory points; manufacturing technologies of partially sintered ZrB2 with designed porosity and cooling 
architectures; material characterisation measurements; measurements of permeability (Darcy-Forchheimer) 
coefficients, spatial distribution of flow; measurement of internal heat transfer coefficients; coupling of coolant 
ejection with a hypersonic cross flow and mixing. Finally, the paper will culminate in the experimental verification 
of this cooling approach through testing of an actively cooled stagnation point coupon which was exposed to a 
high enthalpy plasma wind-tunnel flow. 
 


English

Ultra-high temperature ceramics for transpiration cooling applications in hypersonic vehicles

Lam, Benjamin

McMurray, Jake

Cakmak, Ercan

Lance, Michael

Mitchell, David

THERMODYNAMIC AND EXPERIMENTAL SiC-ZrC CVD PROCESS DEVELOPMENT  
 
Benjamin Lamm, Oak Ridge National Laboratory, USA 
lammbw@ornl.gov 
Jake McMurray, Oak Ridge National Laboratory, USA 
Ercan Cakmak, Oak Ridge National Laboratory, USA 
Michael Lance, Oak Ridge National Laboratory, USA 
David Mitchell, Oak Ridge National Laboratory, USA 
 
 
The codeposition of SiC-ZrC using CVD is of interest for application as ultra-high temperature materials. SiC 
and ZrC have been previously investigated as UHT materials for high-speed and nuclear applications, and the 
study of SiC-ZrC codeposition functions as groundwork for future mixed composition UHTC coatings. 
Computational thermodynamics was used to explore the potential CVD processing space with regards to 
pressure and temperature for a given set of precursor gases.  The Zr process gas was generated via 
chlorination of solid metal pieces.  Coupling thermodynamic calculations with experimental CVD can reduce the 
required number of CVD runs to achieve optimum deposition by refining experimental conditions prior to 
experimentation. Comparison of thermodynamic and experimental results offer insight into mechanistic 
decomposition and deposition behavior, potential for process impurities, and coating properties. A custom 
thermodynamic database developed at ORNL was used to model the cold-wall CVD process to identify an 
optimum region of temperature, pressure, and gas precursor ratios for deposition of the desired species. 
Representative characterization such as Raman microscopy demonstrating the synergy between the model and 
the coating composition will be presented. Targeted process runs will also be presented to improve kinetics 
models for the SiC-ZrC CVD system. 
 
Research sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National 
Laboratory, managed by UT-Battelle, LLC for the US Department of Energy under contract DE-AC05-
00OR22725. 
 
 
 
 
Figure: Surface Raman map of codeposit. White – SiC, Red – ZrC, Green – ZrSi. 
 


Thermodynamic and experimental SiC-ZrC CVD process development

N/A
Please click Additional File below for the presentation

Brander, Christian

Christian Brander 
ICREA Senior Research Professor
IrsiCaixa AIDS Research Institute
AELIX Therapeutics, Barcelona
Vaccine Technology VIII
June  13th 2022
Sitges
Advances in Therapeutic 
HIV Vaccine Development
- The problem with HIV (chronicity)
- T cell vaccines for HIV cure
- Recent advances in HIV therapeutic vaccination 
Advances in Therapeutic 
HIV Vaccine Development
The HIV pandemic is still ongoing 
36.7 Million HIV + (PLWH)
19 million living in Africa
1.1 million deaths every year
Since HIV was first identified, 78 millions estimated infections (35 million deaths)
1 in 4 people who are HIV positive do not know their HIV status
The many hurdles to HIV Cure and Eradication
Kulpa. J. Virus Erad. 2015
Ø HIV establishes a life-long, latent reservoir shortly after acute infection
Ø The decay kinetics of the reservoir under Antiretroviral Treatment (ART) are too 
slow to eliminate the virus from the body.
Ø Latently infected cells are largely invisible to the immune system
Ø Treatment interruptions lead to
rapid rebound of viremia 
Ø Unclear what immune responses
a therapeutic vaccination should
target as functional (!) immune
correlates of virus control remain
poorly defined 
The Rational for Therapeutic HIV Vaccination
Ø Essentially everybody who becomes infected with HIV makes a strong immune 
response to the virus
Ø The vast majority of PLWH show progressive HIV disease if left untreated  
Ø Evidently then, the immune response that we measure upon natural infection 
does not (fully) protect from infection and HIV disease progression
Ø HLA-association studies, CD8 T cell depletion in SIV infected monkeys, viral 
evolution analyses, etc all support a role of CD8+ cytotoxic T lymphocytes in 
virus control
Natural HIV Infection
Time (years after infection) 
Viral Load
CD4 T cells
CTL response
Antibody response
Therapeutic Vaccine
Infection
Viral control
Eradication
Protection from re-infection
Potential components of a therapeutic vaccine:
T cell response to viral proteins
- CD8 CTL “killer T cell” to kill infected cells
- CD4 T-helper cells to maintain functional CTL
- Combination approaches with nAb
- Viral reservoir activators
Ø Modulation of a pre-existing, ineffective immunity 
The Rational for Therapeutic HIV Vaccination
Natural HIV Infection
Time (years after infection) 
Viral Load
CD4 T cells
CTL response
Antibody response
Catalan HIVACAT Vaccine Program 
(2008-2022)
Ø Therapeutic Strategies:  
Ø IL2, AUTOVAC, TIBET, 2X4 (Clotet, Ruiz, Arno,..)
o autologous virus released by treatment interruption, cytokines
Ø (Kick and) kill strategies: 2008 onwards
o DCV-02:  autologous virus on DC (Gil 2011, Garcia 2013)
o RISVAC-03:   MVA/DSF  (Mothe 2015, Rosas-Umbert 2017)
o HIVARNA:  mRNA delivered HTI (Leal, 2021)
o BCN01/BCN02:  ChAd-MVA +Rmd (Mothe 2020, Rosas Umbert 2020)
o AELIX-002:   DNA-ChAd-MVA -HTI (Bailon 2022)
o AELIX-003:   ongoing (ChAd-MVA +TLR7)
o BCN03:   ongoing (ChAd-MVA +SOSIP)
o HIVACAR:   ongoing (conserved epitopes RNA)
DCV-02 (autologous virus-loaded dendritic cell vaccine) 
Garcia et al STM, 2013
Ø Isolation of autologous virus during 1st treatment interruption,  in vitro expansion, 
heat-inactivation, pulsed on autologous, in vitro matured dendritic cells
Ø 55% active vs 9% placebo with >1log lower viral set point 12 and 24w after ART stop
Ø Aside from “complex” development process , still elevated viral loads during 
analytic treatment interruption (ATI), making this unsafe for the patients and their 
sexual partners
Therapeutic HIV Vaccine Program Barcelona
2008
DCV-02
RISVAC 03 (kick-and kill, MVA vaccine and disulfiram LRA)
Mothe et al JAC, 2015
Ø MVA-B (but not MVA-B + DSF) 
vaccination showed modestly 
delayed viral rebound (2 weeks)
Ø Reduced peak viremia related to 
level of virus adaption to host 
genetics  
Ø Proviral HIV-1 DNA  (i.e. measure 
of reservoir size) at study entry 
associated with delayed HIV-1 RNA 
rebound and lower  peak viremia 
Therapeutic HIV Vaccine Program Barcelona
DCV-02
RISVAC 03
Mothe et al Front Imm, 2020
BCN-01/02 (Kick and kill strategy using ChAd and 
MVA vaccines expressing HIVCons plus Romidepsin)
Therapeutic HIV Vaccine Program Barcelona
Insights gained from BCN02
Mothe et al Front Imm, 2020
Rosas-Umbert Front Imm 2020
1) No placebo control, what is the rate of Post-Treatment-Control (PTC: 8-13%)
2) Romidepsin safe yes, but effective ?
- minor peaks in viremia 
- transient increase in apoptotic T cells 
- reduced polyfunctional cells
- in vitro antiviral (VIA) activity preserved 
3) Reservoir possibly important, no reduction up to ATI (like RIVER, AELIX002, etc)
Insights gained from BCN02
1) No placebo control, what is the rate of Post-Treatment-Control (PTC: 8-13%)
2) Romidepsin safe yes, but effective ?
- minor peaks in viremia 
- transient increase in apoptotic T cells 
- reduced polyfunctional cells
- in vitro antiviral (VIA) activity preserved 
3) Reservoir possibly important, no reduction up to ATI (like RIVER, AELIX002, etc)
4) Bacteroidales/Clostridiales ratio predicts HIV-1 reservoir size and virus control 
Mothe et al Front Imm, 2020
Rosas-Umbert Front Imm 2020
Borgogno MBIO, 2022 in press
Insights gained from BCN02
1) No placebo control, what is the rate of Post-Treatment-Control (PTC: 8-13%)
2) Romidepsin safe yes, but effective ?
- minor peaks in viremia 
- transient increase in apoptotic T cells 
- reduced polyfunctional cells
- in vitro antiviral (VIA) activity preserved 
3) Reservoir possibly important, no reduction up to ATI (like RIVER, AELIX002, etc)
4) Bacteroidales/Clostridiales ratio predicts HIV-1 reservoir size and virus control 
5) Pre-ATI (and pre-vaccination) methylation imprints associated with ATI control
Mothe et al Front Imm, 2020
Rosas-Umbert Front Imm 2020
Borgogno MBIO, 2022
Oriol-Tordera EBioM, 2022
Oriol-Tordera Plos Path 2021
Insights gained from BCN02
1) No placebo control, what is the rate of Post-Treatment-Control (PTC: 8-13%)
2) Romidepsin safe yes, but effective ?
- minor peaks in viremia 
- transient increase in apoptotic T cells 
- reduced polyfunctional cells
- in vitro antiviral (VIA) activity preserved 
3) Reservoir possibly important, no reduction up to ATI (like RIVER, AELIX002, etc)
4) Bacteroidales/Clostridiales ratio predicts HIV-1 reservoir size and virus control 
5) Pre-ATI (and pre-vaccination) methylation imprints associated with ATI control
6) T-cell specificity, effector function and T cell receptor (TCR) repertoire may be  
linked to outome (epitope-specific 10Xsc and OMNISCOPE OS-T analyses) 
DCV-02
RISVAC 03
Mothe CROI 2021
Bailon, submitted
BCN-01/02
AELIX-002
Therapeutic HIV Vaccine Program Barcelona
AELIX-002: HTI vaccination mediates improved 
Viral Control in ATI
Mothe CROI 2021
Bailon, submitted
Placebo: 1/12 (8%) ATI of 24w Vaccinees: 8/20 (40%) ATI of 24w
Time off ART (weeks) Time off ART (weeks)
AELIX-002: Time off ART is correlated with the 
strength of the vaccine induced HTI immunity 
Mothe CROI 2021
Bailon, submitted
Placebo: 1/12 (8%) ATI of 24w Vaccinees: 8/20 (40%) ATI of 24w
Time off ART (weeks) Time off ART (weeks)
DCV-02
RISVAC 03
BCN-01/02
AELIX-002
AELIX-003 (HTI + TLR7)
BCN-03 
(combined T and 
B cell vaccination) 
Therapeutic HIV Vaccine Program Barcelona
HIVACAR mRNA
Conclusions - Next steps 
Ø Years of clinical trials of therapeutic HIV vaccination have yielded until 
recently mostly  frustrating results 
Ø Target population (early, chronic, reservoir size)
Ø Immunogen design (T cell specificity, viral evolution, adapted reservoir)
Ø Manufacturing  and up-scale hurdles 
Ø Clinical trials of therapeutic HIV vaccination start showing clinically relevant 
efficacy signals (AELIX-002)
Ø Biomarkers of virus control
Ø Target population definition 
Ø Modulation of pre-existing conditions (epigenetics, microbiota)  
Ø Effective HIV cure strategies will likely require combination strategies to 
harness humoral and cellular immunity and to effectively tackle the latent  
viral reservoir  
Ø Latency reactivators
Ø Combined T and B cell vaccination strategies (BCN03)
Ø May inform prophylactic vaccine setting 
 
Beatrize Mothe
Marta Ruiz-Riol
Alex Olvera
Bruna Oriol
Samandhy Cedeño
Sandra Silva Arrieta
Anuska Llano
Tuixent Escribà
Pep Coll 
Acknowledgments


Advances in therapeutic HIV vaccine development

Milčić, Nevena

Švaco, Petra

Sudar, Martina

Majerić Elenkov, Maja

Findrik Blažević, Zvjezdana

 Poster Number 6 
ASSESSMENT OF C-TYPE HALOHYDRIN DEHALOGENASE STABILITY 
 
Nevena Milčić, Faculty of Chemical Engineering and Technology, University of Zagreb, Croatia 
nmilcic@fkit.hr 
Petra Švaco, Institut Ruđer Bošković, Zagreb, Croatia 
Martina Sudar, Faculty of Chemical Engineering and Technology, University of Zagreb, Croatia 
Maja Majerić Elenkov, Institut Ruđer Bošković, Zagreb, Croatia 
Zvjezdana Findrik Blažević, Faculty of Chemical Engineering and Technology, University of Zagreb, Croatia 
 
 
Key Words: halohydrin dehalogenases, operational stability, kinetic modelling 
 
Halohydrin dehalogenases (HHDHs) are important enzymes that display excellent selectivity and activity in 
reversible dehalogenation of vicinal halohydrins. Furthermore, HHDHs may employ wide range of unnatural 
nucleophiles in ring-opening reactions, extending the possibilities for the synthesis of enantiopure β-alcohols1. 
On account of these desirable properties, the variant of the HheC enzyme is employed in the synthesis of 
Lipitor, one of the best-selling pharmaceuticals in the world2. However, insufficient is known about the enzyme 
kinetic stability that is a prerequisite for further industrial implementations2,3. In this work, we assessed the 
stability wild-type HheC and two mutants with enhanced kinetic properties. First, freeze-thaw cycle testing was 
performed via activity measurements, whereby HheC proved to be very stable, facilitating protein handling 
procedures. The kinetic stability of different biocatalyst forms (whole cells, cell-free extract, and purified enzyme) 
in buffer was determined through activity tests, which led us to the conclusion that the cell wall and other 
proteins in the extract protect the enzyme and keep it active for longer period. Size distribution of purified protein 
was also investigated by dynamic light scattering methods to establish existence of larger protein particles 
formed due structural changes and aggregation. The stability of mutants during incubation with substrates and 
unnatural nucleophiles (azides, cyanides) was monitored to determine whether the components of the reaction 
mixture have an effect on enzyme activity. It was found that at higher concentrations of fluorinated styrene oxide 
derivatives, the enzyme loses activity faster, which was later reflected in the results of enzyme operational 
stability decay in the batch reactor. The co-dependency of operational stability decay rate constant on substrate 
initial concentration (Figure 1) was mathematically described by Eq 1. Understanding and quantifying the 
influence of compounds concentrations on enzyme behavior is of a great importance for larger scale 
processes3. Presented results give a better insight into the limitations of HheC application and help with 
biocatalysts handling for prolonging its longevity in reactors. 
 
0,epoxide
0,epoxide
d
c a
k
c b

=
+
     (Eq 1)  
 
 
1 – A. Schallmey, M. Schallmey, Appl Microbiol 
Biotechnol (2016) 100, 7827–7839. 
2 – Z. Findrik Blažević et al., Adv. Synth. Catal. (2021) 
363 (2), 388-410. 
3 – A. S. Bommarius, F. Paye, Chem. Soc. Rev. 
(2013) 42, 6534. 
 
 
Figure 1 – Dependence of HheC-W249P deactivation 
constant on rac-2-[4-(trifluoromethyl)phenyl]oxirane 
initial concentration. 


Assessment of C-type halohydrin dehalogenase stability

Zukic, Erna

Willrodt, Christian

Ditrich, Klaus

Kroutil, Wolfgang

 Poster Number 4 
BIOCATALYTICAL ACCESS TO AMIDES 
 
Erna Zukic, acib, Krenngasse 37, Austria 
erna.zukic@uni-graz.at 
Christian Willrodt, BASF, Ludwigshafen, Germany 
Klaus Ditrich, BASF, Ludwigshafen, Germany 
Wolfgang Kroutil, Institute of Chemistry, University of Graz, Austria 
 
 
Key Words: Biocatalysis, amide synthesis, aminolysis of ester, SpL  
 
Amide formation is one of the most important reactions in industrial pharmaceutical synthesis.[1] Although the 
synthetic approaches appear to be simple, they suffer in fact from many drawbacks. The synthesis requires 
toxic or hazardous reagents to activate the ester or acid component and have a poor atom economy.[2] So far, 
many strategies to obtain amides have been published, but several problems have not been solved, yet.[3,4] 
Challenges are for example the direct transformation of carboxylic acids or esters with amines. Known chemical 
strategies require the hydrolysis of the ester, activation of the carboxylic acid via SOCl2 leading to a reactive acyl 
chloride which reacts with the amine.  
 
For these challenges, biocatalysis may offer a solution, especially because biocatalysis is known for using 
harmless reagents and working under mild conditions. 
 
 
 
 
Our aim is to identify a biocatalytic access to amide formation, especially by the help of hydrolases starting from 
bulky acyl donors. Screening several hydrolases, one promising candidate is the lipase from Sphingomonas sp. 
HXN-200 also known as SpL.[5] The lipase was discovered and expressed by Li and co-workers in 2018. SpL 
shows high activity in aminolysis towards various acid and esters with amines in organic solvents. New 
substrates have been tested and functional studies on this protein shall be conducted to understand its reaction 
behavior with the help of the Loschmidt tools.   
 
Acknowledgement: The COMET center: acib: Next Generation Bioproduction is funded by BMVIT, BMDW, SFG, 
Standortagentur Tirol, Government of Lower Austria und Vienna Business Agency in the framework of COMET - 
Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian 
Research Promotion Agency FFG. 
 
 
References 
[1] D. J. Constable, P. J. Dunn, J. D. Hayler, G. R. Humphrey, J. L. Leazer, R. J. Linderman, K. Lorenz, J. 
Manley, B. A. Pearlman, A. Wells, A. Zaks, T. Y. Zhang, Green Chem. 2007, 9, 411–420.  
[2] M. Petchey, A. Cuetos, B. Rowlinson, S. Dannevald, A. Frese, P. W. Sutton, S. Lovelock, R. C. Lloyd, I. J. 
Fairlamb, G. Grogan, Angew. Chem. Int. Ed. 2018, 57, 11584–11588. 
[3] J. Pitzer, K. Steiner, J. Biotechnol. 2016, 235, 32–46 2018, 57, 11584–11588. 
[4] V. R. Pattabiraman, J. W. Bode, Nature 2011, 480, 471–479. 
[5] S. Zeng, J. Liu, S. Anankanbil, M. Chen, Z. Guo, J. P. Adams, R. Snajdrova, Z. Li, ACS Catal. 2018, 8, 
8856–8865. 
 
Figure 1 – Amide formation starting from a bulky acyl donor. 


Biocatalytical access to amides

Bornscheuer, Uwe

BIOCATALYSIS AND ENZYME ENGINEERING – A PERSONAL VIEW ON THE LAST THREE DECADES 
 
Uwe Bornscheuer, Greifswald University, Germany 
uwe.bornscheuer@uni-greifswald.de 
 


German

Biocatalysis and enzyme engineering – a personal view on the last three decades

Asano, Yasuhisa

CONTINUITY AND CHANGE IN SCREENING FOR INDUSTRIAL ENZYMES 
AND PROTEIN ENGINEERING 
- A TRIBUTE TO THE LATE PROFESSOR HIDEAKI YAMADA- 
 
Yasuhisa Asano, Toyama Prefectural University; ERATO, JST, Japan 
asano@pu-toyama.ac.jp 
 
 
Key Words: aldoxime-nitrile pathway, hydroxynitrile lyase, 5’-inosinic acid, transcrystallization, soluble 
expression  
 
This talk includes how the idea of the late Professor Hideaki Yamada are reflected in the current enzyme 
engineering, and the concepts which researchers in the new generation have succeeded and evolved from him.  
  
1. Discovery of “nitrile hydratase” and acrylamide synthesis: The industrial process had become a model of 
enzyme engineering, prior to the establishment of Green Chemistry Award in USA (since 1996). We later 
discovered “aldoxime dehydratase”, a nitrile-synthesizing enzyme constituting the bacterial “Aldoxime-Nitrile 
Pathway”. Now optically active b-hydroxynitrile can be synthesized by aldoxime dehydratase-catalyzed Kemp 
elimination reaction. We discovered and solved three new structures of plant and animal hydroxynitrile lyases 
(HNL) among seven HNL structures hitherto known. 
 
2. Umami tastes: 5’-Inosinic acid and 5’-gluanylic acid have been industrial targets. Prof. Koichi Ogata of 
Kyoto University industrialized the ribonucleotide production by rRNA hydrolysis, when he belonged to Takeda 
Pharmaceuticals. Prof. Hideaki Yamada and Prof. Hideo Katagiri of the same laboratory studied 
transphosphorylation of p-nitrophenylphospate to inosine forming 5’-inosinic acid. However, a toxic p-nitrophenol 
remained as a problematic by-product. We screened microorganisms and found very selective bacterial “acid 
phosphatase” using pyrophosphate as a phosphoryl donor. Further directed evolution of the enzyme led to the 
successful industrial production of ribonucleotides. This has been the first case of the use of directed evolution 
in the industrial enzymatic process (2003). 
 
3. Screening for enzymes: Microbial screening for enzymes has been successfully carried out from culture 
collection and by isolation of microorganisms by enrichment culture technique. The screening space has been 
extended not only to plants and animals, but also to database by in silico screening for similar but different 
enzymes using our INTMSAlign-Angler software based on Blast search.  
 
4. Transcrystallization of the products: Enzymatic syntheses up to several 100gs/L of the product have 
been shown to be a good indication of his successful “enzymation” reactions. Prof. Yamada has clearly 
demonstrated that the equilibrium of the b-tyrosinase-catalyzed a,b-elimination reaction has been shifted toward 
DOPA synthesis because of precipitation of DOPA in the reaction mixture. Learning from the case, enzymatic 
“transcrystallization” would be one of the advantageous future methods of enzymatic synthesis as we have 
shown in the production of 12-lauric acid and lumichrome. The database for “transcrystallization” has been 
developed. 
 
5. Crystallization of enzymes (for X-ray crystallography): Prof. Yamada’s experience in the crystallization of 
bovine serum amine oxidase, promoted many students to crystallize their target enzymes. Now, as a part of 
rational protein engineering, structure determination of the enzymes has become a very important step. We 
generate the data by in house X-ray facility and are using software to alter the surface structure of the enzyme 
by INTMSAlign and calculating decrease of entropy of the residues, such as by SERp. Optimization of the 
stereoselectivity is carried out by calculation by MOE, followed by protein engineering. 
 
6. Soluble expression: We generated a new concept of “soluble expression” of genes for enzymes in 
heterologous hosts and developed a-helix rule, HiSol score, INTMSAlign software and machine learning. The 
concepts are used in the improvement of practical enzymatic synthesis. 
 
 
 
 
 


Continuity and change in screening for industrial enzymes and protein engineering- A tribute to the late Professor Hideaki Yamada

Lovelock, Sarah

BIOCATALYTIC APPROACHES TO THERAPEUTIC OLIGONUCLEOTIDE MANUFACTURE 
 
Sarah Lovelock, Manchester Institute of Biotechnology, University of Manchester, UK 
Sarah.lovelock@manchester.ac.uk 
 
 
Key Words: Oligonucleotides, Nucleic Acids, Biocatalysis, Manufacturing 
 
Therapeutic oligonucleotides bind to mRNA to modulate the production of disease related proteins and have 
emerged as a new drug modality for the treatment of a range of disease areas including genetic disorders and 
viral infections. Until recently, approved therapeutics were limited to the treatment of rare diseases, however in 
2020 Inclisiran was approved for the treatment of atherosclerotic cardiovascular disease, which affects 
approximately 30 million people in the USA alone. This emergence of oligonucleotide products for more 
common diseases, creates a significant manufacturing challenge as existing methods of oligonucleotide 
synthesis are restricted to 10Kg batches and are not suitable for large scale applications (>100Kg). Current 
chemical approaches rely on iterative deprotection, coupling, capping and oxidation steps to extend sequences 
on a solid support. They use an excess of synthetically complex building blocks with protecting groups which 
must be removed during the synthesis with washing steps, resulting in poor atom efficiency. The process also 
uses prohibitively large volumes of acetonitrile (1000Kg per Kg of oligonucleotide) and chromatographic 
purification of the final product. Due to limitations of existing synthetic methods, current marketed RNA-based 
therapeutics are typically supplied with ~90% purity and as complex mixtures of diastereoisomers (resulting from 
phosphorothioate modifications). Here we present a biocatalytic strategy for therapeutic oligonucleotide 
synthesis inspired by the polymerase chain reaction, which has the potential to address the limitations of current 
manufacturing processes. Target sequences containing pharmaceutically relevant modifications are produced in 
a single operation, using a catalytic template and unprotected nucleotide triphosphate building blocks, in 
aqueous media. To showcase the approach, we have synthesized the first approved therapeutic oligonucleotide 
Vitravene as a single diastereoisomer in 63% yield and 85% purity, without the need for chromatographic 
purification. 
 
.  
 
 
 


Biocatalytic approaches to therapeutic oligonucleotide manufacture

Green, Anthony

Tuesday, May 24 2022                                                                Session 4:  Novel Enzymes and Enzyme Activity 
DESIGN AND EVOLUTION OF ENZYMES WITH NON-CANONICAL CATALYTIC MECHANISMS 
 
Anthony Green, University of Manchester, United Kingdom 
Anthony.green@manchester.ac.uk 
 
 
Key Words: Enzyme design, genetic code expansion, directed evolution, enzyme structure 
 
Enzyme design and evolution strategies rely exclusively on Nature’s standard amino acid alphabet of twenty 
canonical residues which contain limited functionality. Here we demonstrate that incorporation of non-canonical 
amino acids into enzyme active sites provides a fruitful avenue to probe complex biological mechanisms and 
can lead to the creation of designed enzymes with wholly new catalytic functions. Significantly, optimization of 
enzyme activity can be achieved using directed evolution workflows adapted to an expanded genetic code. We 
are optimistic that this integration of enzyme design, genetic code expansion and laboratory evolution can 
provide a versatile strategy for creating enzymes with catalytic functions not accessible to Nature. 
 
 


Design and evolution of enzymes with non-canonical catalytic mechanisms

Hyster, Todd

Tuesday, May 24 2022                                                                                                                                Keynote 
PHOTOENZYMATIC CATALYSIS - USING LIGHT TO REVEAL NEW ENZYME FUNCTIONS 
 
Todd Hyster, Cornell University, USA 
tkh33@cornell.edu 
 


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