Day 2 :
Keynote Forum
Ibrahim Abdulhalim
Ben-Gurion University of the Negev, Israel
Keynote: Plasmonic biosensors on demand: Tunable penetration depth, compactness, ultrahigh sensitivity and enhanced spectroscopies
Time : 11:00-11:40
Biography:
Ibrahim Abdulhalim is a Professor at the Electro-optical Engineering Unit at Ben-Gurion University of the Negev. He worked in academic institutions and companies such
as the OCSC in University of Colorado Boulder, the ORC at Southampton University, the Thin Films Center of the University of Western Scotland, in KLA-Tencor, Nova and
GWS Photonics. He has published over 200 articles, two books, 10 chapters and has 20 patents. He is a fellow of IoP and SPIE and an Associate Editor for the Journal
of NanoPhotonics and for the Journal of Imaging.
Abstract:
Evanescent wave optical biosensors allow specifi c sensing by using a surface binding layer which enhances the capture
of specifi c bio-entities within the nanoscale neighborhood to the sensor surface. However, this evanescence region is
sometimes too small at the scale of few tens of nanometers which prevents obtaining monotonic signal versus concentration
when the bio-entities are larger than the optical penetration depth. Th e purpose of this study is to describe methods for
sensing both small (molecules, viruses, etc.) and large bioentities (cells, large molecules) using plasmonic sensors with
tunable penetration depth. During the last few years, we have been developing diff erent structural and system confi gurations
for improving the performance of plasmonic biosensors based on improving the reading method and enhancing the local
electromagnetic (EM) fi eld further for the purpose of improving the sensitivity and lowering the detection limit based on
SPR, SERS and SEF. Th e structural improvements include: (i) planar thin metal fi lms combined with dielectric fi lms, (ii)
periodic metallic structures on planar substrate, (iii) nanosculptured thin fi lms prepared by the glancing angle deposition
technique. (iv) long range self-referenced plasmonic confi gurations, and lately, (v) combination of nanostructures with thin
metal fi lms for coupling of extended surface plasmons (ESP) to localized surface plasmons (LSP). Th e system improvements
include: (i) diverging beam approach in the angular mode, (ii) polarimetric spectral mode, (iii) image and signal processing.
Particularly, we have shown recently that even much higher enhancement of the EM fi elds is obtained by exciting the LSPs
through extended surface plasmons generated on a semi-infi nite metallic fi lm surface. Biotechnology applications will be
presented for sensing biomolecules and cells in water and in blood. In spite of the technological advances in optics, the need
for developing molecular binding layer to improve the specifi city is still in demand from the biotechnology community.
Keynote Forum
Jennifer A Littlechild
University of Exeter, UK
Keynote: Thermophilic enzymes with applications for industrial biocatalysis
Time : 11:40-12:20
Biography:
Jennifer A Littlechild is an Emeritus Professor of Biological Chemistry and has established the Henry Wellcome Centre for Biocatalysis at Exeter University in 2003. Her
research studies involve the structural and mechanistic characterisation of a range of enzymes from thermophilic bacteria and archaea that have industrial applications.
She has published over 200 publications in refereed high impact journals and presented her research work internationally. She has coordinated EU related project THERMOGENE
and was a partner in a consortium grant HOTZYME. In UK she is funded from BBSRC and Innovate UK. These grants involve both large industrial companies
and SME enterprises. She has supervised over 40 PhD students and acts as External Examiner for other PhD and Masters Students. She is the UK representative and
Vice Chair of the European Section of Applied Biocatalysis and a Member of EU advisory committees for Industrial Biotechnology.
Abstract:
There is an increasing demand for new enzymes with enhanced performance and/or novel functionalities that provide
savings in time, money and energy for industrial processes in the areas of high value chemical production and other
white biotechnology applications. Only a small proportion of nature’s catalysts have been utilised for industrial biotechnology.
Th e number of enzymes explored to date remains within the range of 1-2% of known biodiversity. A problem with using
enzymes for industrial biocatalysis reactions is oft en their stability under the harsh conditions employed. Th e use of naturally
thermostable enzymes isolated from hot environments are more stable to high temperatures, extremes of pH and exposure
to organic solvents. Th e projects HOTZYME and THERMOGENE have identifi ed hydrolase and transferase enzymes of
industrial interest isolated from high temperature environments around the world. Th ese have been isolated from thermophilic
bacterial and archaeal genomes and metagenomes. A selection of these novel thermostable enzymes including cellulases,
carboxylesterases, lactonases, epoxide hydrolases, transketolases, hydroxymethyl transferases and transaminases have been
characterized both biochemically and structurally. Transaminase enzymes have received special attention for the production
of chiral amines which are important building blocks for the pharmaceutical industries. Th ese enzymes catalyse the reversible
transfer of an amino group from a donor substrate onto a ketone/aldehyde or sugar acceptor molecule. Th ey can be subdivided
into 6 classes. Th e less studied class 4 (branched chain) (R) selective, class 5 (S) selective and class 6 (sugar) enzymes have
been identifi ed. An example of the archaeal class 4 enzyme from Archaeoglobus fulgidus; a thermostable class 5 archaeal
transaminase from Sulfolobus solfataricus and class 6 sugar transaminase from A. fulgidus. Two new enzymes with interesting
substrate specifi city and stereo-selectivity have been discovered which have already been demonstrated at industrial scale for
the production of new chiral chemical building blocks.
Keynote Forum
Kam Bo Wong
The Chinese University of Hong Kong, China
Keynote: How urease accessory proteins coupled GTP hydrolysis/binding to nickel delivery to urease?
Time : 12:20-13:00
Biography:
Kam Bo Wong obtained his BSc and MPhil from the Chinese University of Hong Kong. He then pursued his PhD degree in the laboratory of Prof. Alan Fersht at the University
of Cambridge. After Postdoctoral training in the University of Washington and University of Cambridge, he joined the Chinese University of Hong Kong in 1999, where
he is now a Professor at the School of Life Sciences. His research interests are on the structure-function studies of proteins. His research group uses multi-disciplinary
techniques, including protein engineering, biophysical characterization, computational methodologies, and structure determination by NMR and X-ray crystallography, to
study how proteins function on the atomic and molecular levels.
Abstract:
Urease is a nickel-containing metalloenzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide. Th is
enzymatic reaction, which produces the acid-neutralizing ammonia, is essential for the survival of Helicobacter pylori in
human stomach. In Helicobacter pylori, nickel ions delivery for urease maturation is assisted by four urease accessory proteins,
UreE, UreF, UreG and UreH. Specifi c protein-protein interactions among these urease accessory proteins are essential for the
control of binding/release of nickel along the metal delivery pathway. We have previously determined the crystal structures of
UreF/UreH and GDP-bound-UreG/UreF/UreH complexes. Upon binding of UreH, the C-terminal residues of UreF are induced
to form an extra helix and a loop structure stabilized by Arg-250. Th ese conformational changes facilitate the recruitment of
UreG to the UreG/UreF/UreH complex, which is essential to urease maturation. Recently, we have determined the crystal
structure of the nickel/GTP-bound UreG dimer, which reveals how GTP hydrolysis induces conformational changes that
induce dissociation of UreG from the UreG/UreF/UreH complex and the release of nickel to the urease.
- Industrial Biotechnology| Pharmaceutical Biotechnology |Biotechnology Applications
Location: Armstrong
Chair
Sergey Suchkov
I M Moscow State Medical University, Russia
Session Introduction
Iwona Zur
Polish Academy of Sciences, Poland
Title: Efficiency of the antioxidative system is the first prerequisite for effective doubled haploids production with the use of isolated microspore culture method
Biography:
Iwona Å»ur completed her PhD and Habilitation in the fi eld of Agronomy and Plant Physiology at the University of Agriculture in Kraków, Poland. Since 2010, she has
been the Head of the Department of Cell Biology at the Institute of Plant Physiology Polish Academy of Sciences. She has published 38 papers in peer-reviewed
journals.
Abstract:
The technology of doubled haploids as the fastest route to total homozygosity is highly appreciated in many domains of basic research
and breeding. Among several methods, the one using isolated and in vitro-cultured immature cells of male gametophyte induced
towards embryogenic development (microspore embryogenesis-ME) possesses the highest potential for commercial application.
However, effi cient ME induction requires a precisely balanced stress treatment, strong enough to induce microspore reprogramming
but not exceeding cell stress tolerance threshold. As the general cause of injuries in in vitro-cultured cells is the overproduction of
reactive oxygen species (ROS), an effi cient antioxidative defence was suggested as the fi rst prerequisite for stress survival and eff ective
ME initiation. To establish the role of ROS and the antioxidative system in ME initiation, the generation of hydrogen peroxide, and
the activities of antioxidative enzymes and low molecular weight antioxidants were analysed in isolated microspores of two cultivars
of barley (Hordeum vulgare L.), winter cv. Igri and spring cv. Golden Promise, diff ering signifi cantly with respect to embryogenic
potential. Th e analyses were conducted in microspores redirected towards embryogenic development by low temperature tillers pretreatment
(4 weeks at 4°C). Additionally, the eff ects of compounds known as cellular redox status modifi ers, e.g. glutathione and L-2-
oxo-4-thiazolidinecarboxylic acid (OTC), on microspore viability and ME initiation effi ciency were estimated. Th e received results
suggest that the activity of the antioxidative system is the fi rst prerequisite for successful ME initiation, though in the case of its low
activity, antioxidative defence could be supported by the application of exogenous antioxidants.
Omirbekova N Zh
Al-Farabi Kazakh National University, Kazakhstan
Title: Development of approach to obtain Brachypodium distachyon L. regenerative plants with morphogenetic stability
Biography:
Omirbekova N Zh graduated from Al-Farabi Kazakh National University and Lomonosov Moscow State University and has completed her Doctoral studies from
Al-Farabi Kazakh National University. She is currently a Professor at the Department of Molecular Biology and Genetics, School of Biology and Biotechnology
of KazNU named after Al-Farabi (Republic of Kazakhstan). Her research interests include chemical mutagenesis, genetics and biochemistry of wheat. She has
published more than 30 papers in high valued journals.
Abstract:
The aim of the research is development of eff ective methodological approaches of in vitro cultivation, object 21 line (BD21) B.
distachyon. In order to develop cultivation methods, ability for callus formation, regeneration of generative and vegetative organs
of VD21 was studied. To cultivate, Linsmayer-Skoog and Murashige-Skoog medium, additional introduction of phytohormones
was used. Aseptic culture conditions for callusogenesis cultivation: under dark conditions at a temperature of 24°C, for t shoots
regeneration: 16/8 hour photoperiod and lighting of 3000 lux. Infl orescence and immature embryos isolated from green spikes of
vegetating plants and isolated embryos from mature seeds were used as primary explants to induce callus formation in vitro. During
immature embryo cultivation, callus formation takes place near the corimbe for 20-25 days. During the cultivation of whole caryopsis
with mature embryos, the sprouts grew aft er a week of cultivation on MS medium without hormones. Th e level of maturity of isolated
caryopsis has a signifi cant infl uence on the callus formation and the type of callus tissue. Th e mature caryopsis formed callus on
the 10th day of cultivation with a frequency of 75%. Th e cultivation of the overgrown caryopsis in the dark on medium MS 1 with
2 mg/L 2.4 DPA, led to the formation of a primary shoot in 60% of explants; the formation of callus in the area of the scute, but for
30-35 days. Passage of the callus on the same medium and on the hormone-free medium led to the appearance of greenish pointwise
impregnation of 30% of the calluses. For microclonal propagation, nodal segments of young shoots of plants were introduced into the
culture. To culture introduction, side shoots 5 cm long with 3-4 interstitial sites were cut, the microcrops were planted in inducing
media. Th e shoot-forming capacity of primary explants was about 59%; the multiplication factor for two passages was 5.7.
Biography:
Helmi Mohamed El-bendary is Assistant Professor of Agriculture at Fayoum University. He fi nished his BSc in Plant Protection Department at Cairo University and D.S.P.U at Mediterranean Agronomic Institute, Greece, M.S.c. at Cairo University, LLB at Cairo University, and Ph.D. at Mansoura University.
Abstract:
Naturally occurring micron sized silica has gained enormous popularity as a physically active insecticide. Nano-sized silica has insecticidal properties and would be needed in lesser quantity in comparison with conventional insecticides because of the huge surface to volume ratio of nanoparticles. Nano molecules have been widely used in consumer and industrial applications,
such as medicine, cosmetics and foods, because they exhibit unique physicochemical properties and innovative functions. However,
nanomaterials (NMs) can also be problematic in terms of eliciting a toxicological eff ect by their small size. Th e present study was
designed to examine the toxic eff ects of orally administered pesticide Sil-MATRIX 29% (potassium silicate) and silica nano-particles
(SiO2-NPs) using male albino rats, at sublethal doses [2/5, 1/4 and 1/8 LD50], relative to control on [body, organs weight such as liver, kidney, heart, spleen, and cytotoxic eff ect (such as total protein content levels as biochemical aspects)] for 28 and 45 days’ time exposure period. Orally ingested Sil-MATRIX 29% and silica nanoparticles (SiO2-NPs) [2/5, 1/4 and 1/8 LD50] were not associated with signifi cant changes in the average gain of body and organ weight. On the other hand, total protein content value aft er ingestion
with Sil-MATRIX and SiO2-NPs for all doses and treatments time period were increased signifi cantly in a pattern similar to control rats. Our results suggested that the well-dispersed nano-silica cytotoxic eff ect caused systemic exposure in mouse and induced mutagenic activity. Our information indicated that further studies of relation between physicochemical properties and biological responses are needed for the development and safer form of (NMs).
Jean M Francois
Université Fédérale Toulouse Midi-Pyrénées, France
Title: Engineering carbon-conserving synthetic pathways for assimilation and conversion of C5/C6 carbon sources into added value chemicals
Biography:
Jean M François got his PhD in Biological Science and Agronomy from the University of Louvain (Belgium) in 1988. He is Professor of Industrial Microbiology and
BioNanotechnology at the Federal University of Toulouse, School of Engineer. His research activity concerns integrated physiology and functional genomics in
microbial systems, with a specifi c focus on carbon and energy metabolism in yeast and fi lamentous fungi . He is author of more than 180 papers and 15 patents
and Editor in Chief of BMC Biotechnology for Biofuels.
Abstract:
The development of carbon effi cient pathways for added value (bio)chemicals production is the essence of White Biotechnology.
Th e limit of carbon conservation in all (bio)chemical syntheses is determined by the electron balance in substrate(s) and
product(s). Frequently, natural metabolic networks do not have the stoichiometric capacity to produce a value-added compound
at yields that correspond to the thermodynamic maximum. A good example of natural metabolic networks lacking stoichiometric
effi ciency is the bioproduction of glycolic acid (GA), a two carbon compound of considerable industrial interest notably in cosmetics
and biodegradable polymers. We addressed this objective to approach this maximal conversion yield by employing the following
strategies. Firstly, we reconsider a completely diff erent route of C5 assimilation that by-passes the decarboxylation reaction in the
pentose phosphate pathway and that rely on the carbon-conserving aldolytic cleavage of X1P or R1P to yield the C2 compound
glycolaldehyde and the C3 DHAP compound. Th is metabolic scheme required the expression of human hexo(fructo)kinase(Khk-C)
and human aldolase (Aldo-B). Th en glycoaldehyde can be either reduced by endogenous aldehyde reductase to produce ethylene
(EG) glycol or oxidized into glycolic acid. With this approach, we obtained yield of EG and GA close to maximal theoretical yield of 1
mol/ mol sugar. Interestingly, we found that the engineered strain expressing this synthetic pathway exhibited a remarkable rewiring
of the metabolic networks that culminate with a dramatic reduced metabolites and metabolic energy levels. We then combined this
synthetic pathway with the natural glyoxylate shunt that can be engineered to produce GA from DHAP. Th is combination led to
an optimized production strain that produced ~30 % more GA from a xylose/glucose mixture (66%/33%) than when the natural
pathway is working alone.
Jean M Francois
Université Fédérale Toulouse Midi-Pyrénées, France
Title: Engineering carbon-conserving synthetic pathways for assimilation and conversion of C5/C6 carbon sources into added value chemicals
Biography:
Jean M François got his PhD in Biological Science and Agronomy from the University of Louvain (Belgium) in 1988. He is Professor of Industrial Microbiology and
BioNanotechnology at the Federal University of Toulouse, School of Engineer. His research activity concerns integrated physiology and functional genomics in
microbial systems, with a specifi c focus on carbon and energy metabolism in yeast and fi lamentous fungi . He is author of more than 180 papers and 15 patents
and Editor in Chief of BMC Biotechnology for Biofuels.
Abstract:
The development of carbon effi cient pathways for added value (bio)chemicals production is the essence of White Biotechnology.
Th e limit of carbon conservation in all (bio)chemical syntheses is determined by the electron balance in substrate(s) and
product(s). Frequently, natural metabolic networks do not have the stoichiometric capacity to produce a value-added compound
at yields that correspond to the thermodynamic maximum. A good example of natural metabolic networks lacking stoichiometric
effi ciency is the bioproduction of glycolic acid (GA), a two carbon compound of considerable industrial interest notably in cosmetics
and biodegradable polymers. We addressed this objective to approach this maximal conversion yield by employing the following
strategies. Firstly, we reconsider a completely diff erent route of C5 assimilation that by-passes the decarboxylation reaction in the
pentose phosphate pathway and that rely on the carbon-conserving aldolytic cleavage of X1P or R1P to yield the C2 compound
glycolaldehyde and the C3 DHAP compound. Th is metabolic scheme required the expression of human hexo(fructo)kinase(Khk-C)
and human aldolase (Aldo-B). Th en glycoaldehyde can be either reduced by endogenous aldehyde reductase to produce ethylene
(EG) glycol or oxidized into glycolic acid. With this approach, we obtained yield of EG and GA close to maximal theoretical yield of 1
mol/ mol sugar. Interestingly, we found that the engineered strain expressing this synthetic pathway exhibited a remarkable rewiring
of the metabolic networks that culminate with a dramatic reduced metabolites and metabolic energy levels. We then combined this
synthetic pathway with the natural glyoxylate shunt that can be engineered to produce GA from DHAP. Th is combination led to
an optimized production strain that produced ~30 % more GA from a xylose/glucose mixture (66%/33%) than when the natural
pathway is working alone.
- Structural Enzymology | Enzymology & Biochemistry
Location: Armstrong
Chair
Magali Remaud-Simeon
INSA-Tolouse, France
Session Introduction
Albert Jeltsch
University Stuttgart, Germany
Title: Molecular enzymology of DNA methyltransferases – conformational changes and allosteric regulation
Biography:
Albert Jeltsch completed his PhD working on the mechanism of restriction endonucleases at University of Hannover in 1994. Afterwards, he started to study
DNA methyltransferases at Justus-Liebig University Giessen and at Jacobs University Bremen. Since 2011, he is a Professor of Biochemistry at the University
Stuttgart. He received the Gerhard-Hess award (DFG) and BioFuture award (BMBF). He has long standing expertise in Biochemical study of DNA and protein
methyltransferases, methyl lysine reading domains and in rational and evolutionary protein design. His work has been published in more than 250 publications in
peer reviewed journals and he is in the editorial boards of several journals.
Abstract:
DNA methylation is an essential epigenetic chromatin modifi cation. Th e setup and maintenance of DNA methylation patterns
depends on the coordinated activity of DNA methyltransferases (DNMTs) and their allosteric regulation by interacting proteins,
other chromatin modifi cations and post-translational modifi cations. I will present novel assays for DNMTs including single enzyme
assays to study their mechanism and conformationally locked mutants to study allosteric eff ects. Based on this, recent data regarding
the regulation and targeting of DNMTs by allosteric eff ect will be presented. Moreover, I will present insights into the mechanism of
DNMTs regarding target site location, specifi city and processivity.
David J Merkler
University of South Florida, USA
Title: N-acyltransferases and their role in fatty acid amide biosynthesis
Biography:
David J Merkler obtained a PhD in Biochemistry from Pennsylvania State University in 1985 and completed Postdoctoral Fellowships in Enzymology at Temple
University School of Medicine (1985-1987) and the Albert Einstein College of Medicine (1987-1989). His next position was as Senior Scientist at Unigene
Laboratories, Inc. involved in the in vitro production of a peptide hormone, calcitonin. In 1995, he moved back to academia as a Professor of Chemistry and
Biochemistry fi rst at Duquesne University (1995-1999) and then the University of South Florida (1999-present). His laboratory has been interested in the fatty
amides: identifi cation and characterization of the fatty acid amides (Lipidomics), identifi cation and characterization of the enzymes of fatty acid amide biosynthesis
(Enzymology and Structural Biology), and changes in the fatty acid amidome after targeted enzyme knock-out (subtraction lipidomics).
Abstract:
Fatty acid amides are a family of cell signaling lipids with the general structure of R-CO-NH-Y. Th is structural simplicity belies
a wealth of diversity amongst this lipid family as the R-group is derived from fatty acids (R-COOH) and the Y-group is derived
from biogenic amines (H2N-Y). Th e fatty acid amide family is divided into classes, defi ned by parent amines. Examples include the
N-acylethanolamines (NAEs, R-CO-NH-CH2-CH2OH) and the N-acylglycines (NAGs, R-CO-NH-CH2-COOH). Other classes of
fatty acid amides are known. Th e best known fatty acid amide is N-arachidonoylethanolamine (anandamide), a fatty acid amide found
in the human brain that binds to the cannabinoid receptors. We have a long interest in the enzymes of fatty acid amide biosynthesis.
We identifi ed an enzyme that oxidizes the NAGs to the primary fatty acid amides and showed that inhibiting this enzyme led to the
cellular accumulation of the NAGs. We have characterized several insect N-acyltransferases (from D. melanogaster, B. mori, and T.
castaneum) that catalyze the acyl-CoA-dependent formation of fatty acid amides from an amine acyl-acceptor substrate. Knock-out
experiments in D. melanogaster validate our in vitro substrate specifi c studies demonstrating that one novel N-acyltransferases, arylalkyl
N-acyltransferase-like 2 (AANATL2), does catalyze the formation of N-acyldopamines in vivo. We developed a straightforward
platform technology to rapidly identify substrates for our panel of uncharacterized insect N-acyltransferases. Our application of this
technology leads to identifi cation of an enzyme in D. melanogaster, agmatine N-acetyltransferase (AgmNAT), which catalyzes the
formation of N-acetylagmatine, a virtually unknown metabolite. We have determined the X-ray structure of AgmNAT. Our work on
AgmNAT hints at an unknown reaction in arginine metabolism and points to a novel class on fatty acid amides, the N-acylagmatine.
Th e presentation will also include our results on the kinetic and chemical mechanisms of the novel N-acyltransferases.
Karlo M Lopez
California State University-Bakersfield, USA
Title: Lysyl oxidase: A versatile and elusive enzyme
Biography:
Karlo M Lopez is currently an Associate Professor of Biochemistry at California State University, Bakersfi eld. He received a PhD from Clark University and was a
Howard Medical Institute Fellow at Pomona College. His research focuses primarily on the structural characterization of lysyl oxidase and understanding the role
this enzyme plays in cancer metastasis. He is a member of the Committee on Ethics of the American Chemical Society and was part of the Task Force for Safety
Education Guidelines.
Abstract:
Lysyl oxidase is an extracellular matrix, copper-dependent, amine oxidase that catalyzes a key crosslinking step in collagen and
elastin. Th e enzyme is synthesized as a proenzyme that, upon excretion to the extracellular matrix, is cleaved at the Gly168-
Asp169 bond by procollagen C-proteinase in the mammalian form of the enzyme. Lysyl oxidase is highly regulated and changes in its
regulation have been shown to play a role in fi brosis and several other diseases. More recently, the enzyme has been shown to play a
paradoxical role in cancer. In the early stages of cancer, the cleaved pro-peptide has been shown to inhibit the RAS oncogene, whereas
in late stages of cancer lysyl oxidase has been shown to promote metastasis. Lysyl oxidase is highly insoluble and this has hampered
its full characterization. Recent work in the by our study group has addressed some of the issues associated with the insolubility and
characterization of the enzyme. In particular, this talk will address how plasmids were used to increase enzyme yields over those
obtained directly from bovine aortic tissue, the role solubility tags play on enzyme activity and suitability for characterization studies,
and will end with an innovative new approach to drug delivery that targets lysyl oxidase in cancer cells but remains inactive in normal
cells.
Biography:
Anna V Hine studied at the University of Manchester (UK) and Harvard Medical School. She is a Reader and Associate Dean Enterprise at Aston University (UK).
In March 2013, she was named BBSRC Commercial Innovator of the Year 2013, for her work in transferring ProxiMAX randomization into SME Isogenica Ltd. She
is a Molecular Biologist by training, she relishes interdisciplinary work.
Abstract:
ProxiMAX randomization is the technology that lies behind Isogenica’s Colibra™ off ering. It is a defi ned saturation mutagenesis
process that delivers precision control of both identity and relative ratio of amino acids at specifi ed locations within a protein/
antibody library. Th us unwanted amino acids such as cysteine and methionine can be eliminated from libraries because no constraints
are imposed by the genetic code. Moreover, the process is non-degenerate, which means that encoding DNA libraries are as small as is
physically possible. ProxiMAX relies on a process of saturation cycling comprising ligation, amplifi cation and digestion for each cycle
and is the science behind the commercial Colibra™ technology. Currently focused on antibody libraries but with achieved diversities
of >99% (6 & 11 saturated codons) and the potential to generate libraries of up to 1014 components, we contest that ProxiMAX
randomization is a vital tool in engineering any protein library of the highest quality. Th is presentation will examine the development
of the ProxiMAX process and give examples of libraries created to date.
Sotaro Fujii
Hiroshima University, Japan
Title: Stability and function of a thermophilic cytochrome c’
Biography:
Sotaro Fujii is working on the stability, structure, and function of proteins that are important for microbial energy metabolism. A characteristic aspect of his research
activity is comparison of the homologous proteins isolated from microorganisms living in extreme environments in which humans cannot live and those isolated
from ‘normal’ environments.
Abstract:
Cytochromes c' are classifi ed as heme proteins found in restricted Gram-negative bacteria. Th ey usually form a homo dimeric
structure, and the single subunit typically consists of four helix bundle. Biochemical analysis showed that they can bind diatomic
gasses such as NO or CO, but not O2. Recently we purifi ed cytochrome c' from thermophilic Hydrogenophilus thermoluteolus, and
named it PHCP. H. thermoluteolus grows optimally at 52°C, indicating that PHCP is more stable than homologous proteins from
mesophiles. In this study, we compared stability and function of PHCP with its mesophilic homologue, Allochromatium vinosum
cytochrome c' (AVCP) having 55 % amino acid sequence identity. In order to check the stability, we measured the circular dichroism
spectra with increasing temperature. Th e denaturation temperature of PHCP was 87°C, which was higher than that of AVCP
(52°C). Th e X-ray structure comparison between PHCP and AVCP revealed that the stability diff erence was due to the heme-related
interactions and subunit-subunit interactions, which was also proofed by mutagenesis study. Th ese results indicated that PHCP
advantageously retains the native structure at high temperature. Th e PHCP X-ray structure further revealed a ligand binding channel
and a penta-coordinated heme, as observed in the AVCP protein, indicating PHCP could bind diatomic gasses at high temperature.
Th us, we measured the gas binding affi nity of PHCP and AVCP using absorption spectra. Th e association constant (Ka) of PHCP
with CO was 3 times lower than that of AVCP at 25°C, and PHCP could maintain normal spectral changes up to 60°C. In AVCP, such
spectral changes with CO could not to be detected at 60°C, because of denaturation of AVCP. In conclusion, PHCP has a structure
fulfi lling the requirement for both gas-binding function and thermal stability. Th is stable cytochrome c' will become a model for
protein engineering fi eld.
- Molecular Enzymology | Enzyme Therapeutics |Enzymology in drug discovery | Industrial Biotechnology|Biotechnology Applications
Location: Armstrong
Session Introduction
Shree Kumar Apte
Bhabha Atomic Research Centre, India
Title: Engineering cyanobacterial nitrogen bio-fertilizer for rice cultivation in stressful environment
Biography:
Shree Kumar Apte obtained his Master’s in Botany from Jiwaji University, Gwalior, India with a Gold Medal in Science Faculty in 1972. He researched at the Bhabha
Atomic Research Centre (BARC), Mumbai, India for 42 year, before retiring in 2014 as a distinguished Scientist and Director of the Bioscience Group, BARC. He is
an elected fellow of all three National Science Academies and the National Agriculture Science Academy in India. Currently he serves as Emeritus Professor, Homi
Bhabha National Institute, J C Bose National Fellow (DST) and Raja Ramanna Fellow (DAE) at BARC, Mumbai.
Abstract:
As a naturally abundant, photosynthetic, nitrogen-fi xing microbe, the cyanobacterium Anabaena contributes signifi cantly to
the nitrogen and carbon economy of tropical soils, especially in cultivation of rice paddy. However, its nitrogen bio-fertilizer
potential is adversely aff ected by common abiotic stresses. Engineering enhanced nitrogen fi xation and stress tolerance capabilities
in this microbe through genetic manipulation is seriously limited due to the unavailability of appropriate tools and techniques
and knowledge of suitable candidate genes. In recent years, our laboratory has devised an electroporation protocol for genetic
transformation that achieves high frequency gene transfer and overcomes problems associated with the current practice of triparental
conjugation between E. coli strains and Anabaena. We have also constructed (a) a suitable vector for new gene discoveries,
and (b) a novel integrative expression vector pFPN, placing desired genes at a defi ned locus in Anabaena genome and facilitates their
high level expression from an eco-friendly light-inducible promoter. Using these tools we have identifi ed several genes responsible
for enhanced heterocyst formation and nitrogen fi xaton (hetR), chaperones (groESL, cpn60) for protein folding and homeostasis, and
several oxidative stress tolerance genes (superoxide dismutases, catalases and peroxiredoxins) which confer superior stress tolerance
to Anabaena. Th e approach has proved very useful for constructing recombinant Anabaena strains capable of nitrogen fi xation in
stressful environments.
Mamuka Kvaratskhelia
University of Colorado School of Medicine, USA
Title: The dual role of integrase in HIV-1 replication
Biography:
Mamuka Kvaratskhelia began his independent research career at the Ohio State University in 2003 and has focused on better understanding of the structure and
function of HIV-1 integrase as a therapeutic target. He has recently (2017) moved to University of Colorado Denver as a Professor of Medicine (Infectious Diseases)
to further extend his studies on HIV-1 integrase. By employing innovative biochemical, biophysical, structural biology, molecular biology and virology approaches,
his research team has made many important contributions to the fi eld, which include the discovery of second, non-catalytic role of integrase in HIV-1 biology and
elucidating the mode of action of ALLINIs.
Abstract:
A key HIV-1 enzyme integrase catalyzes irreversible insertion of a viral DNA copy of its RNA genome into human chromosome,
which is essential for viral replication. Th erefore, integrase is an important therapeutic target. Productive integration into host
chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined viral and target DNAs. We
have used cryo-EM coupled with biochemistry and virology experiments to obtain high-resolution structures for STCs and to
characterize the integrase multi-subunit assemblies into large, nucleoprotein complexes. We are currently extending these studies
to elucidate structural basis for the mode of action of clinically used integrase strand transfer inhibitors (INSTIs), which bind to the
enzyme active site in the context of the integrase-viral DNA complex and block the strand transfer reaction. Our parallel eff orts are
focused on studying allosteric HIV-1 integrase inhibitors (ALLINIs), which are currently undergoing clinical trials (2-5). Unlike
INSTIs, ALLINIs bind at the integrase dimer interface and induce aberrant protein multimerization. Unexpectedly, in infected cells
ALLINIs were signifi cantly more potent during virion maturation rather than during integration. ALLINIs markedly altered virus
particle morphogenesis by misplacing the ribonucleoprotein complexes outside the protective viral capsid shell and yielded inactive
virions. In turn, these fi ndings have suggested that integrase has a second function in HIV-1 biology. Our follow up studies have
revealed that integrase directly binds the viral RNA genome in virions. Th ese interactions have specifi city, as integrase exhibits
distinct preference for select viral RNA structural elements. ALLINIs impair integrase binding to viral RNA in virions of wild-type,
but not escape mutant, virus. Th ese results reveal an unexpected biological role of integrase binding to the viral RNA genome during
virion morphogenesis and elucidate the mode of action of ALLINIs. Collectively our fi ndings indicate that viral integrase plays a dual
role during HIV-1 replication.
Alessandra Astegno
University of Verona, Italy
Title: Characterization of cystathionine γ-lyase from T. gondii: A target for drug development?
Biography:
Alessandra Astegno is interested in diff erent aspects of Protein Chemistry and Enzymology, including folding, evolution and structure-function relationship of
proteins and macromolecular assemblies. She is currently an Assistant Professor in Biochemistry at the Department of Biotechnology of the University of Verona.
She has a solid background in recombinant protein expression and purifi cation, functional and structural characterization of pyridoxal phosphate-dependent
enzymes as well as metallo-proteins.
Abstract:
Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As there
is currently no vaccine available for human, the identifi cation of good target candidates for future drug development is urgently
required. A recent proteomic analysis of partially sporulated oocysts of T. gondii showed that oocyctes have a greater capability of
de novo amino acid biosynthesis, shedding light on a stage-specifi c subset of proteins whose functional profi le is consistent with
the oocyst need to resist various environmental stresses. Among these putative oocyst/sporozoite-specifi c proteins, three enzymes
involved in cysteine metabolism, i.e., cystathionine β-synthase, cystathionine γ-lyase (CGL) and cysteine synthase, were found.
However, despite the central metabolic roles of these enzymes, the functionality of none of them has so far been investigated. Herein,
CGL from T. gondii (TgCGL) has been cloned, expressed and physiochemically and enzymatically characterized. Th e purifi ed TgCGL
is a functional enzyme which splits L-cystathionine almost exclusively at the CγS bond to yield L-cysteine. Th is fi nding likely implies
that the reverse transsulfuration pathway is operative in the parasite. Th e enzyme displays only marginal reactivity toward L-cysteine,
which is also a mixed-type inhibitor of TgCGL activity, therefore indicating a tight regulation of cysteine intracellular levels in the
parasite. Structure-guided homology modelling revealed two striking amino acid diff erences between human and parasite CGL active
sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of these two residues to the corresponding residues
in human revealed their importance in modulating both substrate and reaction specifi city of the parasitic enzyme. Our fi ndings might
have far-reaching implications for the use of TgCGL as anti-toxoplasmosis drug target.
Yungui Yang
Northwest A & F University, China
Title: Infulence of four kinds of additives and concentration on oats silage effect
Biography:
Yungui Yang graduated from China Agricultural University in 1987 with a Master's degree in Grassland Science. In August of the same year, he taught at the
Northwest A&F University. In 2006, he received a Doctorate in Soil Resources and Information Technology direction. Now he is a member of Lawn Professional
Committee of China Grass Society and Director of Grassland Resources and Management Committee. In October 2012, he went to the United States to attend
the international annual conference jointly organized by the American Society of Agricultural Sciences, the Crop Science Society and the Soil Science Society. In
October 2013, he went to Mongolia to attend the Eurasian Pacifi c Union Symposium. He is currently a Member of Comprehensive Utilization of Straw Resources of
China Agronomy, a review expert of Life Science Division of NSFC, a fellow of American Society of Agricultural Sciences, Soil Society and Crop Society.
Abstract:
Statement of the Problem: Th e aim for this research was to study the eff ect of diff erent additives and concentration on oat silage.
Selected Dancer as material was used for silage in milk stage, and four kinds of additives respectively at diff erent concentration were
used. Th ey were lactobacillus (0 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg), formic acid (0 ml/kg, 1 ml/kg, 5 ml/kg, 10 ml/kg), sucrose (0%,
1%, 2%, 4%) and cellulose (0 mg/kg, 50 mg/kg, 100 mg/kg, 150 mg/kg). Th e materials were ensiled at room temperature and opened
60 days later, and the fermentation quality and the chemical composition were analyzed. Results showed that it had a positive impact
on Dancer silage with four kinds of additives. Considering nutritional value index(crude protein, ether extract and crude fi ber) When
cellulase were applied at 50 mg/kg, the oats silage were excellent. Considering silage quality indexes of pH value, AN/TN, soluble
sugar content and lactic acid content, sucrose at 2% level was the best concentration. Th e best concentration of diff erent additives
were that: lactobacillus (5 mg/kg), formic acid (5ml/kg), sucrose (2%) and the infl uence on silage quality have no close connection
with concentration when added cellulase.
Biography:
C Gopinathan is working as a Associate Professor in the Department of Biotechnology at the University of Calicut. He has fi nished his MSc, MTech in Biotechnology.
His specialization is towards Bioprocess Technology/Fermentation Technology. He is the former member of Academic Council, University of Calicut, American
Society for Microbiology and Association of Microbiologists of India.
Abstract:
Mosquito borne diseases not only cause loss of lives but also impose heavy health and economic burdens. Extensive use of
chemical insecticides for the control of malaria and other mosquito borne diseases has led to the development of resistance in
mosquitoes to these insecticides and are hazardous to the environment. Biolarvicides of the strain Bacillus thuringiensis israelensis
(Bti), serotype H-14 is highly eff ective against mosquito larvae. Even though Bti products are effi cient controls for mosquito and black
fl y larvae, their use in developing countries is limited by their cost. Th us, there is a need to reduce the overall production cost of Bti in
order to make it competitive in the market. It depends on many factors; however, the raw material cost is one of the most important
criteria which may comprise >70% of the overall production cost. Fruit wastes are available in plenty and contain mainly fructose as
the carbon source, which is easily fermentable and can substitute costly substrates like glucose. Channelizing huge quantities of rotten/
waste pineapples which otherwise are discarded can substantially reduce production cost of Bti. Similarly fi sh-amino acid produced
by fermenting rotten fi sh and jaggery/molasess has proved to be excellent as a medium supplement; especially to overproduce the
much wanted delta endotoxin produced by Bti. India is one of the countries leading in fruit and vegetable production. It is also blessed
with one of the longest coastline in the world of approximately 7516.6 km. Th e total annual catch is around 4 million metric tons. In
addition it is second aft er Brazil in sugarcane cultivation with an annual yield of 3412 million metric tons. Th e massive availability
of fruit wastes (pineapples) and huge quantities of rotten /discarded fi sh , which are freely available, all can be channelized for cost
eff ective production of this value added product, substantially lowering the media cost of Bti production when scale- up is attempted.
Results show biomass increase of up to 27% compared to control when pineapple juice was used as the main carbon source. Th e
toxicity improvements with fi sh-amino acid supplemented medium, shows considerable reduction in killing time of Aedes aegypti
larvae.
- Posters
Location: Armstrong
Session Introduction
Kathryn A Johnston
California State University, USA
Title: Inhibition of Lysyl oxidase in breast cancer cells by small-molecule inhibitors
Biography:
Kathryn A Johnston is a fourth–year Senior Student at California State University, Bakersfi eld and has worked in Dr. Lopez’s laboratory for the past two and a
half years. During this period, she has published three papers. Her work deals primarily with the inhibition and reduction of viability of breast cancer cells using
derivatized inhibitors of β-aminopropionitrile. She has presented her work as posters and invited talks at regional meetings, as well as national meetings of the
American Chemical Society.
Abstract:
Lysyl oxidase (LOX) is an extracellular matrix, copper-dependent, amine oxidase that catalyzes a key crosslinking step in
collagen and elastin. Th is enzyme has also been shown to play a role in promoting metastasis. Th e correlation between
high LOX activity and cancer metastasis is strong enough that upregulated LOX activity can be used as a diagnostic marker for
the severity of cancer in patients. β-aminopropionitrile is a known potent inhibitor of lysyl oxidase; however, this inhibitor is
not selective and, therefore, cannot be used as a therapeutic agent. β-aminopropionitrile has been derivatized using aromatic
sidechains and has been used to selectively target lysyl oxidase in breast cancer cells. Th e inhibitor LP-1-2 has been shown to
reduce breast cancer cell viability with a 100 μM dose and 72-hour incubation period. Th e eff ect on cell viability increased
with increasing amounts of inhibitor. Th e selective targeting of lysyl oxidase was verifi ed using western blot analysis and lysyl
oxidase activity assays. Th e activity assays showed that addition of increasing amounts of inhibitor decreased the activity of
lysyl oxidase. Th e highest level of inhibition detected was with lysyl oxidase isolated from cells treated with 5000 μM of LP-1-2
for 3 days, which decreased the activity three-fold as compared to lysyl oxidase isolated from untreated cells.
Brian Effer
University of La Frontera, Chile
Title: Design, construction and extracellular expression of L-asparaginase from Dickeya chrysanthemi in yeast
Biography:
Brian Eff er is a PhD candidate at the University of La Frontera and University of São Paulo in Cell and Molecular Biology and Biochemical and Pharmaceutical
Technology areas, respectively. He has published seven papers in reputed journals and has been serving as a referee in several journals.
Abstract:
L-asparaginase (L-ASNase) is an important enzyme used as a biopharmaceutical to treat acute lymphoblastic leukemia (ALL).
Currently there are two L-ASNase approved by FDA: native and of bacterial origin, both from E. coli and D. chrysanthemi.
Due to L-ASNase’s immunogenic eff ects, it is necessary to seek alternatives such as recombinant expression in yeast. Th is
expression system can provide extracelullar secretion and glycosilation process, which can decrease immunogenicity and
facilitate downstream process. We report the construction of three diff erent expression vectors in order to obtain extracelullar
L-ASNase from D. chrysanthemi using eukaryotic exrpression system. asnB gene from D. chrysanthemi was cloned in pJAG-s1
plasmid in fusion with endogenous signal sequence (SS), that addresses protein to bacterial periplasm, and with or without
histidine tag (His). SuperMan5 yeast strain was transformed with pJAG-s-asnB constructs in order to be able to express the
recombinant protein. Aspartic acid β-hydroxamate method was applied for activity determination of L-ASNase recombinant
in culture supernatants. When both SS and His-tag were removed (expression of mature protein), protein expression and
secretion process were improved considerably compared to other constructions, indicating that for this gene, additional
structures added to the recombinant protein may interfere with the expression, fi nal enzyme activity and cell secretion.
Purifi cation processes are being executed.
Short S
Universidad de La Frontera, Chile
Title: Study of the potential use of antifreeze proteins of Deschampsia antarctica in the cryopreservation of Salmo salar spermatozoa
Biography:
Short S completed her Biotechnology Engineering at Universidad de La Frontera and started her Doctoral studies in Applied Cellular and Molecular Biology at
the Universidad de La Frontera. She is currently TA of Enzymology, Protein Structure and Immunology at the same institution. She has published fi ve articles in
reputed journals.
Abstract:
Cryopreservation allows to preserve genetic resources in aquatic species, such as Atlantic salmon (Salmo salar). However,
freezing may cause cell damage aff ecting the sperm quality. New procedures including antifreeze proteins (AFPs) seem
to improve sperm quality aft er cryopreservation. AFPs have the ability to bind to ice crystals inhibiting their growth, and ice
recrystallization (IRI) in vitro. Deschampsia antarctica is a freezing tolerante vascular plant species (LT50 -27°C) exhibiting
apoplastic antifreeze activity. We hypothesize that AFPs from D. antarctica favor the sperm quality of cryopreserved S.
salar spermatozoa. Th e aim of this work is to evaluate cryoprotection of AFPs from D. antarctica in S. salar spermatozoa.
Cryopreservation of S. salar spermatozoa has been made with a standard freezing medium (C+) and diff erent treatments with
protein extracts (20 μg/ml) of D. antarctica supplemented with permeating, DMSO 1.3 M, glucose 0.3 M, and non-permeating,
BSA 2% w/v cryoprotectants. Post-thawing plasma membrane integrity (PMI) by SYBR-14/PI and mitochondrial membrane
potential (MMP) by JC-1 markers were assessed using fl ow cytometry. Th awed cells in the presence of protein extracts from
D. antarctica without BSA maintained PMI as well as C+ and showed signifi cant diff erences respect to the other treatments.
Th e percentage of cells thawed with protein extracts of D. antarctica and with cryoprotectants showed higher MMP than C+.
While, treatments without permeating and non-permeating cryoprotectants maintained a similar MMP to C+. AFPs from
D. antarctica showed a cryoprotective eff ect in S. salar spermatozoa and these would act as non-permeating cryoprotectant,
replacing BSA in standard freezing medium.
FarÃas J G
Universidad de La Frontera, Chile
Title: Characterization of antifreeze activity in apoplastic extract of Deschampsia antarctica
Biography:
Farías J G is the Associate Professor and Director of the Chemical Engineering Department, Universidad de La Frontera. His research interest is in Pharmaceutical
Biotechnology, focusing on Molecular Therapeutics and Drugs Production. He has published 51 articles in reputed journals.
Abstract:
Deschampsia antarctica Desv. is a vascular plant species that colonized maritime Antarctica exhibiting extreme freezing
tolerance (-27°C). Th is has been associated with apoplastic antifreeze activity. Antifreeze proteins (AFPs) have the ability
to bind to the growing surface of ice crystals inhibiting their growth; however, this activity has been poorly characterized in
this species. Th erefore, the aim of this work is to characterize the antifreeze activity of apoplastic extracts from D. antarctica.
To understand how this plant can tolerate freezing temperatures year-around, and in order to evaluate the potential antifreeze
activity of apoplastic proteins from D. antarctica as future applications, experiments have been developed in cold-acclimated
and non-aclimated plants. To identify the best apoplastic proteins accumulation aft er plant cold-acclimation, apoplastic
extracts were quantifi ed every four days for 21 days of low temperature exposure. Antifreeze activity was determined by ice
recrystallization inhibition (IRI), thermal hysteresis activity (TH) and ice crystal growth in dilution series of apoplastic extracts.
Th e results indicate that the minimum IRI activity was evident in extracts with a concentration equal to 0.005 μg/μl at coldacclimated
condition, while in non-aclimated plants the IRI activity was lost at 0.05 μg/μl. At concentration equal to 2.5 μg/μl,
ice crystals showed a bipyramid shape and a TH equal to 0.4°C. In conclusion, we observed that cold-acclimation increased
apoplastic antifreeze activity, obtaining higher IRI but low TH in these apoplastic extracts. Th is high IRI is remarkable and
further studies are needed to characterize the apoplastic extract to associate this activity to apoplastic antifreeze proteins which
could be of interest for later studies as a cryoprotectant.
Shahid ullah
Shenzhen University, China
Title: Computer aided screening of mangrove ecosystem derived compound against acetyl-cholinesterase
Biography:
Shahid ullah is from Shenzhen University, China.
Abstract:
Alzheimer’s disease (AD) is considered as the most common type of dementia among older people. Almost 9 million
people are suff ering from AD in china and increasing with the course of time. Currently many diff erent herbs are used
for the treatment of AD including six Flavors Rehmannia Pills , Gastrodia and Uncaria Drink. It is been suggested that some
Acetyl-cholinesterase inhibitors induced molecular and cellular change that directly infl uence AD pathogeneses. In our study
literature search was perform to fi nd Mangrove eco-system phytochemical structures by using Builder soft ware implemented
in Molecular operating environment (MOE 2009). Acetyl-cholinesterase (PDB ID 1EVE) structure with bound ligand was
retrieved from protein data bank. Molecular docking was performed by triangular matcher placement method and rescore
by London dG parameter. Th e crystal structure has bound ligand which was active against acetyl-cholinesterase. It can be
concluded by docking analysis of diff erent compounds that mangrove ecosystem compound may serve as good inhibitors
against Acetyl-cholinesterase