Earticle

Efficient immobilization of glucose oxidase (GOx) has been explored for many biological applications. In this work, we describe the constitutive expression of R5 tagged GOx in Pichia pastoris X-33 for biosilicification reaction. The gene encoding the GOx from Aspergillus niger KTCT 6278 was cloned into the pGAPZαC including saccharomyces cerevisiae α-factor secretion signal. Several transformants were selected on YPD medium with zeocin and the one showed excellent enzyme activity. Expressed recombinant proteins in the medium were concentrated and analyzed as a single band on SDS-PAGE. Influence of carbon sources, both glucose and glycerol was observed with growth rate, pH and enzyme activity. It is available high expression (1.96 U/ml) when glucose was consumed in a day. Although glycerol based medium expended in four days was obtained high cell density but was expressed low (0.41 U/ml). Production of GOx through fermentation using glucose was optimized in a 1L reactor with controlling pH, DO, and aeration rate

Comparative Study of Enzyme Thermostability using Torsion Potentials in the Region of β-Turn Hong Seung Yeon and Young Je YooSchool of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Korea Thermostable enzyme has been a key research topic because it industrially gives a number of beneficial advantages. Various strategies to enhance thermostability of enzymes have been proposed, however, an integrated strategy has not yet reported. In this study, torsion potentials in the region of β-turn, as one of strategy to explain enzyme thermostability, were analyzed between thermophilic and mesophilic enzymes. β-turn is an element of secondary structure which consists of four residues and plays an important role in protein structure directing folding pathways. The hypothesis of this study is that it might act to enhance thermostability of enzymes by introducing favorable torsion angle in the region of β-turn because β-turn is passively formed by other secondary structures. Thus, it may be assumed that torsion potential of β-turns in mesophilic enzyme is bigger than thermophilic one. To demonstrate this hypothesis, thirty sets of thermophlic and mesophilic enzyme pair were chosen through several qualified criteria and intrinsic torsion potential equation was used. The statistical analysis showed that torsion potential of mesophilic enzymes in the region of β-Turn is bigger than thermophilic one at less than 5% level of significance. From this statistical result, optimizing torsion angle in the region of β-turn can be used as a strategy for enhancing thermostability of enzymes. ReferencesVijaya Parthiban, M. Michael Gromiha, Christian Hoppe, and Dietmar Schomburg. PROTEINS:Structure, Function, and Bioinformatics 66:41-52(2007)Young Kee Kang, Kyoung Tai No, and Harold A. Scheraga. J. Phys. Chem 1996, 100, 15588-15598Saul R. Trevino, Stephanie Schaefer, J. Martin Scholtz, and C. Nick Pace. J Mol Biol. 2007 October 12; 373(1): 211-218

Direct electron transfer (DET) between oxidoreductases and electrode surfaces is very important part in bioelectrocatalysis, which can be applied to electroenzymatic synthesis, electrochemical biosensors, and enzymatic biofuel cells. The DET does not require any mediators between enzymes and electrodes, eliminating problems related to mediator stability, selectivity, and mass transfer limitations. In addition, DETbased enzyme electrodes theoretically functions at a potential range that is close to the redox potential of the enzyme itself. In this study, the DET of redox enzymes was attempted using two types of immobilization methods: One was enzyme immobilization based on self-assembled monolayers; the other was entrapment with a matrix of polymers in which conducting nanomaterials such as carbon nanotubes (CNTs) and metal nanoparticles were co-entrapped. As a model enzyme was selected laccase, which is a multicopper oxidase to hold four copper ions and can perform a one-electron oxidation of phenols and similar molecules with concomitant reduction of molecular oxygen to water. We checked that the DET between laccase and electrodes took place in buffers with different pH values saturated with argon as well as with oxygen using cyclic voltammetry.

Spring model for enzyme deformation is proposed for the calculation of residual force as a new mechanical approach. This new modeling method assumes that enzyme moves like elastic body doing substrate docking and product release. It was validated by selection of forced and twisted region in cases of HIV-1 protease and bacteriophage T4 lysozyme. Enzyme flexibility has relation to enzyme activity because enzyme does flexible motion for catalysis. The selected regions using spring model were compared with activity-enhanced mutation regions in bacteriophage T4 lysozyme and Candida antarctica lipase B. It was found that the mutations at the helix edges located far away from twisted region increased enzyme activity in T4 lysozyme. In case of Candida antarctica lipase B, mutations at the most twisted region make enzyme activity increase. It will be discussed about relationship between enzyme flexibility and activity based on these data.

A recombinant L-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band of 76 U mg–1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximal activity for l-fucose isomerization was observed at pH 7.0 and 75ºC in the presence of 1 mM Mn2+. The half-lives of the enzyme at 60, 65, 70, 75, and 80ºC were 62, 13, 6, 2, and 1 h, respectively. Among the monosaccharide substrates, the enzyme displayed activity only for L-fucose, with a kcat of 11,910 min–1 and a Km of 141 mM, d-arabinose, D-altrose, and L-galactose in decreasing order. The enzyme produced 80 g D-altrose l–1 from 500 g D-psicose l–1 under the conditions of pH 7.0, 65ºC, 1 mM Mn2+, and 20 U enzyme ml–1 within 7 h with a productivity of 11.4 g l–1 h–1.

This study investigated that Aeropyrum pernix K1 chaperonins (ApCpnA and ApCpnB) can efficiently prevent the thermal aggregation and inactivation of foreign model protein, citrate synthase (CS, 48.9 kDa, dimer) from porcine heart. ApCpnA and ApCpnB, chaperonin genes were amplified by PCR from the A. pernix K1 genomic DNA, and subcloned into pET3d and pET21a vectors, respectively. Each of the constructed pET3d-ApCpnA (6.1 kb) and pET21a- ApCpnB (6.9 kb) was transformed into E. coli Rosetta and BL21 Codonplus (DE3), and the transformant cells successfully expressed ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa). Each of the recombinant ApCpnA and ApCpnB was purified by heat treatment and anion-exchange chromatography. ApCpnA and ApCpnB prevented the thermal aggregation and inactivation of foreign model protein, CS at 43℃. Especially, the addition of ATP or CTP with ApCpnA and ApCpnB more extensively prevented thermal aggregation and inactivation of CS. Based on these results, we propose that ApCpnA and ApCpnB from A. pernix K1 can be utilized to enhance the durability and cost effectiveness of high-temperature biocatalysts.

Saxagliptin is a pharmaceutical chemical used for treatment of type 2 diabetes. We prepared 2-(3-hydroxy-1-adamantyl)-(2S)-amino ethanoic acid which is an intermediate of saxagliptin, by coupling two enzyme reactions. Both Branched-chain amino transferase (BcAT) from Enterobactor sp. and aspartate amino transferase(AAT) from E.coli were transformed into the E.coli BL21(DE3). 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid was applied as the substrate for the transaminase reaction. The activity of the enzyme was analyzed with HPLC after 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl isothiocyanate(GITC) derivertization. This enzyme reaction performed high (s)-form enantioselectivity which was 99.9% ee. Though BcAT alone showed activity toward the substrate, coupling AAT shifted the equilibrium constant and made 1.7 fold higher product yield.

α-ketoglutarate is a kind of ketone derivatives of glutaric acid, an intermediate of Krebs cycle and therefore the function of this chemical is biologically very important in metabolism. It is known that the functions of α-ketoglutarate are various such as prevention of nitrogen accumulation in the body, medicine to cure malnutrition, control lipid level in serum, and delay wrinkle formation. α-ketoglutarate can be synthesized by deamination of cheap L-glutamate by glutamate dehydrogenase. The glutamate dehydrogenase is cofactor-dependent enzyme and therefore it is needed to continuous addition of cofactor for the synthesis. The introduction of regeneration method for the cofactor regeneration is necessary due to high cost of the cofactor. In order to recycling cofactor, electrochemical method was used in this study. Continuous type of electrochemical bioreactor was designed for the continuous α-ketoglutarate production. For this setup, NAD+-dependent glutamate dehydrogenase from recombinant E.coli which contains thermophilic glutamate dehydrogenase from Thermus sp. was purified. NAD+ was modified with polyethylene glycol (PEG) to retain cofactor in the continuous electrochemical bioreactor. Details will be presented and discussed.

Uncharacterized genes from the mesophilic bacterium Clostridium difficile and hyperthermophilic bacterium Thermotoga maritima that were previously proposed to be ribose-5-phosphate isomerases (RpiBs) were cloned and expressed in Escherichia coli. The RpiBs were compared to RpiB from the thermophilic bacterium C. thermocellum. The maximal activity of l-talose isomerization was observed at pH 7.5 and 40 °C in C. difficile RpiB, at pH 7.5 and 65 °C in C. thermocellum RpiB, and at pH 8.0 and 70 °C in T. maritima RpiB. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as d-ribose, d-allose, l-talose, llyxose, d-gulose, and l-mannose. In contrast, C. thermocellum and T. maritima RpiBs displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the d- and l-forms of ribose, talose, and allose.

Production of phenolic polymers catalyzed by peroxidases has been studied for decades and drawing much interest due to the environmentally friendly production process and mild reaction conditions. Various substituted phenols, aminophenols, and anilines have been polymerized by peroxidases in organic solvents-water mixtures (1, 2). Phenolic polymers thus produced have potentials as alternative materials in a wide range of fields where conventional phenol-formaldehyde polymers, resols and novolak, have been used. In this study, various substituted phenols and aminophenols were polymerized by horseradish peroxidase in a dioxane-aqueous buffer (100 mM sodium acetate, pH5) mixture (80% dioxane by volume) with H2O2 as an oxidant. DSC(differential scanning calorimetry) was used to investigate the structural features of the polymer products. The two types of phenolic products, substituted phenols and aminophenols, resulted in the distinctive DSC curves. Phenylphenol polymer shows complex structural transitions between 150-250℃ while phenylenediamine polymer shows a strong single endothermic peak near 150℃. These DSC curves imply that the two types of phenol polymers possess different structures; more amorphous nature for phenylphenol polymer and an homogeneous oriented nature for phenylenediamine polymer.

Human α-synuclein, a small unfolded protein, has been implicated to play a significant role in Parkinson's disease (PD) and other related diseases. The N-terminal regions of α-synuclein contains seven imperfect repeat sequences (KTKE(Q)GV), which are involved in the aggregation and fibrillation process with their aptitudes to form α- helices. The missense mutants (A30P, E46K, A53T) in the α-synuclein have been discovered in PD patients. In this study, we made three Nterminal deletion mutants (1-35, 1-50, 1-60), and they were expressed in Eschrichia coli BL21, and purified by ion exchange chromatography. Their structural properties were investigated by using circular dichroism, spectrofluorometer, dynamic light scattering, chemical cross-linking, and size exclusion. In addition, ionic liquids (ILs), which are organic salts with high polarity and negligible vapor pressure are used to form amyloid fibrils of α-synuclein.

Candida antarctica lipase B(CALB) is an important enzyme for biodiesel reaction. The use of E.coli for the production of CALB has faced many practical and biological problems such as insoluble aggregates and proteolytic degradation. And CALB shows low organic solvent stability while having reaction with methanol. numerous approaches have been tried to solve these problems including optimizations of expression conditions. But the methods have been mostly specific for their targets and are not highly effective. In this work, the effect of the fusion partner on the protein expression and solubility were investigated as well as the effect of different E.coil expression systems on the expression level. Skp, pelB, GST were used as the N-terminus fusion partners to enhance the solubility of CALB in cytoplasm for the direct fusion expression of CALB. We demonstrated that Skp was a powerful solubility enhancer. Details will be presented and discussed.

β-Lactam antibiotics such as penicillins and cephalosporins have a four-atomring as a common element in their structure. The β- lactamases, which catalyze the inactivation of these ntibiotics, are of great interest because of their high incidence in pathogenic bacteria. A novel oligomeric class C β-lactamase (Est-Y29) from a metagenomic library was expressed, purified and crystallized. Est-Y29 possesses the Ser-X-X-Lys motif which is highly conserved among β-lactamases and site-directed mutagenesis of Ser to Ala resulted in complete loss of activity(1). To our knowledge, Est-Y29 is the first multimeric class C β-lactamase with broad hydrolytic activity. The recombinant protein was expressed in Escherichia coli with an N-terminal 6×His tag and purified to homogeneity. EstY-29 was crystallized and X-ray intensity data were collected to 1.49 A ° resolution using synchrotron radiation. The crystal structure of Est-Y29 will provide an invaluable framework for understanding its unique features such as its oligomeric state, high stability and (S)-specific stereoselectivity, as well as its significance for the development of antibiotic drugs against class C β-lactamases.

A recombinant putative glycoside hydrolase from Bacillus lichemiformis was purified with a specific activity of 18 U mg -1 by His-Trap affinity chromatography. The purified enzyme showed a single band on SDSPAGE with an apparent molecular weight of 34 kDa. The native enzyme was a monomer with a molecular mass of 34 kDa as determined by gel filtration. The optimal pH and temperature for arabinan endo-1,5-α-l-arabinanase (Abn) activity were pH 6.0 and 35°C, respectively. The enzyme activity exhibited the highest for debranched arabinan (1,5-α-l-arabinan) among sugar beet arabinan, arabinoxylan, arabinogarlactan, starch, xylan and p-nitrophenyl-α-l-arabinofuranoside, with a km of 10.7 mg ml–1, kcat of 10751.3 s–1, kcat/km of 998.8 mg ml–1 s–1. No activity was observed for arabinoxylan, arabinogarlactan, starch, xylan or p-nitrophenyl-α-l-arabinofuranoside. These results indicated that the putative enzyme was an arabinan endo-1,5-α-larabinosidase.

Fluorinated alpha-hydroxyacids and alpha-amino acids are considered to be highly versatile chiral building blocks in asymmetric synthesis for the production of several compounds of pharmacological interest. 3-fluoro-L-alanine is recognized as an antibiotic via irreversible inactivation of bacterial alanine racemase. It also acts as an inhibitor of serine palmitoyl transferase and it is a potential precursor for the synthesis of fuoroamino compounds. Asymmetric synthesis of an unnatural amino acid was demonstrated by omega-transaminase from Vibrio fluvialis JS17. 3-fluoro-L-alanine was synthesized from 3- fluoropyruvate and (S)-alphabenzylamine with an enantiomeric excess higher than 99%. The reaction showed severe product inhibition by acetophenone, which was overcome by employing a biphasic reaction system to remove the inhibitory product from the aqueous phase. When a biphasic reaction was carried out with 100 mM 3-fluoropyruvate and 125 mM (S)-alphabenzylamine using isooctane as an extractant, conversion of 3-fluoropyruvate reached 98%.

Gamma aminobutyric acid (GABA) is not only an essential inhibitory neurotransmitter which has a lot of physiological functions in humans but also a major monomer for a biobased and biodegradable polymer, polyamide 4. From an industrial point of view, the production of GABA from the cheap source such as L-glutamate (L-MSG) is a completely reasonable and valuable process. The process can be well catalyzed by the enzyme glutamate decarboxylase (GAD) gained from various microorganism resources. In this work, we have conducted the α-decarboxylation of glutamate to yield GABA using pyridoxal phosphate-dependent GAD extracted from Escherichia coli. In order to obtain the intracellular enzyme, the strain was cultivated for 24 hours at 37 oC in LB medium and then sonicated, and the GAD was partially purified from the cell extract. The GAD showed a catalytic activity toward the conversion of L-MSG into GABA. With this glutamate decarboxylase from E. coli, effects of reaction conditions such as temperature, pH, and substrate concentration were examined.

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the cloning and characterization of panK from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acid with a calculated molecular size of 33.2 kDa, which share high homology with panK from S. coelicolor A3(2). To elucidate the putative function of panK, it was cloned into pET- 32a(+) to construct pPKSP32a expression recombinant plasmids. Expression was carried out using E. coli BL21 (DE3) expression host with His-tag fusion protein and enzyme assay was carried out based on coupling assay. Assay was performed with different concentration of pantothenate to assess the phosphorylating activity of panK with varying substrate concentration at different time

α-glucosidase of Thermotoga maritima belongs to family 4 of the Glycosyl hydrolases. The enzyme α-glucosidase has the unusual property of requiring NAD+, divalent metal cations Mn2+ and reducing conditions (DTT) for activity. The gene aglS encoding 459 amino acid with 51 kDa was expressed under the control of the T7 promoter in Escherichia coli. It was found soluble form and thermo-stable up to 70oC. It showed hydrolyzing activity towards para-nitrophenyl-α-Dglucoside (pNP-α-Glc). The transglycosylation activity was tested using maltose and sucrose as donor and cyclodextrin as acceptor. The molecular mass of cyclodextrin containing two glycosyl moieties is seen in ESI/MS when maltose and sucrose was used as donor. The further analysis is yet to be confirmed.

There is continuing interest in self-sufficient P450 engineering for the biotransformation of aromatic compounds like flavonoid, chalcone and lignin monomers with the object of increasing antioxidant effect. However there is no efficient screening system to analyze large number of mutant library against that substrate. In this study, we developed the high throughput screening system using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to screen the P450 BM3 mutant library towards daidzein. The hydroxylation activity of the P450 BM3 F87A mutants saturated at Y51 has been shown to occur to varying degrees with small alterations in the structure of substrates. Reaction and extraction of mutant library were carried out in 96 deep-well plates. Reaction sample containing more than one substrate were spotted on the 384 MALDI target plate with a matrix and surfactant. To reduce matrix ion interference, small molecule analytes have been investigated with respect to their suitability for use in analysis by surfactant applied MALDI-TOFMS. Hydroxylated metabolites were automatically detected within 2 hours. In this way developed method for the detection of the hydroxyated flavonoid and lignin monomers can be applied to screen the large number of mutant library against the different substrates at a same time, which is less time consuming and more accurate than the conventional screening methods.

Targeting particular mRNAs has become an excellent approach for gene silencing. Here, we showed a cell-penetrating antibody (transbody) that specifically hydrolyzes targeted mRNAs in the cellular cytosol and leads to targeted gene silencing. We generated a synthetic library on the yeast surface based on 3D8 VL, which is a cell-penetrating and nucleic acid-hydrolyzing single domain antibody of the light chain variable domain. With the cell-penetrating activity, 3D8 VL into he cytosol of living cells can selectively decrease the amount of target sequence-carrying mRNAs. We selected 3D8 VL variants had higher affinity and greater selective hydrolyzing activity for target single-stranded (ss)-DNAs than for off targets in the library by using 18bp-ssDNAs as target substrates. In particular, one 3D8 VL variant targeting the Her2 sequence showed more efficient downregulation of Her2 expression than a smallinterfering RNA targeting the same sequence, and the variant caused apoptotic cell death of Her2-overexpressing breast cancer cells. Our results demonstrate that the 3D8 VL variants with cell penetrating, nucleic acid-hydrolyzing activity and sequence-selectivity could degrade target mRNAs in the cytosol, which suggests that they would be potential tools in anti-cancer or anti-viral therapies.

the addition of methyl group to any biologically active molecules brings change in physico-chemical properties of that molecule. The methylations may take place in biosynthesis, metabolism, detoxification, signal transduction, amino acids or nucleotide processing. In majority of the cases, methylation reactions are catalyzed by Sadenosylmethionine- dependent methyltransferases (SAM-MTs). Aminoglycoside antibiotics also contain methyltransferase enzymes which are SAM-dependent and may function during the biosynthesis or post modification of these compounds. Here, we have investigated and compared some of the SAM-dependent methyltrasferases in silico, and overexpressed them in E. coli for enzymatic characterization.

In the present study, the characteristics of the group ІІ chaperonin (PhCpn) from the hyperthermophilic archaeum P. horikoshii OT3 were investigated using NTPs (ATP, CTP, UTP, and GTP) and inorganic phosphatase (PPase) as model substrates. The recombinant PhCpn was overexpressed in E. coli and purified to 91% by heat treatment at 80°C for 10 min and HiTrap Q anion-exchange chromatography. The purified PhCpn showed hyperthermophilic NTPase activity at 85°C, and could effectively protect PPase from thermal inactivation at 85°C and 110°C. Especially, the addition of Mg2+, Co2+, and Mn2+ ions significantly facilitated thermal protection activity of PhCpn on PPase. This result indicated that divalent metal ions such as Mg2+, Co2+, and Mn2+ are crucial to the thermal protection effect of PhCpn on PPase and enhances the protein holding efficiency. The holding activity of PhCpn will provide a reliable and relevant strategy for prolonging the process stability of recombinant biocatalysts.

Ice binding proteins (IBPs) are diverse proteins with the capability of inhibiting ice crystal growth against in subzero environments for their survival. Some Antarctic algae are known to produce IBPs. In this study, two Antartic microalgae, named AnF0046 and AnF0048, were used for identifying IBPs. Extraction of genomic DNA and total RNA was conducted for confirmation of gene existence related to IBPs. After amplification of DNA by PCR with specially designed primers, single bands at 2500 bp and 1047 bp were found on the two agarose gels loaded DNA and synthesized cDNA from mRNA, respectively. When performing Blastn and Blastx for the homology search after sequencing, the sequences were similar to IBP isomer 1 of Chlamydomonas sp. CCMP681 with high identity. The amounts of IBP coding mRNA were expressed differentially under various period and/or temperature of cold shock treatments. It means that the amount of expressed IBPs were dependent upon cold shock period and cold shock temperature. This result suggests that AnF0046 and AnF0048 seems to have the putative IBP coding genes, whose sequeces have high identity with one of the known IBP isomer and expression of the genes were functions of cold shock temperature and period.

A recombinant α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus was purified by heat treatment and Hi-Trap anion exchange chromatography with a specific activity of 28.2 U mg−1. The native enzyme was a 58-kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α-l-arabinofuranosidases were completely conserved in α-l-arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5.5 and 80ºC with a half-life of 49 h at 75ºC. Among aryl-glycoside substrates, the enzyme displayed activity only for p-nitrophenyl-α-l-arabinofuranoside, with a maximum kcat/Km of 220 mM–1 s–1, and p-nitrophenyl-α-l-arabinopyranoside. No activity was observed for arabinan or debranched arabinan. This substrate specificity differs from those of other α-l-arabinofuranosidases. In a 1 mM solution of each sugar, arabino-oligosaccharides with two to five monomer units were completely hydrolyzed to l-arabinose within 13 h in the presence of 30 U ml−1 of enzyme at 75ºC. The novel substrate specificity and hydrolytic properties for arabino-oligosaccharides demonstrate the potential of α-l-arabinofuranosidase from C. saccharolyticus for use in the commercial production of l-arabinose.

The Strep 13 encodes as sesquiterpene alcohol (germacredinol) synthaseencoding from Streptomyces peucetius ATCC27952. Complete open reading frame (ORF) of Strep 13 shows the production of germacredinol/geosmin. Here, we have explored the independent function of N-terminal and C-terminal protein of strep 13. Both terminal recombinant proteins were expressed at high level in Escherichia coli and analysis were carried out. N-terminal protein (~58.15 kDa) showed the Mg2+ dependent activity for the conversion of conversion of Farnesyl diphosphate to Germacrene D. In contrast, the expressed C-terminal domain of Strep13 has no farnesyl diphosphate cyclase activity.

Green fluorescent protein (GFP) Nobel Prize protein from jellyfish is a versatile reporter protein for monitoring gene expression and protein localization in variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral prosperities, including several blue emission variants, all of which contains the single point mutation in tyrosine analogue in green fluorescent protein chromophore. Generally, GFP variants will be generated through replacement of naturally occurring amino acids by site directed mutation or directed evolution methodology and disadvantages of this methodology is limited with 20 naturally occurring side chains. In other hand unnatural amino acid mutagenesis has been used to selectively substitute target amino acids with our interesting side chains in our objective protein. Current studies have explored incorporation of unnatural amino acid analogues into green fluorescent protein and alter the spectral emission and excitation properties. We have selected different tyrosine analogues and successfully incorporated into GFP to alter the specific characters. We have selected the tyrosine analogues due to the important role in the chromophore and structure formation. Current study not only demonstrated the importance of unnatural amino acid mutagenesis and also demonstrated new approach to generate tailormade proteins with interesting and useful spectral properties.

Biodiesel is becoming an area of high concern because of high petroleum prices, increasing environmental concerns and the fact that it comes from renewable resources. It is a natural substitute for petroleum-derived diesel fuel consisting of the alkyl monoesters of fatty acids from vegetable oils of animal fats. Biodiesel is industrially produced by transesterification of riglycerides with short chain alcohols over various catalysts. Canola oil is one of the major vegetable oil resources for biodiesel production due to its high oil content. In this study, the transesterificaion reaction of canola oil with methanol in the presence of a novozym 435 (immobilized lipase) was investigated using supercritical carbon dioxide as a solvent. The reaction was conducted under ultrasound-assisted supercritical conditions in a batch reactor. Various parameters such as temperature, catalyst concentration, ultrasonication power, supercritical carbon dioxide density, oil to methanol ratio, amount of oil, and reaction time were investigated in order to evaluate the feasibility of converting canola oil to biodiesel.

Monooxygenases are very useful enzymes because they can introduce one oxygen of molecular oxygen into the non-activated carbon atom. However, almost all of them require cofactors such as NAD(P)H, Fe-S cluster, FAD or FMN for electron transfer, which is the main bottleneck for the synthesis with isolated enzymes. In this study, direct electrochemical FADH2 regeneration for asymmetric synthesis of (s)-styrene oxide with a styrene monooxygenase from Pseudomonas putida SN1 was investigated. The concept of electrochemical generation of FADH2 was reported elsewhere, but the productivity of the system is too low to be used for practical application. We found from the cyclic voltamograms that when super P® carbon black material was used for electrode fabrication, those electrodes showed higher reduction current for FAD than most of the other electrodes. But in real synthetic application, the productivity of (s)- styrene oxide was not that high compared to the the fast FADH2 generation rate due to the low coupling efficiency between FADH2 generation and monooxygenation. Therefore, the carbon black electrode was modified with various materials and we found that zinc oxide (ZnO) substantially enhances the reaction rate when it was incorporated into the electrode, increasing the abovementioned low coupling efficiency. In addition, electroenzymactic reaction conditions were optimized for additional increase of the productivity. Details will be presented and discussed.

An alginate degrading microorganism was isolated from airborne bacteria. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the species Pseudomonas fluorescence. The alginate lyase gene of the bacteria was cloned by using PCR with the specific primer designed from homologous nucleotide sequences and expressed in Escherichia coli. We attempted to improve the stability and activity of the enzyme by deleting the signal peptide and improving cloning condition. The culture condition for protein expression was on 150C for 24 hour under 0.1 mM IPTG. The enzyme was later purified using a Ni-sepharose affinity column. The purified enzyme was characterized and the degradation product was further analyzed using a Biogel P2 column. The alginate lyase is found to be a specific poly MG-lyase, degrading the glycosidic bond between guluronate and mannuronate fraction. One of the degraded products is found to be a low molecular substance which exhibit no absorbance value at 235 nm but gave a slow reaction with TBA. Acknowledgment: This work is financially supported by Korea industrial Technology Foundation (KOTEF) through the Human Resources Training Project for Strategic Technology

Lactide, diolide of lactic acid, as a monomer for ring-opening polymerization of PLA (poly lactic acid) is key compound for industrial production of PLA. PLA is one of biodegradable plastics from nature material.[1] Because physical property of PLA is affected by ratio of enantiomer, synthesis of enantioseletive lactide is needed to produce PLA with high physical property.[2] However, enantioseletive synthesis from lactate to lactide using lipase has not been reported until now. In our study, commercial CALB called Novozym 435 was found to be able to synthesize lactide from lactate and showed R-specific enantioselectivity. As reaction temperature was changed from 5℃ and 60℃, the rate of consumption and conversion is gradually increased, but rate of conversion was decreased over time in fixed temperature. When lactide was used as a substrate in reaction system, it was found that lactide was reduced about 40%. In this result, CALB can not only convert from lactate to lactide but also break ring conformation of lactide.[3] Actually, when water activity of enzyme was controlled, lactide was reduced drastically at high water activity.[4] This result demonstrates that water activity influences both formation and reduction of lactide and concern with water content of enzyme.