Earticle

Candida antartica lipase B (CALB) has been widely applied in industrial field such as pharmaceutical, food processing. For the production of biodiesel using lipase, reaction condition needs to operate at high temperature because both triglyceride and fatty acid methyl ester have high viscosity in mild temperature.[1] Novel route for immobilized autosilcificated CALB-R1, which is fusion protein between CALB and R1 silaffin peptide, was suggested for improvement of thermostability. Target enzyme was entrapped as over 90% entrapment efficiency in produced silica gel.[2] Additionally, this result shows that thermostability of CALB-R1 was not improved critically and influenced by environmental pH value. pH value was main factor for biosilicification because this reaction was carried out between protein pI value and pH of biosilicification. CALB-R1 was adsorbed in VPOC 1600 (commercial support material) and immobilized CALB-R1 was successfully coated with silica film using autosilicification. Immobilized autosilicificated CALB-R1 showed be much improved thermal stability owing to restricted conformational variation by novel entrapment immobilization method.

The GDSL enzymes are composed of various structural α/β hydrolases fold and they have an activity in lipases, esterases, tioesterases, arylesterases, proteases and lysophospholiases. This family has conserved amino acid sequences which is composed of glycine (G), aspartate (D), serine (S), and leucine (L) in N-terminal region in blocks I, II, III, and V in five sequence. Most especially, the nucleophilic serine is located in active site nearby N-terminal region. This serine is found in the sequence G-X-S-X-G, which accounts for the turn and is considered to be as one of the identifying features of these enzymes. Here, we investigated the structural and functional properties of GDSL enzymes with specific emphasis on the recently-known Sm23. Structural and functional comparisons with other GDSL enzymes and alpha/beta hydrolase were done for its engineering applications.

The demand for biopolymers-based materials has recently gained worldwide attention in the biomedical fields such as tissue engineering, drug delivery systems, dialysis membranes, and biosensors owing to their inherent biocompatibility and biodegradability. However, the insolubility of unmodified native biopolymers in most organic solvents has limited the applications of biopolymer-based materials and composites. Ionic liquids have a great potential to dissolve biopolymers and develop biopolymer-based materials, because of their synthetic flexibility by changing the combinations of cation and anion, and green solvent properties such as non-volatility, non-flammability and recyclability. In this work, biocompatible ionic liquid, [Emim][acetate], was used to encapsulate lipase into various biopolymer-based composite beads. Cellulose, cellulose/chitosan, cellulose/agarose, and cellulose/agar beads containing lipase from Candida rugosa were prepared by co-dissolution of biopolymers and lipase in [Emim][acetate] followed by reconstitution with distilled water. Cellulose/chitosan was the most efficient composite to encapsulate and retain activity of lipase.

The chaperonin α-subunit gene (ApCpnA, 1,871 bp ORF) from the hyperthermophilic archaeon Aeropyrum pernix K1 was amplified by PCR and subcloned into vector pRSFDuet-1. The constructed pRSFDuet-ApCpnA (5.4 kb) was transformed into E. coli BL21 Rosetta (DE3). The E. coli transformant cell successfully expressed ApCpnA as 60.7 kDa protein in both soluble and insoluble fractions of cell lysate. The purified ApCpnA showed ATPase activity and its activity was dependent on temperature. When the alginate lyase gene (aly, 1.19 kb) from Pseudoalteromomas elyakovii was coexpressed with ApCpnA, alginate lyase was produced as a soluble and active form, speculating that ApCpnA facilitates the correct folding of alginate lyase. These results suggest that ApCpnA has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

Agar is a structural polysaccharide found in cell walls of red algae and is generally considered to be a mixture of agarose and agaropectin. Enzymatic hydrolysis of sulfate groups in agaropectin or agar simplifies the production process of high-quality or low sulfate-content agarose. In this work, arylsulfatase gene (astA, 984 bp ORF) from Pseudoalteromonas carrageenovora was cell-surface displayed in S. cerevisiae and applied to desulfatate agar. The arylsulfatase gene was fused in frame with GAL1 promoter and AGA2 gene. To prepare the desulfatated agar, the commercial agar was treated with arylsulfatase displayed S. cerevisiae cell. The sulfate content in agar was decreased to 0.2% as a yeast biocatalyst concentration-dependent manner. Electrophoresis of λDNA HindⅢ marker and 1 kb DNA ladder with the arylsulfatase treated agar and commercial agarose showed the similar pattern of DNA migration and resolution. In addition, the gel strength of arylsulfatase treated agar and commercial agarose were compared.

Proteins composed of twenty amino acids play important biological roles in a cell and their biological activities have been employed in many academic and industrial fields. Recently, great efforts have been devoted to expanding the genetic code for the in vivo biological incorporation of non-canonical amino acids into proteins. This methodology will be useful to manipulate the protein with non natural amino acids that possess unique side chains, which could be used for probing protein function and structure. Here, we report the global incorporation of 3, 4-dihydroxy L-phenylalanine (L-DOPA) into green fluorescent protein (GFPdopa) which altered the spectral and functional properties of the protein. The successful incorporation L-DOPA further facilitates the selective chemical cross-linking of protein-polysaccharide interaction in vitro. GFPdopa protein were oxidized upon sodium periodate (NaIO4) which lead to the formation an orthoquinone intermediate that is subsequently attacked by nucleophilic side chains of amine group interacting polysaccharide, through either Michael addition or Schiff base formation. In general, we have developed an efficient bioconjugants by selective covalent cross linking using genetically encoded unnatural amino acid. Developing such a bioconjugants having lot of potential application in tissue engineering, regenerative medicine and developing a novel drug carrier. Finally, our current approach could be a valuable and efficient addition tool to the protein engineering field as well as for developing of protein-polysaccharide based therapeutics for mankind

A mutated soluble epoxide hydrolase (sEH) from Danio rerio was developed and characterized. We obtained a mutated D. rerio sEH possessing five-point mutations. The catalytically important amino acids of Asp331, Tyr379, Tyr460, Asp496, and His524 were maintained, indicating that the mutated sEH could possess hydrolytic activities. Kinetic characteristics of the mutated sEH were analyzed, and the activity was proved to be enhanced. Enantiopure 99 %ee styrene oxide was prepared by using the mutated D. rerio sEH as the biocatalyst.

A recombinant non-characterized protein previously proposed as putative l-rhamnose isomerase (RhaA) from Thermotoga maritima was purified by His-Trap affinity chromatography with a specific activity of 55 U/mg. The enzyme was identified as a single 46 kDa band on SDS-PAGE and the native enzyme was a tetramer with a molecular mass of 184 kDa by the gel filtration chromatography. The half-lives of the enzyme at 75, 80, 85, 90 and 95°C were 773, 347, 187, 118, and 65 h, respectively, indicating that it is the most thermostable of the known RhaAs. Among all aldopentoses and aldohexoses, RhaA displayed activity only with aldose substrates that possess hydroxyl groups oriented in the right-handed configuration at the C-2 and C-3 positions, such as L-rhamnose, L-lyxose, L-mannose, D-allose, D-gulose, and D-ribose in decreasing activity order. Under the optimum conditions of pH 8.0, 85°C, and 1 mM Mn2+, RhaA with 100 U enzyme/ml converted 500 L-xylulose/l to 225 g/l L-lyxose after 3 h, and converted 500 L-fructose/l to 175 g/l L-mannose after 5 h.

Long isomalto-oligosaccharides (IMOs) were generated via an engineered fusion enzyme of dextransucrase and dextranase (DSXR). To increase the expression level, response surface methodology (RSM) was utilized for optimization of protein expression conditions for enhancement of protein production by the effects of three-level-three-factors and their mutual interaction in Escherichia coli. Seventeen experiments were designed and conducted for investigation of cell density to start induction, induction temperature, and induction time. Optimal induction conditions included a cell density to start induction (A600) of 0.76 at 12.16℃ for 18 h for dextransucrase activity and a cell density to start induction (A600) of 0.75 at 10.5℃ for 20.9 h for dextranase activity. The produced dextransucrase or dextranase activity was obtained at 120.1±7.2 U or 871±58 U, respectively, from 1 L cultures.

Pasteurella m ultocida is a m ultifunctional sialyltransferase (PST) which transfer sialic acid from cytidine 5’-monophosphonuraminic acid (CMP-NeuAc) to an acceptor sugar. Multifunction of PST was controlled by temperature and pH. Multifunction of PST is associated with its conformational change and binding with lactose acceptor according to pH and temperature. By analyzing the protein-substrate binding structure, Arg313, Asn85 and Ser62 are selected for potential sites affecting lactose binding. We generated mutants library for each sites through the site directed saturation mutagenesis. And mutagenic primers were constructed by rational design. To identify combinatorial effect of 3 sites, multi-site mutagenesis method was used using the anchor sequence and gateway method. To select mutants having an increased activity, pH-color assay method is used. Screening is based on the color changes of the pH indicator cresol red when proton is released during the transfer of NeuAc from CMP-NeuAc to acceptor substrate. By using the method, mutants were selected and combinatorial mutagenesis was done using the selected amino acid sites.

ω-Transaminase can be applied to asymmetric synthesis of chiral amines and unnatural amino acids from ketone precusors. Even though the ω-transaminase shows excellent enzyme properties for chiral synthesis via kinetic resolution, the asymmetric synthesis suffers from various limitations such as low substrate reactivity, severe product inhibition and enzyme instability. Here, we explored how the substrate selectivity is controlled to determine optimal reaction conditions for the asymmetric synthesis. We examined how Michaelis constants and specificity constants were affected by pH and analyzed the effect of structural differences of substrates on the enzyme activity. Aided by the kinetic information, we found that binding between substrate and enzyme is a rate-determining step and the ionic and hydrophobic binding interactions are differently affected by the medium pH. This study suggests that the optimal reaction conditions could have different pH values depending on the substrate type. This work was supported by BK21 program from the Korean Ministry of Education and Seoul R&BD Program (NT080612, KU080657).

An enzyme of the glycosidase hydrolase family 10 endo-1,4-β-xylanase C from Phaenerocheate chrysosporium was cloned into the pPICZ and pPICZα vectors and expressed in Pichia pastoris under the control of the methanol inducible alcohol oxidase I (AOXI) promoter. Enzyme assay indicated that both intrinsic signal peptide and P. pastoris α-factor secretion signal peptide were efficient in mediating Xylanase C secretion. Xylanase C activity reached 2.5 unit/ml in medium culture after induction for three days with 1% methanol. The enzyme activity in this study is four to five times higher than previously reported Xylanase expression in Aspergillus niger. The optimal temperature and pH the enzyme are 70OC and 4.5, respectively.

Long isomalto-oligosaccharides (IMOs) were generated via an engineered fusion enzyme of dextransucrase and dextranase (DSXR). To increase the expression level, response surface methodology (RSM) was utilized for optimization of protein expression conditions for enhancement of protein production by the effects of three-level-three-factors and their mutual interaction in Escherichia coli. Seventeen experiments were designed and conducted for investigation of cell density to start induction, induction temperature,and induction time. Optimal induction conditions included a cell density to start induction (A600) of 0.76 at 12.16℃ for 18 h for dextransucrase activity and a cell density to start induction (A600) of 0.75 at 10.5℃ for 20.9 h for dextranase activity. The predicted maximal enzyme activity by the obtained optimization model equations was estimated to 115.8 U/L for dextransucrase and to 863 U/L for dextranase activity. The maximum DSXR activity obtained experimentally under the optimized conditions was found to be 120.1±7.2 U/L for dextransucrase and 871±58 U/L, which was in close agreement with the model prediction.

Highly stable and active nanoscale enzyme reactor (NER) [1] has been developed by adsorbing and crosslinking glucose oxidase (GOx) in polyaniline nanofibers (PANFs). PANFs, prepared by oxidative polymerization with ammonium persulfate as an oxidant [2], contains nanometer-scale pores that enable the NER approach. The simple addition of enzyme crosslinking step resulted in highly stable and active enzyme systems. For example, the activities of adsorbed GOx (ADS) and NER were 0.040 and 0.382 (A500/min) per 0.1 mg PANFs, respectively. This 9.6-fold increase of enzyme activity can be explained by the improved enzyme loading of NER because the enzyme crosslinking can effectively prevent the enzyme leaching from PANFs. Stability-wise, ADS retained only 50% of initial activity after 5 day incubation at room temperature while NER maintained more than 90% of initial activity in the same condition for two months. To demonstrate the feasible application of highly stable and active NERs in conductive PANFs, the operation of biofuel cells (BFCs) were tested with enzyme anodes based on ADS and NER. The detailed result will be given in the presentation.

Cellulose-based materials have many potential applications in biomedical fields, because of their biocompatibility and biodegradability. Carbon nanotubes (CNTs) have been reported as ideal electrode materials due to their high electrical conductivity and large surface area. Recently, it was also reported that CNTs facilitate the direct electron transfer (DET) from redox enzymes to electrodes.1 DET between glucose oxidase (GOx) and electrodes is essential for GOx-based biosensors, biofuel cells, and bioelectronic devices, although GOx generally requires mediators for electrical communications.2 In this work, cellulose-CNT composites were prepared by vacuum filtration of CNTs and coagulation of cellulose dissolved in ionic liquid. Furthermore, GOx was immobilized on cellulose-CNT composite. Cyclic voltammograms of cellulose-CNT-GOx electrodes showed a pair of well-defined peaks. The formal redox potential peak was -460 mV, which agreed well with that of FAD/FADH2. This result clearly shows that the DET between the GOx and the cellulose-CNT electrode was achieved. It was also found that the GOx immobilized on the electrode retained catalytic activity for the oxidation of glucose.

Several pathogenic organisms, such as Vibrio Colera, E. coli, Helicobacter pylori, and influenza virus A and B, recognize a sialic-acid-containing carbohydrate receptor structure on target cells. Therefore, sialyl compounds may function as soluble receptor analog to attachment of the pathogens on the cell surface receptors. In this study, Biosynthesis of sialyllactose was investigated using calcium alginate blends with gelatin beads. The trypsin entrapment using Ca2+-alginate and gelatin mixture was experimentally optimized under various conditions. The substrate, N-α-benzoyl-DL-arginine-4-nitroanilide (BAPNA), was used for the confirmation of the trypsin activity. The biosynthesis for sialyllactose using several glycosyltransferases of the pathway has been built up. Five enzymes involving the biosynthesis were over-expressed in E.coli BL21 (K-12) and entrapped by Ca2+-alginate and gelatin mixture. The entrapment of five enzymes may guarantee the high stabilities of the enzymes in biosynthesis

Enzyme flexibility is thought to be related to enzyme activity conceptually. But there has been no attempt to enhance enzyme activity by modulation of enzyme flexibility. In this study, enzyme mutations of Candida antarctica lipase B were performed with the aim of activity enhancement. In the analysis using spring model developed in our laboratory and the molecular dynamics simulation in the organic solvents, the forced or twisted residues and residues showing solvent-dependent flexibility change were mostly located at the active site surroundings. Accordingly, the target sites were selected the edges of helices surrounding active site. The flexible amino acids, D and E, were preferentially chosen as the alternatives. In the E.coli expression system, three mutations among seven target sites showed activity enhancement. And two mutants among three activity enhanced mutants were obtained in the Pichia pastoris expression system and they all showed enhanced activity.

Galactooligosacchride (GOS) as a prebiotic is known to be beneficial to the growth of human intestinal microbial flora, and thereby led eventually to control the human diseases. Typically GOS is synthesized by β-galactosidase (β-gal)-catalyzed enzymatic reaction on lactose. During this reaction (transgalactosylation), galactose moiety is known to be transferred to hydroxyl group of lactose, and thereby GOS is synthesized. There have been lots of reports related to GOS production by β-galactosidase and immobilized β -galactosidase. Recently, in order to increase the productivity of GOS, various reactor systems and operational strategies have been studied. In our previous studies, it was demonstrated that active Escherichia coli (E.coli) β-gal inclusion bodies (IBs) were produced by the addition of a repressor (α-methyl D-glucospyranoside) or an inducer analog (D-fucose) after induction of the araBAD promoter system in E.coli. Inaddition, we have operated successfully a packed-bed reactor for hydrolysis of o-nitrophenyl-ß-D-galactoside using immobilized β-gal IBs-containing E.coli cells. Previously, it has been reported that enzyme IBs in E.coli expression system were expressed as biologically active IBs. However, there have been little numbers of report that have explored an enzyme reactor using active IBs. The aim of this study is to investigate whether β-gal IBs-containing E.coli cell can synthesize GOS by transgalactosylation reaction, and, in addition, to explore the optimal condition of GOS synthesis by this β-gal IBs-containing E.coli cell. Interestingly, this study is the first trial for GOS synthesis by β-gal IBs. Because, if we can use active β-IBs as biocalayst, β-IB utilization will be more convenient and economic in enzymatic GOS synthesis, active β-gal IBs might be applied to the enzymatic transgalactosylation reaction without any purification steps. That is, a whole E.coli cells containing active β-gal IBs will be able to be directly used for GOS synthesis.

Quorum sensing (QS) is a cell density-dependent signaling system used by bacteria to coordinate gene expression within their population. In the QS mechanism of many gram-negative bacteria, acyl homoserine lactones (AHLs) are known to be the triggering molecules which form a complex with a transcriptional activator protein and promote the binding of the complex to DNA regulatory site activating transcription of many virulence genes. In this study, we describe the development and characterization of in vivo cell-based bioassay systems for detecting QS inhibitors based on three members of the LuxR family proteins, TraR, LasR and the recently identified QscR. Three different gram-negative bacteria, Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa, were used as reporter strains to over-produce one of the QS activator proteins and respond to AHLs and/or their inhibitors. The nine different in vivo assay systems (3 reporter strains × 3 QS proteins) were evaluated for their applicability and reliability by studying quantitative responses to various AHLs and furanones, the latter of which were observed as potent inhibitors against AHLs. The results indicate that, although they do not detect direct binding between QS proteins and AHLs and/or inhibiting molecules, the cell-based in vivo bioassay systems are a sensitive and reliable tool for screening of QS activators and inhibitors. This study also suggests that furanones are potentially important QS inhibitors for many LuxR-type activator proteins.

Conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) from glycerol by a coenzyme B12-dependent glycerol dehydratase (DhaB) often limits the economic viability of the 3-hydroxypropionic acid (3-HP) production process. Therefore, it is important to develop coenzyme B12 independent process for the production of the same. In this study, the genes dhaB, encoding coenzyme B12 independent DhaB, from Clostridium butyricum and Pelobacter carbinolicus were cloned and expressed E. coli BL21 for the production of 3-hydroxypropionic acid production

Alginate is widely used in food additives, medicals, cosmetics, and other applications, and oligosaccharides derived form alginate have been investigated for new functional materials. Alginate lyases catalyze the degradation of alginate through β-elimination of the glycosidic bond. Many alginate lyases with various substrate specificities have been isolated from algae, marine invertebrates, and a wide range of microorganisms. In this study, we tried to analyze alginate-hydrolyzing patterns by alginate lyases from different marine microorganisms such as Pseudoalteromomas elyakovii, Streptomyces sp. ALG-5, Flavobacterium sp., and Bacillus sp. JS-1. Alginate-hydrolyzed patterns by alginate lyases were strain-specific. The major end-product of alginate hydrolysis by the enzyme from Bacillus sp. JS-1 was monosaccharide. The degraded products of alginate by the enzymes Flavobacterium sp., P. elyakovii, and Streptomyces sp. ALG-5 were disaccharides.

Two enzyme mixtures of α-l-arabinofuranosidase and endo-1,5-α-l-arabinanase from C. saccharolyticus produced l-arabinose from debranched arabinan or sugar beet arabinan. The optimum ratio for debranched arabinan was α-l-arabinofuranosidase(42 U ml–1) and endo-1,5-α-l-arabinanase (14 U ml–1) of 3:1 at pH 6.5 and 75 ºC for 1 h. Under the optimum ratio, 16 g l–1 l-arabinose was obtained from 20 g l–1 debranched arabinan after 1h, with a conversion yield of 80% and a volumetric productivity of 6.2 g l–1 h–1. The optimum ratio for sugar beet arabinan was α-l-arabinofuranosidase (20.4 U ml–1) and endo-1,5-α-l-arabinanase (3.1 U ml–1) of 8:1 at pH 6.0 and 75 ºC for 1.5 h. Under the optimum ratio, 16.4 g l–1 l-arabinose was obtained from 20 g l–1 sugar beet arabinan after 2 h, with a conversion yield of 82% and a volumetric productivity of 8.1 g l–1 h–1. The hydrolytic properties for arabino-oligosaccharides of α-l-arabinofuranosidase and endo-1,5-α-l-arabinanase from C. saccharolyticus demonstrated the potential in the commercial production of l-arabinose.

Lignin is an abundant renewable polymer that is a byproduct of biofuel synthesis. To use lignin as feedstock for chemicals and polymers, it can be fragmented to p-coumaric acid and other aromatic fragments Phenolic acid decarboxylase (PDC) catalyzes the non-oxidative decarboxylation of p-coumaric acid (pCA) to p-hydroxystyrene (pHS), which is a polymer monomer PDC from Lactobacillus plantarum is a well known enzyme and we used it as a template to search sequence databases for other, potentially more active, PDCs. The retrieved sequences were arranged in a phyologenetic tree and representativesequences from each of the five groups were cloned and over-expressed in PDC from Bacilllus amylolichefaciens (BaPDC) had 1.5-fold higher specific activity for p-coumaric acid than PDC from Lactobacillus plantarum and a 10-fold higher kcat/Km The initial rate of BaPDC-catalyzed formation of p-hydroxystyrene was 1.44 mmol/min/g DCW, but slowed due to product inhibition (Ki = 0.5 mM). Using a compatible organic solvent we can extract high concentration of the pHS seperated from water in biphasic reaction.

Ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum has been reported that converted d-psicose into d-allose as pharmaceutical compound without byproduct. Moreover, ribose-5-phosphate isomerase from Clostridium thermocellum has effective thermal stability among d-allose conversion enzymes. A docking study of ribose-5-phosphate isomerase from Clostridium thermocellum with d-psicose in the real 3D structure was performed. The 11 active site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 132 was correlated with an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity among mutants modified with Ala, Gln, Ile, Lys, Glu or Asp. The maximal activity of wild type and R132E mutant enzymes towards d-psicose was observed at pH 7.5 and 80°C. The thermal inactivation with half-lives of wild type enzyme was 15, 10, 6.6, 3.5, 1.2, and 0.5 h at 55, 60, 65, 70, 75, and 80°C, respectively. Whereas, that of R132E mutant enzymes was 21, 13, 8.3, 5.1, 2.6, and 0.8 h at 55, 60, 65, 70, 75, and 80°C, respectively. The specific activity and catalytic efficiency (kcat/Km) for d-psicose using the R132E mutant were 1.3- and 1.5-fold higher than those of the wild-type enzyme, respectively. The production rate of d-allose from d-psicose using the R132E mutant enzyme was higher than that using the wild-type enzyme. After 80 min, the conversion yield of d-allose from d-psicose using the R132E mutant enzyme (32%) was 7% higher than that using the wild-type enzyme (25%).

The SG(A)NH superfamily is characterized by four highly conserved sequence blocks containing serine (S), alanine (A), asparagine (N), and histidine (H) residues, each of which is incorporated in active sites or oxyanion holes. High specificities of SG(A)NH hydrolases could be used for the preparations of chiral compounds and enantiomeric resolutions. Here, a novel SG(A)NH hydrolase (XAC833) from Xanthomonas axonopodis pv, which has a SANH fold (Ser34, Ala68, Asn51, His135) was expressed, purified, and characterized using biochemical and biophysical methods. A sequence comparison of XAC833 with other SG(A)NH members confirmed the presence of a catalytic triad and functionally important amino acids. The wild type enzyme was able to hydrolyzed p-nitrophenyl acetate, p-nitrophenyl butyrate and p-nitrophenyl valerate. Structural and functional properties of XAC833 were investigated using circular dichroism (CD), fluorescence, dynamic light scattering (DLS), electron microscopy (EM), and time of flight (TOF) mass spectrometry with site-directed mutagenesis. Site-directed mutagenesis and crystallographic analysis is currently under progress for its industrial applications.

The SGNH superfamily is characterized by four highly conserved sequence blocks containing serine (S), glycine (G) asparagines (N), and histidine (H) residues, each of which is incorporated in active sites or oxyanion holes. High specificities of SGNH hydrolases could compounds and enantiomeric resolutions. Here, a novel SGNH hydrolase (BG42) from Burkhoderia glumae, which has a SGNH fold (Ser184, Thr266(Asn), His369) in its C-terminal domain, was expressed, purified, and characterized using biochemical and biophysical methods. A sequence comparison of BG42 with other SGNH members confirmed the presence of catalytic triad. The wild type enzyme was able to hydrolyzed p-nitrophenyl acetate, α-and β-naphthyl acetate. Structural and functional properties of BG42 were investigated using circular dichroism (CD), fluorescence, dynamic light scattering (DLS), electron microscopy (EM), and time of flight (TOF) mass spectrometry with site-directed mutagenesis. Sitedirected mutagenesis and crystallographic analysis is currently under progress for its industrial applications.

Cadaverine can be used a monomer for a biobased polyamide with dicarboxylate such as adipic acid. Latest research environment has driven the sustainability as a major goal of chemical production in next generation. In such a viewpoint, production of polymers using cadaverine, which can be produced from L-lysine, is highly preferred. In this work, we researched the screening of highly active lysine decarboxylase. L-lysine was used as limiting C-source and N-source in minimal media culture. As a result, we have screened Achromobacter sp. which is from soil source and shows high activity in the a-decarboxylation reaction of L-lysine. In whole cell reaction, A. sp. showed 4.5x10-3μmol/min×mg(dry cell weight) while Escherichia coli K-12 showed 2.6x10-3 μmol/min×mg(dcw). Optimum pH and temperature of lysine decarboxylase reaction using A. sp was pH 5.8 and 30℃, respectively.

Glucose inhibition by β-galactosidase from Caldicellulosiruptor saccharolyticus is the lowest among the currently known β-galactosidases. However, the enzyme activity for lactose hydrolysis is inhibited at low galactose concentrations. In order to reduce galactose inhibition, the predicted galactose-binding residues, which were determined by sequence alignment, were replaced separately with Ala. The activities of the Ala-substituted mutant enzymes were assessed with the addition of galactose. As a consequence, amino acid at position 349 was correlated with the reduction in galactose inhibition. The F349S mutant exhibited the highest activity in the presence of galactose relative to the activity measured in the absence of galactose among the tested mutant enzymes at position 349. The Ki of the F349S mutant, which was 13-fold that of the wild-type enzyme, was the highest among the reported values of β-galactosidases. The wild-type enzyme hydrolyzed 62% of 100 g lactose L−1 with the addition of 30 g galactose L−1, whereas the F349S mutant hydrolyzed more than 99%. This is, to the best of our knowledge, the first report regarding the reduction of product inhibition via the mutation of β-galactosidase.

It has been demonstrated that the approach of enzyme coatings on various nanomaterials is successful in stabilizing the enzyme activity in an unprecedented way [1,2,3]. For example, the trypsin coatings on electrospun polystyrene-poly(styrene-co-maleic anhydride)(PS-PSMA) nanofibers showed no decrease of enzyme activity under recycled uses and rigorous shaking for one year [3]. In the present work, trypsin was covalently attached or coated onto PS-PSMA nanofibers with varied concentrations of maleic anhydride group, which enables an easy conjugation of enzymes onto nanofibers. The concentration of maleic anhydride group correlated well with the enzyme loading and activity of covalently-attached trypsin. Trypsin-coated nanofibers, prepared via covalent attachment and follow-up enzyme crosslinking, resulted in highly stable nanobiocatalytic systems, irrespective of added PSMA amounts. The variation of PSMA amounts enables the facile control of various properties of trypsin-coated nanofibers, such as enzyme loading, activity, structure, and mass transfer limitation. The property control of highly stable enzyme coatings would provide a powerful tool for more successful applications of enzyme coatings in various fields.

Due to the depletion of fossil fuels, climate changes, growing world population, future energy supply from the renewable biomass is one of the important challenges in biotechnology. Biobutanol, which has been produced from the solventogenic microorganism such as Clostridium acetobutylicum, is considered as valuable biofuel because it has more heat energy and is less corrosive and evaporative than bioethanol. The goal of this research is the development of the biocatalytic process for conversion of levulinic acid to 2-butanone in order to utilize levulinic acid is be produced via the acidic saccharification of the red algae. The product 2-butanone can be easily converted to 2-butanol via hydrogenation using chemical catalysis. Herein, acetoacetate decarboxylase was selected as biocatalyst for the conversion of levulinic acid to 2-butanone. The kinetic parameters and optimum condition of the biocatalyst was determined using levulinic acid as substrate. For higher turnover number, structure-based molecular docking and binding energy calculation were studied. The product analysis will be discussed.