Earticle

Phospholipase D (PLD, EC 3.1.4.4) catalyzes hydrolysis of phospholipids resulting phosphatidic acid (PA) and alcohols. PLD catalyzes alcohol head group exchanging reaction known as transphosphatidylation reaction in presence of appropriate alcohol. Because of transesterification propensity, PLDs are proved to be bioindustrially important and a significant attention is being given in the search for such enzymes from wide variesties of sources, especially from microbes. More importantly, it produces scarce and commercially important phospholipids such as phosphatidylserine. Although there are a huge number of literatures found dealing with purification of extracellular PLD, only a few are from intracellular PLD. a membrane-bound phospholipase D was partially purified from the strain using sequential chromatographic steps after ammonium sulfate precipitation. The enzyme activity was found maximum at pH 8.0 and 60 °C. It was highly stable at or below 60 °C and between pH 8.0 and 10.0, indicating that it is an alkaline thermostable enzyme. Almost all detergents, except, Triton X-100 and CHAPS, suppressed the enzyme activity by various extents. Metal ions and detergents showed varied effect in the enzyme activity. Triton X-100, slightly enhanced the activity at 0.25%, however, beyond this concentration it suppressed the in concentration dependent manner.

Synthetic wood composite films containing various contents of cellulose, xylan, and lignin, the three major components of lignocellulosic biomass, were prepared in a room temperature ionic liquid solvent, 1-ethyl-3- methylimidazolium acetate, [Emim][Ac]. Various synthetic wood composites were obtained by dissolution of individual wood components together with additives, including polyethylene glycol (PEG), chitosan, and multi-wall carbon nanotubes in [Emim][Ac]. The addition of water affords a gel that was dried in either a low humidity environment or under vacuum. Synthetic wood films had improved physicochemical properties compared with cellulose, xylan, and lignin films. Synthetic wood films showed smoother surface textures, higher water resistance, and higher tensile strengths than cellulose films formed by the same methods. The advantages of synthetic wood composites include low production cost by using renewable biomaterials, reduced biodegradability compared with cellulose composites because of the presence of lignin, greater water resistance, and potential for modification and functionalization.

One Streptomyces strain, which was recently isolated from Korean soil, produced extracellular lipase in the OSYM culture medium. The enzyme was purified using a single step gel permeation chromatography on Sepharose CL-6B. The enzyme was nearly homogenous in SDS-PAGE analysis with a single band. The activity was optimum at 40 °C and pH 7.0 and was stable between pH 5.0 and 8.0 and below 50 °C. Most of the tested metal ions as well as detergents showed inhibitory effect on the enzyme activity, but with various extents.. The enzyme showed highest hydrolytic activity with p-nitrophenyl palmitate (pNPP), a long chain substrate and Km and Vmax of the enzyme for the substrate were determined to be 0.24 mM and 4.6 mM/min mg, respectively. Furthermore, the enzyme hydrolyzed triolein producing 1,2- and 1,3-diolein along with oleic acid. More importantly, it catalyzed biodiesel production in presence of methanol and various oils; therefore, it can be applicable in bio-energy industry.

In this study, two organic solvent-tolerant lipases were purified from two Streptomyces strains using ammonium sulfate precipitation followed by various chromatographic techniques, and compared characteristics of two enzymes. Molecular mass of the purified lipase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the lipase from strain CS268 were found to be 8.0 and 30 °C, respectively. The lipase was stable in the pH range 4.0 - 9.0 and at temperatures below 50 °C. It showed the highest hydrolytic activity towards p-nitrophenyl decanoate (C10) and Km and Vmax for the substrate were 0.594 mM and 319.48 mM min-1mg-1, respectively. It hydrolyzed triolene at all positions, suggesting that it is a non-specific lipase. The lipase showed higher stability in the presence of various organic solvents, such as octane, hexane, toluene and benzene with various extents. Similarly, the optimum pH and temperature for lipase activity from the strain 133 were pH 8.0 and 40 °C, respectively. While assessing the stability, the enzyme remained unaltered when treated at 40°C for 120 min and was stable in alkaline range. The enzyme activity was enhanced 5-fold by Triton X-100 compared to the control without any detergents. MgSO4 and KCl slightly enhanced the enzyme activity. The enzyme showed a very high stability in the presence of various organic solvents when evaluated after 48 h of incubation. Lipase enzyme from CS 133 acts as a potential biocatalyst to produce biodiesel using soybean oil and olive oil

A Thermococcus onnurineus NA1 has been studied for growth and hydrogen (H2) production from fomate and carbon monoxide (CO) as a main carbon source. The effect of amino acid and flue gas on growth and H2 production was examined in MM1 medium. And stability of H2 production experiment was employed. In addition, H2 production under resting cell conditions in different pH and temperature was investigated. The maximum specific growth rates in formate and CO were found to be 0.33 h-1 and 0.30 h-1, respectively. The addition of three amino acids (leucine, isoleucine and valine) to formate MM1 medium, showed an increase in maximum specific growth rate to 0.50 h-1 and final cell density was increase double. Among flue gas, only CO2 shows a little inhibition on initial growth and H2 production. The stability of H2 production activity with formate (100mM) and CO (100%, head space) has been study in 4 days. There was no decrease H2 production. Under resting cell conditions, optimum pH was around 6 to 7 and temperature was 80℃. NA1 produced H2 by water-gas shift reaction from CO with a theoretical yield of 1(mol/mol). T. onnurineus NA1 has good potential to be used for H2 production given the fact that H2 productivity of 20 mmol/mg/h. NA1 cells have the capability for repeated H2 production giving it an edge for commercial water-gas shift reactions.

A lipase (RL74) able to catalyze biodiesel production from Ralstonia sp. CS274, recently isolated from Kora soil, was purified to electrophoretic homogeneity. Molecular weight of RL74 was estimated to be 28,000 Da by SDS-PAGE. The activity was highest at 50-55 °C and pH 8.0-9.5 and was stable at pH 7.0-12.0 and up to 45 °C. It was resistant to oxidizing and reducing agents and the activity was enhanced by detergents. RL74 was 1,3 specific and Km and Vmax for p-nitrophenyl palmitate were 2.73±0.6 mM and 101.4 ± 1.9 mM/min mg, respectively. N-terminal amino acid sequence showed partial homology with that of Penicillium lipases. RL74 produced biodiesel more efficiently in palm oil than in soybean oil; and the production was highest at pH 8.0, at 5% methanol and at 20% water content.

With the aim of isolating a microbial biocatalyst with potential application in the field of bio-fuel, a lipase from actinomycetes strain recently isolated from Korean soil, was purified using sequential chromatographic steps. Molecular weight of the enzyme was estimated to be 32,400 Da by SDS-PAGE. The enzyme was highly active at low temperature with maximum at 30°C and pH 8.0. The activity at 3°C was about 43% of the maximum activity, thus the enzyme showed the characteristics of low temperature-active enzymes. All the tested detergents, except Triton X-100, inhibited the enzyme activity by various extents. The enzyme preferentially hydrolyzed p-nitrophenyl deconate, a medium chain substrate. More importantly, it catalyzed biodiesel production using palm oil and methanol. The enzyme thus could be potentially applicable in bio-fuel industry.

With the aim of isolating a microbial biocatalyst with potential application in the field of bio-fuel, a lipase from actinomycetes strain recently isolated from Korean soil, was purified using sequential chromatographic steps. Molecular weight of the enzyme was estimated to be 32,400 Da by SDS-PAGE. The enzyme was highly active at low temperature with maximum at 30°C and pH 8.0. The activity at 3°C was about 43% of the maximum activity, thus the enzyme showed the characteristics of low temperature-active enzymes. All the tested detergents, except Triton X-100, inhibited the enzyme activity by various extents. The enzyme preferentially hydrolyzed p-nitrophenyl deconate, a medium chain substrate. More importantly, it catalyzed biodiesel production using palm oil and methanol. The enzyme thus could be potentially applicable in bio-fuel industry.

Flavonoids are classified as Chalcone, Flavone, Flavanon and etc. Apigenin is a kind of Flavone. It is contained in apples, bean, broccoli, cerely, cherry, grape, onion and etc. It has effect on inflammation, oxidation stress decrease and carbohydrate metabolism increase. According to previous study, flavonids hydroxylation or methylation increased their efficiency. For this reason, we tried biotransformation of apigenin by Streptomyces ceolicolor, Streptomyces avermitilis and Streptomyces grisues. Microorganism incubated in R2YE culture and then added Apigenin 5 mM. And the product was analyzed by HPLC and GC Mass.

Recently, Candida antarctica lipase B (Cal B) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, Cal B, which is stable under harsh condition, is in demand from many industries. Cal B was expressed in the methylotrophic yeast Pichia pastoris X-33 and was produced in a large scale fermentor for the commercial application. Using 5L fermenters, cultivation conditions were investigated for the high expression of Cal B in a 500L fermentor. During the cultivation, glycerol and methanol were added to obtain high cell density and for the induction of cal B, respectively. When DO rebounded from the minimum value, the glycerol feeding was started and 8L glycerol was pumped into the fermentor. Thereafter, the methanol feeding was started for the induction of Cal B. After 70 hours cultivation, OD600 for cell density was reached 277, the activity of lipase was 1.5 × 105 U/L, and 2.8 g/L protein was obtained. After the freeze dry, the residual activity of lipase was 5 × 106 U/g.

Lipoxygenases (LOXs) constitute a family of lipid-peroxidizing enzymes that catalyze the oxidation of unsaturated fatty acid containing a (1Z,4Z)-pentadiene structural unit, leading to formation of conjugated (Z,E)-hydroperoxydienoic acid. Recently, several microbial LOXs were reported to be involved in the production of hydroperoxy fatty acids. Among the microorganisms that produce hydroxy fatty acids, Pseudomonas aeruginosa PR3 is known to convert linoleic acid to trihydroxy fatty acid, which suggests the involvement of a LOX enzyme. Based on these reports, we identified a novel thermostable LOX from P. aeruginosa PR3 strain. The protein was purified 34.3-fold with a recovery rate of 5.14%. The Km and Vmax values of the purified enzyme were 3.57 mM and 0.73 μmol/min/mg, respectively. Heat stability of the purified enzyme was unexpectedly high with an LD50 of 90 min at 80oC, although P. aeruginosa PR3 is known as a mesophilic bacterium. Substrate specificity of the purified enzyme was restricted only to unsaturated fatty acids carrying a (1Z,4Z)-pentadiene unit.

Engineering of cellulosic biomass-degrading enzymes are important for the purpose of cost-efficient bioenergy production. Cellulose- and hemicellulose-degrading enzymes are structurally consisted of two domains, so called a catalytic domain and a cellulose-binding domain (CBD). It has been thought that the catalytic efficiency of cellulases could be improved by protein engineering of CBDs as well as catalytic domains. Both a large size of CBD mutational library and high-throughput screening system were required for CBD engineering. First, we constructed fusion proteins composed of both alkaline phosphatase and CBD, to monitor the cellulose-binding property of CBDs. Next, microwell plates containing paper discs with different kinds of cellulosic materials were designed. Binding activities of fusion proteins could be measured quantitatively using 4-methylumbelliferyl phosphate (4-MUP) as fluorescent substrates. In this poster, we report the high-throughout fluorescence-based method for the engineering of CBDs.

Much interest has been recently focused on the reducing of carbon dioxide (CO2). CO2 is a kinetically and thermodynamically stable molecule. It is easily formed by the oxidation of organic molecules, during combustion or respiration, but is difficult to reduce. The production of reduced carbon compounds from CO2 is an attractive proposition, because carbon-neutral energy sources could be used to generate fuel resources and sequester CO2 from the atmosphere. However, available methods for the electrochemical reduction of CO2 require excessive overpotentials (are energetically wasteful) and produce mixtures of products. Formate dehydrogenase H (FDH-H) from Escherichia coli containing selenocysteine that oxidizes formate to carbon dioxide with the release of hydrogen is a component of the anaerobic formate hydrogen lyase complex of E. coli. In this approach, we investigated the effect of pH on FDH-H stability and observed the effect of selenite and formate concentration on the activity of FDH-H. Additionally, coexpression of selenocysteine insertion genes were tried to improve the expression of FDH-H. The highest level of FDH-H expression was achieved by coexpression of selenocysteine insertion genes (pSUABC) as well as by the addition of 10 μM selenite and 10 mM formate. At this optimized condition, a 2.6 fold elevation of expression of FDH-H was achieved.

Stability and activity of immobilized forms of Candida antarctica lipase B and Thermomyces lanuginosus lipase were studied for enzymatic biodiesel production in a non-solvent system at atmospheric pressure and in supercritical carbon dioxide (ScCO2). The lipases immobilized by several methods(entrapment in alginate and silica sol-gel, cross-linking, adsorption, and covalent binding) as well as commercial immobilized enzymes(Novozym 435 and Lipzyme TL IM from Novozymes) were used for a comparative study. The model reaction used was the transesterfication of canola oil with methanol. From the experimental result, it was found that among tested enzymes Lipozyme TL IM and the Thermomyces lanuginosus lipase immobilized in silica sol-gel were most stable under the reaction conditions and the conversion could be enhanced by using ScCO2. These results should help in determining suitable biocatalysts for the enzymatic biodiesel production.

Cellulosic materials are abundant natural resources, however, it is very hard to hydrolyze them due to their structural recalcitrance. Therefore, cellulose engineering has been extensively studied in recent years. We tried to characterize and improve cellulases from Cellulomonas fimi in terms of catalytic activity, substrate specificity, and protein solubility. First, we tested the solubility and functional expression of recombinant cellulases in E. coli. Out of seven catalytic domains of cellulases, four proteins were expressed as inclusion bodies in E. coli. Only Cex (encoding an exo-cellulase) among the four proteins showed a detectable catalytic activity, which was measured using a colorimetric method with p-nitrophenyl-β-D-celloglycoside. Solubility and activity of Cex will be improved by random mutagenesis. Furthermore, based on three-demensional structural analysis, plasticity residues in Cex were rationally selected and engineered using site-specific saturation mutagenesis for the improvement of catalytic activity and modification of substrate specificity. In this poster, we report a rational strategy for enhancement of catalytic efficiency and substrate specificity of cellulases.

Klebsiella pneumoniae converts glycerol to the specialty chemical 1,3- propanediol (1,3-PDO), which is used for the production of polytrimethylene terepthalate. Here, an NAD+-dependent gamma-glutamyl-gammaaminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae, which oxidizes 3-hydroxypropionaldehyde to a platform chemical 3-hydroxypropionic acid (3-HP), was overexpressed in K. pneumoniae DSM 2026 for the co-production of 3-HP and 1,3-PDO from glycerol. In addition, the gene dhaT, encoding NADH-dependent 1,3-propanediol oxidoreductase, was deleted from the chromosome for the balanced production of 3-HP and 1,3-PDO. The recombinant K. pneumoniae ΔdhaT, expressing PuuC, produced 3.6 g/L 3-HP and 3.0 g/L 1,3-PDO with an average yield of 81% on glycerol carbon in shake flask culture under microaerobic conditions. When a fed-batch culture was carried out under microaerobic conditions at pH 7.0 in a 5 L bioreactor, the recombinant K. pneumoniae ΔdhaT (puuC) strain produced 16.0 g/L 3-HP and 16.8 g/L 1,3-PDO with a cumulative yield of 51% on glycerol carbon in 24 h. The production of 1,3-PDO in the dhaT-deletion mutant was attributed to the expression of NAD(P)H-dependent hypothetical oxidoreductase. This study demonstrates the feasibility of obtaining two commercially valuable chemicals, 3-HP and 1,3-PDO, at a significant scale.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced in nature as an intracellular carbon and energy storage material. The surface layer of PHA granule contains various proteins embedded in or attached to a phospholipid membrane, and among them, only the PHA synthase is joined tightly to the granules by covalent linkage. We investigated in vivo immobilization of psychrophilic lipase from Photobacterium lipolyticum M 37 (M37L) on the surface of polyhydroxybutyrate (PHB) in Escherichia coli. M37L were genetically fused to the PHA synthase (PhaCCn) from Cupriavidus necator, and the M37L-PhaCCn fusion protein were expressed along with ß-ketothiolase and acetoacetyl-CoA reductase in E. coli. The functionality of PHA synthase and lipase of the M37L-PhaCCn fusion protein was shown by PHB accumulation and halo formation in recombinant E. coli, respectively. After the PHB granules were simply recovered from the cells, the functional attachment of M37L to the recovered PHB granules were proved. Also, we characterized in more detail the enzymatic properties of the immobilized M37L lipase and evaluated the possibility of using the immobilized enzyme for biodiesel production.

The production of deglycosylated isoflavones from glycosylated isoflavones and soy extract was performed using a recombinant β- glucosidase from Pyrococcus furiosus. The activity for genistin was optimal at pH 6.0 and 95 °C with a half-life of 59 h at 95 °C. The kinetic properties Km, kcat, and kcat/Km for genistin were determined to be 0.5 mM, 6050 s-1 and 12100 mM-1 s-1. The kcat and kcat/Km of the enzyme exhibited the highest values to date. The deglycosylated isoflavones genistin, daidzin, and glycitin at 1.0 mM were completely hydrolyzed to the glycosylated isoflavones genistein, daidzein, and glycitein, respectively, after 100, 140, and 180 min, respectively, by P. furiosus β-glucosidase. The soy flour extract of 7.5% (w/v) containing 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin were absolutely converted to genistein, daidzein, and glycitein, respectively, within 120 min. The productivities of the glycosylated isoflavones in the present study are the highest ever reported among biological methods. These results suggest that the thermostable enzyme may be useful for the industrial hydrolysis of deglycosylated isoflavones.

For the removal of 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 (160 mM), which was 13-fold that of the wild-type enzyme, was the highest among the reported values of β- galactosidase. The wild-type enzyme hydrolyzed 62% of 100 g lactose/l with the addition of 30 g galactose/l, whereas the F349S mutant hydrolyzed more than 99%.

L-Homoalanine is an unnatural amino acid that is used as a building block for synthesis of pharmaceutical drugs. Here, we report an efficient biocatalytic process for production of L-homoalanine from L-threonine using coupled enzyme reactions. L-homoalanine is asymmetrically produced from 2-oxobutyrate using ω-transaminase with benzylamine as an amino donor. The expensive precursor, i.e. 2-oxobutyrate, in the transaminase reaction is produced from L-threonine through dehydration and deamination reaction using threonine deaminase. For the coupled enzyme reactions, threonine deaminase from Escherichia coli and an (S)-specific ω-transaminase from Paracoccus denitrificans were cloned and overexpressed. We performed enzyme characterization to study substrate specificity, substrate inhibition and product inhibition of the ω- transaminase form P. denitrificans. In the presence of 10 mM 2-oxobutyrate, 20 mM benzylamine and ω-transaminase, the conversion of 2-oxobutyrate into L-homoalanine was 94 % ( ee >99 % ). The coupled reaction consisting of ω-transaminase and threonine deaminase resulted in 91 % conversion of L-threonine to L-homoalanine using 10 mM L-threonine and 20 mM benzylamine. [This work was supported by the Advanced Biomass R&D Center funded by the Ministry of Education, Science and Technology (ABC-2010-0029737) and Seoul R&BD Program (KU080657).]

Lignin biodegradation is a key step for carbon recycling in terrestrial ecosystems, where white-rot basidiomycetes fungi degrade this recalcitrant wood polymer enabling cellulose utilization by microbial populations. Three peroxidases involved in lignin degradation are known as lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP). The cDNA encoding manganese peroxydase isozyme H4 (MnPH4), isolated from Phanerochaete chrysosporium, was cloned into the pPICZα vectors and expressed in Pichia pastoris under the control of the methanol inducible alcohol oxidase I (AOXI) promoter. Enzyme assay indicated that the recombinant MnPH4 is efficiently secreted into the medium upon hemoglobin supplementation, at a maximum concentration of 500 U l-1. The purified recombinant manganese peroxidase (rMnP) exhibited a 60 kDa molecular mass considerably larger, because of their glycosylation, and showed optimum pH of 4.5 and temperature at 35 oC.

The S213C, I33L, and I33L-S213C mutant psicose 3-epimerases from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, had 0, 5, and 10 °C increases in optimal temperature, 3.3-, 7.2-, and 29.9-fold increases in half-life at 50 °C, and 3.1, 4.3, and 7.6 °C increases in melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement of thermostability was due to the formation of new aromatic stacking interaction in the I33L mutant enzyme and the increases of putative hydrogen bonds in the S213C mutant enzyme. The activity of the immobilized wild-type enzyme was decreased after 9 days with borate and 17 days without borate in a packed-bed bioreactor, but the activity of the immobilized double-site mutant enzyme was not decreased during the operation time of 30 days. These results suggest that the double-site mutant enzyme will be useful as an industrial producer of D-psicose.

Deracemization is an important tool for the industrial production of single enantiomeric compounds. It has a significant advantage over the kinetic resolution approach by allowing the complete recovery of a single enantiomer from the racemic mixtures. In this study, we have developed a novel approach for deracemization through the combination ω-transaminase and DAAO with improved oxygen usage by the help of a hemoglobin fused DAAO form. This combination of D-selectivity of amino acid oxidase with ω-transaminase has been applied for deracemization of the racemic mixture of 2-amino butyric acid, a non-natural amino acid and some drug candidates. The catalytic activity of D-amino acid oxidase (DAAO) was enhanced by improving the oxygen usage of DAAO by fusing it with Vitreoscilla hemoglobin (VHb) which helps the binding of free oxygen. Using this approach, we successfully obtained a quantitative yield of L-2-aminobutyric acid from D, L-2-aminobutyric acid. This novel approach can help for the scalable production of valuable compounds by deracemization from the racemic mixtures

UDP-galactose-4-epimerase (GalE, EC 5.1.3.2) catalyzes the 4-epimerization for free monosaccharides such as galactose, glucose, fructose, tagatose, psicose and sorbose in a low activity while it catalyzes the reaction for nucleic acid-activated sugar in a high activity. To enhance the 4-epimerization activity for free monosaccharides, random mutations were introduced into GalE by error-prone PCR and clones showing furctose 4-epimerization activity higher than 115% were selected. Two clones carrying five mutation points (D58E, N100S, P193S, I196N and T317S) were selected among 3,060 mutant clones. To investigate the effect of the mutation points on the desired characteristics, 5 mutant clones carrying single mutation were constructed and the variant proteins were characterized after purifications using Ni+ affinity chromatography. The variant D58E was found to contribute the twice as much activity enhancement (21 nmol/mg-protein) while the other variants showed less than 15% contribution. The possible reason of the activity enhancement by the Asp58 substitution into Glu58 is discussed based on the 3D-structure.

The stabilization of the proteins by various techniques is acquiring significance because of its applications in various fields of biotechnology. Over the years, different stabilization protocols as well as novel strategies were evolved for stabilizing the proteins. These stabilization offers over a vast number of applications in medical and industrially important proteins. A method to achieve the stabilization of the proteins is cyclization of the polypeptide back bone and it has been shown that cyclization of peptides frequently improves their stability and biological activity. Protein splicing mechanism provided the novel tools to cyclize the back bone of the desired protein. Inteins are the protein elements join the two ends by catalyzing the chemical steps acyl migration, leading to the formation of a thioester, forms the peptide bond and cyclize the protein. However, the cyclization process will not occur to complete extent either invitro or invivo leading to the mixture of both linear and cyclic proteins which are difficult to selectively separate. In the present investigation the efforts were made to study the process of cyclisation in vitro and in vivo using the well studied protein GFP (Green Fluorescent Protein). In the study various constructs of EGFP protein having variable numbers of HIS tags at N and C terminals express along with inteins to make it cyclize and selective purification of stabilized cyclic EGFP.

Lipase (LP) was immobilized and stabilized in the form of enzyme precipitate coatings (EPCs) on electrospun and ethanol-dispersed polystyrene-poly (styrene-co-maleic anhydride) (PS-PSMA)nanofibers (EtOH-NF). LP precipitate coatings (EPCs-LP) were prepared in a three-step process, consisting of covalent attachment, LP precipitation, and cross-linking of precipitated LPs onto the covalently attached LPs via glutaraldehyde treatment. EPCs-LP improved the LP activity and stability when compared to covalently attached LPs (CA-LP) and the enzyme coatings of LPs (EC-LP) without the LP precipitation. For example, the use of 40% (w/v) ammonium sulfate resulted in EPC40-LP with the highest activity, which was 4.0 and 3.6 times higher than those of CA-LP and EC-LP, respectively. After 165-day incubation under rigorous shaking at 200 rpm, the residual activities of EPC50-LP were 0.5 μ M/min per mg of EtOH-NF, representing 113 and 75 times higher than those of CA-LP and EC-LP, respectively. When LP was partially purified via a simple ammonium sulfate precipitation and dialysis, both activities and stabilities of EC-LP and EPC-LP could be marginally improved. It is anticipated that the improved LP activity and stability in the form of EPCs would allow for their potential applications in various bioconversion processes such as biodiesel production and ibuprofen resolution.

Previous experimental and simulated results reported that mutations of protein cavity could improve the thermal stability. Many comparative studies between thermophilic and mesophilic proteins have been conducted to understand which cavity properties were related to the protein thermostability. However, previous statistical studies compared simple one dimensional information of cavities, e.g., surface area, volume, number of cavity and did not identify important differences of cavity properties between thermophilic and mesophilic proteins responsible for protein thermostability. In this study, we attempted to investigate the role of cavity properties in protein thermostability using three dimensional information of cavity properties. Protein structures were classified into three areas, i.e., core, boundary, and surface and cavity properties were compared using this structure index. According to statistical results, cavity flexibility is closely related to protein thermostability. Thermophilic proteins had less cavities in boundary and surface areas and in particular, cavities of mesophilic proteins in all areas were more flexible than those of thermophilic proteins. Compared to the thermophilic proteins, the flexible cavities of mesophilic proteins in boundary and surface can be deleterious to the stability. Based on this study, rational or computational design of flexibility cavity in surface or boundary areas could be a good strategy to improve the thermostability of mesophilic proteins.

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). EstIM1 was active over a temperature range of 1-50ºC, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1-40ºC. The activity of EstIM1 was about 60% of maximal even at 1ºC, suggesting that EstIM1 is efficiently cold-adapted. Further characterization indicated that the esterase may be very valuable in industrial applications. (This work was supported by a HTS-based Integrated Technology Development grant and by Basic Science Research Program from the MEST.)

cDNA sequence encoding CWPO_C from Populus alba L was heterologously expressed in E. coli as inclusion bodies. Insoluble products were solubilized and reactivated via refolding procedure. The efficiency of refolding was estimated throughout oxidation rate of ABTS. The optimal conditions for refolding CWPO_C with systematically optimized parameters are: 100 mM Tris-HCl at pH 8.5, 0.6 mM GSSG, 5 μM hemin, 0.6 M GmdCl and 5mM CaCl2. Property of substrate preference to sinapyl alcohol rather than coniferyl alcohol was characterized with refolded CWPO_C. This unique property of refolded CWPO_C was confirmed, like native CWPO_C when relative oxidation rate of these monolignols catalyzed by refolded CWPO_C and HRP-C were compared in this study. Successful expression of CWPO_C in E. coli provides a valuable tool for elucidate the structure - function relationship of CWPO_C known as a S-peroxidase having important role in the lignification of angiosperm woody plant cell walls.

To explore the effects of the ionic liquids on enzymatic catalyses, the horseradish peroxidase catalyzed oxidation of 2-methoxyphenol with H2O2 was studied in the aqueous mixtures of the three ionic liquids, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), N-butyl-3- methypyridinium tetrafluoroborate ([BMPy][BF4]), and 1-butyl-3- methylimidazolium methylsulfate ([BMIM][MeSO4]). [BMIM][BF4] and [BMPy][BF4] share an anion (BF4 -) while [BMIM][BF4] and [BMIM][MeSO4] have a cation (BMIM+) in common. On the addition of [BMIM][BF4] or [BMPy][BF4], Km increased while kcat decreased. In the aqueous mixtures of [BMIM][ MeSO4], however, both Km and kcat decreased. Equilibrium partitioning of the substrate with n-octanol as a standard solvent revealed that the three ionic liquids stabilize the free substrate resulting in the diminished binding affinity of the substrate onto the enzyme. Based on these kinetics and thermodynamics investigations, anionic parts of the ionic liquids are presumed to exert a major role to determine how the enzymatic catalysis is affected by the ionic liquids; BF4 - inhibits the enzyme non-competitively, but MeSO4 - uncompetitively.