Earticle

The elastin-like polypeptides (ELPs) are artificial polypeptides based on the amino acid sequence of elastin. ELPs have a distinctive thermal property which leads to reversible phase transition within a very narrow temperature. At a temperature below transition temperature (Tt), ELPs are highly soluble in aqueous solutions. However, upon a temperature shift to above Tt, ELPs become insoluble and form an aggregate of polypeptides. The epoxide hydrolases (EHs) catalyze the addition of water molecule to an epoxide to form the corresponding vicinal diol. A marine epoxide hydrolase from Mugil cephalus (McEH) has enantioselective hydrolysis activity toward (R)-enantiomer of racemic epoxide, resulting in the preparation of enantiopure (S)-enantiomer. McEH showed higher enantioselectivity and stability at lower temperature. In order to develop a novel EH biocatalyst system that catalyzes enantioselective hydrolysis reaction at low temperature and can be recycled by thermo-responsive reversible aggregation at high temperature, ELP-tagged McEH was developed. The recombinant McEH fused with elastin-like polypeptides was cloned and characterized at the molecular level.

Enantioselective kinetic resolution of recombinant epoxide hydrolase (EH) from a marine fish, Mugil cephalus, was investigated. The lyophilized recombinant whole-cell E. coli expressing the M. cephalus EH gene was used as the biocatalyst. The problem of instability and low solubility of styrene oxide substaret was overcome by using an organic solvent as the reaction medium. The effects of water contents, cell concentration, nature of solvent, adding the various lyoprotectants and substrate concentration on activity, stability and enantioselectivity of EH in an organic solvent were optimized. Various organic solvents with log P value between - 0.7 to 4.5 were screened as the reaction solvent. To select the most suitable lyoprotectant, the cells were lyophilized in the presence of various lyoprotectants and detergents. Enantioselective kinetic resolution of 20 mM styrene oxide was carried out in a shaking incubator at 30 ℃ and 250 rpm, and enantiopure (S)-strene oxide with 98% ee was readily obtained.

Glycosyltransferase can convert lipophilic secondary metabolites such as flavonoids, alkaloids, and terpenoids into glycosylated forms. The bioavailability of secondary metabolites can be improved by glycosylation. An oleandomycin glycosyltransferase (OleD GT) gene from Streptomyces antibioticus was functionally expressed in Escherichia coli BL21 (DE3). The purified recombinant OleD GT was employed as the biocatalyst for the glycosylation of amentoflavone as the acceptor via the transfer of glucose moiety from uridine-diphosphate-glucose (UDP-glucose) as the donor. The glycosylation reaction conditions were optimized and the time-course of the glycosylation of amentoflavone was analyzed. Amentoflavone was successfully converted into the corresponding glycosides by OleD GT-catalyzed batch glycosylation reaction.

Uniform magnetic separable branched polymeric micro-hydrogel beads for enzyme immobilization was successfully developed by sequentially combining electrospraying and chemical chelation. The amine groups on the enzyme react with the aldehyde groups on the glutaraldehyde polymer so that the stable conjugation is formed. The specific activity of the immobilized enzyme after conjugation was found to be retained more than 50%, but, interestingly, the Km value is not changed compared to the free enzyme. In addition, the enzyme immobilized in the microhydrogel beads was found to be highly stable for more than 50 days, pertaining over 60% of its initial activity and maintained even after being reused more than 15 times repeatedly. Furthermore, the magnetic-driven controllability provided facile separable characters for the repeated recycling. It is expected that these magnetic separable branched microhydrogel beads can be efficiently utilized as a key tool for successful realization not only in enzyme process but also in extended fields; bio-based sensor or analytical devices, bioprocessing, bioremediation, to name only a few of the numerous areas.

An unusual, internally duplicated, 60 kDa alpha-type carbonic anhydrase of Dunaliella tertiolecta, a unicellular green alga, (Dt-aCA), has been expressed successfully in E. coli system. Here, we demonstrated to express the N- terminal and C-terminal carbonic anhydrase domains of Dt-aCA as distinct proteins using a pET system to increase the expression levels more than one expressed as a duplicated form. The N- and C-terminal domain were expressed using a vector containing an octa-histidine (8 ⨯ His) tag at the C terminus. The purified N- terminal CA domain (residues 1–279) had an apparent molecular mass of; 35 kDa, and retained no activity to hydrolyte p-nitrophenyl acetate (p-NA) into p- nitrophenol and acetate and CO2 hydration. The purified C-terminal CA domain (residues 280-557) had an apparent molecular mass of; 31kDa and retained the ability to catalyze p-NA hydrolysis and CO2 hydration, which were enhanced by the presence of N-terminal domain. The expressions of both domains were increased as compared to that of duplicated dimeric form as one protein. The CA activity was successfully reconstituted by mixing the two domains, N- and C- terminal domains, although N-terminal domain did not show catalytic activity as distinct protein. These results demonstrate that the two domains of CA that has a duplicated dimeric structure can be expressed separately and functionally reconstituted in vitro.

GST fusion proteins not only act as solubility enhancing partners but also offer an important biological assay for direct protein-to-protein interactions. A protein of sponge, called silicatein, can catalyze silica deposition in vitro at an environment friendly condition. The soluble expression of silicatein was enhanced when expressed as a GST fusion protein in E.coli. Furthermore, this fusion protein was immobilized on GSH -coated plate via GST-tag and remained active to form silica layer on surface by means of biosilification in the presence of TEOS at an ambient condition, room temperature and neutral pH. Simultaneously, GFP or HRP protein was immobilized on silica surface by simple adding it during biosilification. Immobilized proteins retained their activity and were released gradually. This technique can be applied to form biocompatible silica coating for catalytic, diagnostic and sensing surface, and matrix for tissue cultures.

Carbonic anhydrases (CAs) are zinc-containing metallo-enzymes that catalyze the inter-conversion of carbon dioxide and bicarbonate. The α -class CAs are found predominantly in vertebrates, but they are also found in Bacteria, algae and cytoplasm of green plants. α-CA from Duneliala sp (DspACA) shows to share high sequence homology to Dunaliella salina (dCAI). The codon optimized DspACA was cloned, expressed in E. coli BL21DE3, purified and characterized. The optimal induction concentration of IPTG and growth temperature was found to be 1.0 mM and 20oC.The optimum temperature of enzyme activity is 350C and pH 7.6 and 10.0. Apparent Km, Vmax and were 0.9095 mM, 3.303 x 10-8 mM min-1 for p-nitrophenylacetate substrate, respectively. Inhibitory effect on CA activity were determined using the esterase method under in vitro conditions. The inhibitory activity metal ions was in the order Fe2+ >Al3+ >Ag+ >Hg2+ >Cu2+ >Pb2+ >EDTA. Some metal ions show maximum enhancement in activity: Co2+ >Cd2+ >Zn2+. Acetazolamide shows maximum inhibition in the order : Acetazolamide >Sulphonamide >CN- >SCN- >F- >NO2- >Cl- >NO3- >SO42- >I- >Acetate.

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B-factors of residue pairs in CalB wild type (CalB-WT) followed by simple flexibility analysis of residues in CalB-WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162-K308, N169-F304, Q156-L163, S50-A273 and S239CD252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C-K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB-WT. Remarkably, the temperature at which 50% of its activity remained after 60-min incubation (T50 60) of CalB A162C_K308C was increased by 8.5oC compared to that of CalB-WT (55oC and 46.5oC, respectively). Additionally, the half-life at 50oC of CalB A162C-K308C was 4.5-fold higher than that of CalB-WT (220 minutes and 49 minutes, respectively). The improvement of thermostability of CalB A162C-K308C was elucidated at the molecular level by molecular dynamics (MD) simulation.

Candida antarctica lipase B (CALB) is one of the most versatile hydolases used in industrial processes. Despite of its heat resistance, further thermostabilization of the CALB is desirable to catalyze effectively biotransformation operated at high temperature. In this study, remarkably improved thermostabilization of the CALB was achieved by simple R1 silaffin-mediated biosilicification rather than laborious protein engineering approach. The CALB was fused with the R1 silaffin peptide and the CALB-R1 fusion protein was expressed successfully in Pichia pastoris expression host. The CALB-R1 fusion protein was immobilized on a macroporous polyacrylate carrier through physical adsorption and then subsequently coated with tetramethyl orthosilicate (TMOS). After R1 silaffinmediated biosilicification, the thermostability of the immobilized CALB-R1 was improved successfully. The T50 60 of the biosilicificated and immobilized CALB-R1 was increased drastically from 45℃ to 72℃, which was 14℃ and 10℃ higher than that of immobilized CALB-R1 and commercial Novozym 435, respectively. The optimum pH condition for biosilicification was determined to be pH 5 by comparison of the T50 60 values at various pH values. This simple R1-mediated biosilicification method for CALB could be useful to improve the thermostability of industrial enzymes which are not easily stabilized through protein engineering.

In this study, for the first time, the combined effect of both the enzyme Carbonic Anhydrase (CA) and polyethylene glycol (PEG) assisted on the crystallization behavior of magnetically responsive calcium carbonate (CaCO3) was studied under the natural diffusion of gaseous CO2 in a CO2 incubator. The characterization of CaCO3 crystals such as morphology, size, and polymorphs conducted by Transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD) as well as elemental analysis (EDAX), strongly indicated that both the enzyme CA and PEG assist to form the ellipsoidal structure of micron-sized CaCO3 crystals as one of calcite polymorphism. The rigidity of PEGs (straight PEG or branched PEGs) used was found to take an important role in the formation of a certain morphological changey of the CaCO3 crystals. In addition, the mixing speed of the reaction solution was also found to be a parameter to control the morphology of CaCO3 crystals. CaCO3 crystallization in the presence of organic additives (both the enzyme CA and PEG) and the magnetic nanoparticles was found to be successfully occurred to form very smooth ellipsoidal structure harboring all additives inside the crystals formed. Due to its convenience, low-cost, and easy fabrication using the present method, this new method may be utilized to fabricate micro-sized calcite particles, which can be not only utilized with many industrial aspects. but also inspired to the study of the bio-mineralization.

In biotechnology, proteomics can have practical applications, for example, through the identification of proteins that may be potential targets for the biotechnology industry, and through the extension of our understanding of the physiological action of these proteins. In this study, outer membrane proteomes were analyzed and then used to identify potential candidates for applying anchoring motifs. We found a few of proteins that fit specific requirements, such as an efficient signal peptide or transport signal to that allows a premature fusion protein to be transported across the inner membrane and a strong anchoring structure that keeps fusion proteins on the cell surface without detachment. Thus, this study shows proteomics is very important tools for the discovery of targets that have potential biotechnological applications. [This work was supported by the Basic Science Research Program (2010-0008826) and Converging Research Center Program (2009-0093652) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology]

Xylan is a major hemicellulose component of the cell walls of monocots and hardwood, representing up to 30% up the dry weight of these plants. To efficiently and stably hydrolyze xylan, the endoxylanase (endo-β-1,4-xylanase) from Bacillus sp. was cloned and expression plasmid, pRSδ-xylp (YIp type) was constructed. This plasmid contains mating factor α signal sequence (MFαs.s) under GAL10 promoter for secretory expression of endoxylanase and δ- sequence for target integration on yeast genome. The pRSδ-xylp was transformed into Saccharomyces cerevisiae SEY2102△trp and the endoxylanase activity was detected by azo-xylan plate assay, meaning that the recombinant endoxylanase was efficiently secreted in yeast cell. In flask culture, the activity of the recombinant endoxylanase reached 4.4 unit/㎖ and the mitotic stability was 100%. The pRSδ-xylp was sequentially transformed in SEY2102△trp/pRSδ -xylp transformant by selective marker rescue and repeated integration, and the endoxylanase activity was also sequentially increased. These results suggested that the endoxylanase from Bacillus sp. was stably expressed and secreted in S. cerevisiae by using genome integration and the MFα s.s, respectively.

Glutamate decarboxylase (GAD : EC 4.1.1.15) is a pyridoxal 5’- phosphate (PLP) dependent enzyme, which catalyses the α- decarboxylation of L-glutamate to produce γ-aminobutyrate (GABA). GADB from E. coli is multimeric structure (hexamer) and its N-terminal residues 1-15 form the triple helix bundle at acidic pH. In this study, we showed that the thermostability depends on the structure of N-terminal changed by pH. And the thermostability of GADB was improved through N-terminal residues protein engineering. Eight mutants located at near N-terminal of GAD were designed considering the ionic interaction and hydrophobic interaction between the N-terminal α-helix residues. The GADB-WT and its N-terminal mutants were expressed successfully in E. coli expression host. The thermostability was determined by T50 10 value. The T50 10 value is the temperature at which 50% of initial enzymatic activity remains after 10 min heat treatment. The T50 10 of mutants were increased compared to that of GADB-WT. Additionally, the double mutant of N-terminal residues showed synergy effect on the thermostability.

A β-1,3-glucanase was widely used in various biotechnological processes and overproduction of its was required for versatile industrial utilization. We subcloned β-1,3-glucanase gene (exgA) from Aspergillus oryzae and examined overexpression of β-1,3-glucanase by using δ sequence-mediated integration. We constructed plasmid, pRSδH-exgA for repetitive integration of exgA gene. This plasmid contains ADH1 promoter for constitutive expression, exoinulinase signal sequence for secretion, loxP-CgHIS3-loxP selective marker for repetitive selection and δ target sequence for integration of β-1,3-glucanase. The pRSδH-exgA plasmid was transformed into S. cerevisiae BY4742△exg1 strain and transformant showing β-1,3-glucanase activity was selected. Subsequently, the CgHIS3 marker was removed for marker recycling and the pRSδH-exgA plasmid was repeatedly transformed to induce overexpression of β-1,3-glucanase. By repetitive transformation, the activity of β-1,3-glucanase was sequentially increased and these results suggested that δ-mediated integration was efficient for enhancement of β-1,3-glucanase activity.

The fixation of carbon dioxide is one of the most important issues for global sustainability. Formate dehydrogenase could be used for direct reduction of carbon dioxide to formate. Most formate dehydrogenases used in formate production were selenium-dependent and were not industrially feasible due to rapid inactivation upon exposure to oxygen. Recently, NAD-dependent formate dehydrogenases were utilized for reduction of carbon dioxide with advantages of oxygen stability, easy recombinant expression. Formate dehydrogenase from Candida boidinii (CbF) is commercially available and is most widely used. The reduction of carbon dioxide requires proton and reduction reaction at acidic pH is more efficient than at neutral pH. However, commercial CbF showed low reduction efficiency at acidic pH and it is necessary to look for efficient formate dehydrogenases for reduction of carbon dioxide at acidic pH. In this study, reduction activity of five formate dehydrogenases with acidic optimum pH was investigated. Four formate dehydrogenases showed higher reduction activity than the CbF and most efficient formate dehydrogenase had 3.2, 3.3, and 4.4 times higher reduction activity than the CbF at pH 5.5, 6.0, and 7.0, respectively. Further discussion will be presented.

The aim of this study is to isolate a novel cold-adapted lipolytic enzyme from the activated sludge of uncultured microorganisms. The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among approximately 100,000 recombinant libraries showed lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni-NTA affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35.6 kDa. Typical residues essential for lipolytic activity such as penta-peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 exhibits low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487) from Pseudomonas mendocina ymp and esterase (31%, AAY45707) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7.5 and 10°C. Thermal stability analysis showed that lipo1 was more unstable at 40°C than at 10°C. An activity-based strategy is an effective method for fishing out a low temperature-adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the HSL family due to the enzyme’s oxyanion hole by the sequence HGGG. Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry.

Benzoate-CoA ligase (BCL) from Burkholderia xenovorans LB400 has narrow substrate specificity toward benzoate. Compared to the activity with benzoate, specific activity of BCL is much less than that with m-/p-hyroxybenzoate. In order to find BCL mutants which have higher activity, site-directed mutagenesis within substrate binding pocket based on rational design were carried out. The comparative structural analysis revealed that active site of 2,3-dihydroxybenzoate AMP ligase(DhbE) and 4-chlorobenzoate CoA ligase(4-CBAL) with that of BCL has shown absolutely different amino acids. Especially, substratebinding pocket of BCL showed relatively narrow circumstanse than that of BCL from Bulkholderia xenovorans due to the surrounding residues(i. e, HisXXX) located at the bottom of the active sites. Their structural analysis allows us to generate thirteen mutants and in-vitro analysis of specific activities using indirect assay monitoring NADH consumption were examined for understanding their structure-activity relationship. In conclusion, BCL mutants screened by above mentioned structural understanding and mutagenesis showed higher activity against benzoate as we are targeting.

Flavonols possess antioxidant and free radical scavenging activity in foods, with myricetin being one of the most active. In plant, kaempferol converts to myricetin by 3’, 5’-hydroxylase which is cytochrome P450. The cytochrome P450s belong to a family of heme containing monooxygenases and catalyze diverse reaction, such as hydroxylation, epoxidation and O-demethylation. CYP102D1, bi-functional cytochrome P450:NADPH-P450 reductase, from Streptomyces avermitilis can hydroxylate the saturated and unsaturated fatty acids. First, three positions, which are L84A/F96V/L448A, were done saturation mutagenesis to increase the size of active site pocket and substrate access channel. Second, A273/G274/T277 was selected among the amino acid within 7Å from heme to saturated mutagenesis. For high throughput screening, aldehyde sensing method was applied with schiff’s test. Finally, we screened six CYP102 mutants towards permethylated kaempferol using schiff’s reagent agar plate assay and expressed.

The production of biodiesel by enzymatic transesterification has attracted much attention for high purity product and enables easy separation. Candida antarctica lipase B is one of the most important enzyme in the biodiesel reaction. In the reaction, CALB shows low organic solvent stability because of methanol. In this study, we concerned about enhancing organic solvent stability in hydrophilic solvents. In watermiscible solvents, enzyme loses its stability due to deformation of enzyme surface by solvent penetration effect. M olecular dynamic simulation was proceeded to predict how the enzyme actually motions in organic solvents. RMSD value of each residue was calculated and distinguished for predicting solvent affecting site. By RosettaDesign, target residues were predicted for further mutagenesis. After in vitro experiment of mutants, we obtained organic solvent stability enhanced CALB variants. The detail will be explained and discussed.

Lactide is a precursor for poly-lactic acid (PLA), a promising biodegradable thermoplastic. Its synthesis is currently carried out in a chemical process which involves complex purification and prepolymerization. Biocatalysts have moderate operating temperature, high activity and high enantioselectivity, and could be a replacement to the conventional method for an one-step process. Rhizomucor miehei lipase (RML) is suitable for such purposes, but the enzyme lacks enantioselectivity for the substrate S-methyl-lactate to synthesize L-lactide. Enantiopurity is a key factor for good physical characteristics of the PLA product. In this study, rational design of RML was conducted to enhance the enantioselectivity of the enzyme using computational docking of the enzyme and lactides. By comparing L-lactide-RML and D-lactide-RML complexes several mutation site candidates were identified that could specifically enhance the interaction between RML and L-lactide while diminishing the interaction with D-lactide. RML mutants have been expressed in Escherichia coli for a confirmation of the desired enantioselectivity change. Further results will be discussed.

Oppositely charged residues often form ion pairs in proteins. Ion pair play important role in oligomerization, molecular recognition, allosteric regulation, domain motions, thermostability and α-helix capping. Most of the ion pair forming residues is present in the secondary structural elements in the proteins. What is clear that, of all the structures, α-helix contains most of the ion pair forming residues and the most of the ion pairs are involved in stabilization of α-helix. But its role in enzyme catalysis remains unclear. Here, in this study, we introduce ion pair at the end of α-helix in Bacillus circulans xylanase to see whether it has any role in catalytic efficiency enhancement. Site directed mutagenesis experiment was conducted to confirm the role of ion pairs. The results show a dramatically increased catalytic efficiency in the mutants. The details will be presented and discussed.

In the past few decades, a novel approach named residue specific incorporation was developed to incorporate non-canonical amino acids into the protein with the help the endogenous translational apparatus. In general, the accuracy of the protein translation is maintained by the fidelity nature of amino acyl tRNA synthetases (AARS). Although, the AARS discriminate the cognate amino acid from the other 19 canonical amino acids, it fails to discriminate the close structural analogs of the cognate amino acid. So, structural insight into the active site of AARS and interaction between the non-canonical amino acid and active site will help to expand the non-canonical amino acid analogs for the residue specific incorporation. So, here we applied an in silico approach to evaluate the polyspecificity nature of Escherichia coli methionyl tRNA synthetases towards the reported methionine analogs

Enantiomerically pure amino acids are widely used as building blocks for the synthesis of a number of chiral drugs. Generally, enantiopure amino acids can be accomplished in two ways: (i) asymmetric synthesis of a pro-chiral compound, and (ii) kinetic resolution of racemic amino acid. However, there are disadvantages to be resolved for efficient preparation of enantiopure amino acids in these approaches. First, in case of asymmetric synthesis, pro-chiral compounds are not readily available compared to racemic amino acid. Second, the theoretical yield of kinetic resolution has a limit of 50%. Here, we report an efficient biocatalytic process for deracemization of amino acids, starting with racemic amino acid to obtain enantiopure amino acid, employing a coupled enzyme strategy to overcome the limitation of two approaches as described above. In this study, the deracemization process consist of kinetic resolution of racemic amino acid employing D-amino acid aminotransferase and asymmetric synthesis of L-amino acid employing ω-transaminase. The coupled reaction consisting of D-amino acid aminotransferase and ω-transaminase resulted in 99 % ee of L- homoalanine using 100 mM DL-homoalanine, 70 mM α-ketoglutarate, and 70 mM isopropylamine. This work was supported by the Advanced Biomass R&D Center (ABC-2010-0029737) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology.

The production of unnatural amino acids is important in chemical industry due to an increase in demand for optically pure non-proteinogenic amino acids as building blocks for pharmaceutical compounds. Given the significance of the unnatural amino acid, its efficient synthesis to optically pure form has become an attractive challenge to organic chemists and biologists. Among the different unnatural amino acids, l-homoalanine is of high pharmacological significance as it is used as a key chiral intermediate for the synthesis of several important drugs such as brivaracetam, levetiracetam and ethambutol. We developed a deracemization method to generate optically pure l-homoalanine from racemic homoalanine using d-amino acid oxidase and ω-transaminase. A whole cell reaction using biphasic system converted 500 mM racemic homoalanine to 485 mM l-homoalanine (> 99% ee).

Electrostatic interactions are important in protein folding, binding, flexibility, stability and function. When we look at the enzyme function, one of the key modulators of the active site residues are electrostatic interactions. The pH at which the enzyme is maximally active is determined by the pKa of the active site residues, which are modulated by several factors including by the change in electrostatics in its vicinity. Here, in this study, we focus on charged only substitutions to modulate the pKa of the active site residues in Bacillus circulans xylanase. Neutral residues are substituted by the charged ones (Glu, Arg) in such a way that the substituted residue can make direct interaction with the catalytic residues. Especially, the cases with other titratable residues (Asp, Tyr, Ser, Arg, Lys and His) present in native xylanase in between the catalytic sites and the substituted sites are avoided. And all the mutation sites are chosen closer to acid/base catalyst. Site directed mutagenesis was conducted to confirm the strategy. The results show the shift in pH optima of the mutants towards acidic side by 0.5 to 1.5 unit. The details will be presented and discussed.

Cytochrome P450 monooxygenases (CYP) are a superfamily of ubiquitous heme proteins and performs various oxidative reactions such as C-H bond hydroxylation, N-dealkylation, N- hydroxylation, O-dealkylation, S-oxidation and epoxidation of numerous endogenous and exogenous compounds. Indeed, the P450 enzyme plays a crucial role in fungi for the adaption of diverse ecological niches by its hydrophobic conversion of the primary and secondary metabolites and in the degradation of ecological pollutants. Genome sequencing projects has revealed the presence of several thousands of putative P450 genes from various families in the fungal kingdom. Bioinformatic annotations showcase the presence of 169 putative P450 genes and 2 putative Cytochrome P450 reducatase (CPR) genes from the whole genome sequence of the filamentous fungus Fusarium oxysporum f. sp. lycopersici. To elucidate the molecular diversity and functional capabilities of the Cyp P450 monooxygenases from F.oxysporum, we initiated the functional genomic research by comprehensive screening of the putative novel P450s by using Bioinformatic approaches and by analyzing the catalytic potential of P450s against a wide variety of compounds. The functional targets includes the Sterol 14α-Lanosterol demethylase, Flavonoid Hydroxylase, ω-Fattyacid Hydroxylase, Benzoate Hydroxylase, Alkalene Hydroxylase, Polycyclic aromatic hydrocarbons (PAH) oxidase, Phenyl Acetate Hydroxylase, and Steroid 11α-Hydroxylase, etc. For the construction of P450 gene library various parameters such as mRNA expression, cDNA synthesis, gene amplification and cloning followed by transformation into E.coli cells as well as Yeast expression system are been optimized. The functional characterization includes the isolation of Microsomes from the Saccharomyces cerevisiae cells harboring the recombinant P450 co- expressed with F.oxyxporum NADPH-P450 oxidoreductase and the enzymatical analysis with various substrates. Hence, the FoCYP P450 gene library will drive us towards an intensive approach for the functional characterization of various novel P450s and facilitates the comprehension of metabolic diversity in ascomycetes and has future biotechnology applications.

Chitosan (CS) nanofibers with a diameter of 150-200 nm were fabricated from mixed chitosan/poly (vinyl alcohol) (PVA) solution by the electrospinning method. The nascent CS/PVA nanofibers were treated with 0.5M NaOH solution for making stable CS nanofibers by removing PVA in aqueous conditions. Hen egg-white lysozyme was immobilized on electrospun CS nanofibers via cross-linked enzyme aggregates (CLEA) and used to effective and continuous antibacterial applications. The maximum amounts of lysozyme immobilized on the CS nanofibers were determined to be 62.3 mg/g of nanofibers under optimum condition. The immobilized lysozyme-CLEA retained more than 75.4% of its initial activity after 80 days of storage at room temperature, while the free lysozyme lost all under same condition. In addition, the immobilized lysozyme-CLEA also retained more than 92.4% activity after 50 consecutive uses. Finally, the durability of lysozyme-CLEA immobilized CS nanofibers showed 82.4% and 79.8% of bacteriostasis ratio after 10 cycles against 2 pathogenic bacteria including Staphylococcus aureus, Bacillus subtilis, respectively. These results demonstrated the lysozyme-CLEA immobilized CS nanofibers could be used as a promising material for enhanced and continuous antibacterial applications.

Degradation and conversion of lignocellulosic biomass is attracting attention because of its potential for the development of a sustainable environmentally friendly bioenergy, biorefining and biomaterials industry (1). It can be converted into useful products such as soluble sugars, ethanol, and industrially useful chemicals. As a linear polymer of D-glucose residues linked by β-1,4- glycosidic bonds, cellulose is hydrolyzed by enzyme systems such as endo-β-1,4- glucanases (EG) (E.C.3.2.1.4), cellobiohydrolases (CBH) (E.C.3.2.1.91) and β-glucosidases (BGL) (E.C.3.2.1.21). These enzyme components, when acting synergistically, affect the maximum hydrolysis of native cellulose (2). In this study, fungal strains with cellulase (BGL and EG) activity were isolated from rotten citrus peel, identified as Penicillium italicum based on ITS region analysis. P. italicum BGL and EG were purified and its biochemical properties, including optimum pH and temperature, thermostability, determination of molecular mass, substrate specificity and kinetic parameters, were investigated. To elucidate the functions of EG and BGL, we have conducted gene cloning studies of cellulases and expression of recombinant proteins from these strains.

Glutamate decarboxylase(GAD) has been purified from several brain tissues and it is generally accepted that the dimeric protein possesses a relative molecular mass of around 110,000. The stochiometry of binding of the cofactor to the protein has not been elucidated, though the presence of non-equivalent binding sites has been postulated to explain the catalytic behavior of the decarboxylase. The substrate L-glutamate, at concentrations slightly higher than Km value, promotes slow inactivation of the decarboxylase. This inactivation process has been attributed to a secondary reaction, i.e. decarboxylation-dependent transamination. Gamma-aminobutyric acid(GABA), a four-carbon non protein amino acid, acts as the major inhibitory neurotransmitter of the central nervous system. Other physiological functions of GABA　are induction of anti-hypertensive, prevention of diabetes, diuretic and tranquilizer effects. GABA is extensively used in pharmaceutical preparations and functional foods. In this study, the optimum temperature, pH, stability and reuse efficiency was studied for immobilized GAD. And For the increase productivity of GABA, investigated buffer concentration and reaction into cation exchange resin. As the results, free enzyme and immobilized enzymes were increased final GABA concentration by changed buffer concentration.

Single-molecule fluorescence resonance energy transfer (smFRET) analysis provides a variety of information including conformational and structural dynamics and localized or colocalized position of the tagged protein. For effective smFRET analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. We have shown that the site-specific labeling of protein via incorporation of an unnautural amino acid provides a greater contrast between the folded and unfolded states of the protein in smFRET analysis than the conventional labeling using double cysteines. As a model study, maltose-binding protein (MBP) was dually labeled via incorporation of ρ-azido-L-phenylalanine and cysteine at specific positions, immobilized on a surface, and subjected to smFRET analysis under native and denaturing conditions. The resulting histograms show that site-specific dual-labeling results in a more homogeneous distribution in protein populations. Considering this case of MBP, site-specific dual-labeling of the protein might offer a new chance for more precise smFRET analysis of the protein.