• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1375-1382
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• 2019

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPAH) is a monomeric enzyme that converts phenylalanine to tyrosine. It shares high amino acid identity and similar structure with a subunit of human phenylalanine hydroxylase that is a tetramer, resulting in the latent application in medications. In this study, semirational design was applied to CvPAH to improve the catalytic ability based on molecular dynamics simulation analyses. Four N-terminal truncated variants and one single point variant were constructed and characterized. The D267P variant showed a 2.1-fold increased thermal stability compared to the wild type, but lower specific activity was noted compared with the wild type. The specific activity of all truncated variants was a greater than 25% increase compared to the wild type, and these variants showed similar or slightly decreased thermostability with the exception of the $N-{\Delta}9$ variant. Notably, the $N-{\Delta}9$ variant exhibited a 1.2-fold increased specific activity, a 1.3-fold increased thermostability and considerably increased catalytic activity under the neutral environment compared with the wild type. These properties of the $N-{\Delta}9$ variant could advance medical and pharmaceutical applications of CvPAH. Our findings indicate that the N-terminus might modulate substrate binding, and are directives for further modification and functional research of PAH and other enzymes.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.40-45
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• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• Toxicological Research
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• v.17
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• pp.145-155
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• 2001

Cytochrome P4502C19 (CYP2C19) is one of human polymorphic xenobiotic-metabolizing enzymes. The enzyme has been reported to catalyze more than 70 substrates, involving more than 100 reactions. These include several classes of therapeutic agents (e.g. anti-microbial. cardiovascular, psycho-active, etc.), sex hormones and insecticides. Associations of the CYP2C19 genotype/phenotype with individual differences in drug efficacy (e.g. diazepam, omeprazole, proguanil) and toxicity (e.g. mephenytoin, barbiturates) have been documented by many investigators. At least 11 allelic variants of CYP2C19 gene were reported to date. Most of the mutant alleles found in the poor metabolizer (PM) led to the production of truncated and/or inactive proteins. Except for the exon 6, single-nucleotide mutations were reported in all nine exons of the gene. Genetic polymorphism of CYP2C19 shows marked interethnic variation with the population frequencies of PM phenotype ranging from 1∼2% up to more than 50%. The prevalence of CYP2C19 PM tends to be higher in Asian and certain Pacific Islanders than other race or ethnic specificity. Genotyping results of CYP2C19 also revealed that there are different proportions of individual mutant alleles among ethnic populations. This may, in part, explains the interethnic difference in the metabolism of certain drugs (i.e. diazepam), though they were from the same CYP2C19 phenotype. Recently, our research group has studied the genotype and phenotype of CYP2C19 and found that the PM frequency (7∼8%) in Thais is lower than other Asian populations. Molecular and clinical impacts of this finding warrant to further investigation.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.73-78
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• 2012

We previously reported that the truncated Cel44C-$Man26A_{P558}$ ${\beta}$-glycosyl hydrolase protein exhibits multifunctional activities, including cellulase, xylanase, and lichenase. DNA shuffling of the truncated Cel44C-$Man26A_{P558}$ enzyme was performed to enhance the enzymatic activity of the multifunctional ${\beta}$-glycosyl hydrolase. Two mutant enzymes, M2Cel44C-$Man26A_{P558}$ that carries one mutation (P438A) and M21Cel44C-$Man26A_{P558}$ that carries two mutations (A273T and P438A) were obtained. The enzymatic activity of the M21Cel44C-$Man26A_{P558}$ double mutant was lower than enzymatic activity of the single mutant (M2Cel44C-$Man26A_{P558}$). However, both mutants displayed the enhancements in their enzyme activities that were ${\approx}1.3$- to 2.2-fold higher than the original enzymatic activity in Cel44C-$Man26A_{P558}$. In particular, the mutant M2Cel44C-$Man26A_{P558}$ exhibited an approximate 1.5- to 2.2-fold increase in the cellulase, xylanase, and lichenase activities in comparison with the control (Cel44C-$Man26A_{P558}$). The optimum cellulase, linchenase, and xylanase activities of ${\beta}$-glycosyl hydrolase were observed at pH 7.0, pH 7.0 and pH 6.0, respectively. These results, therefore, suggest that the amino acid residue Ala438 plays important roles in the enhancement of the activity of multifunctional ${\beta}$-glycosyl hydrolase.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• Animal Bioscience
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• v.34 no.5
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• pp.867-879
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• 2021

Objective: Fibronectin 3 (FN3) and immunoglobulin like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researches have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase belonging to glycosyl hydorlase (GH) family 5 were determined. Methods: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete open reading frames (ORFs). The alkaline, thermostability, tertiary structure of deduced enzymes were predicted by AcalPred, TBI software, Phyre2 and Swiss models. Then, whole and truncated forms of a selected gene were expressed in Escherichia coli and purified by His-tag affinity column for assessment of FN3 ability to enhance enzyme activity, solubility and conformation. Results: From 297 complete ORFs coding for cellulases, 148 sequences containing FN3, Ig were identified. Mostly FN3 appeared in 90.9% beta-glucosidases belonging to glycosyl hydrolase family 3 (GH3) and situated downstream of catalytic domains. The Ig was found upstream of 100% endoglucanase GH9. Rarely FN3 was seen to be situated downstream of X domain and upstream of catalytic domain endoglucanase GH5. Whole enzyme (called XFN3GH5 based on modular structure) and truncate forms FN3, XFN3, FN3GH5, GH5 were cloned in pET22b (+) and pET22SUMO to be expressed in single and fusion forms with a small ubiquitin-related modifier partner (S). The FN3, SFN3 increased GH5 solubility in FN3GH5, SFN3GH5. The SFN3 partly served for GH5 conformation in SFN3GH5, increased modules interaction and enzyme-soluble substrate affinity to enhance SXFN3GH5, SFN3GH5 activities in mixtures. Both SFN3 and SXFN3 did not anchor enzyme on filter paper but exfoliate and separate cellulose chains on filter paper for enzyme hydrolysis. Conclusion: Based on these findings, the presence of FN3 module in certain cellulases was confirmed and it assisted for enzyme conformation and activity in both soluble and insoluble substrate.

• Microbiology and Biotechnology Letters
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• v.41 no.1
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• pp.17-25
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is an attractive model organism for various fundamental studies, such as the genetic control of enzymes involved in methanol metabolism, peroxisome biogenesis, nitrate assimilation, and resistance to heavy metals and oxidative stresses. In addition, H. polymorpha has been highlighted as a promising recombinant protein expression host, especially due to the availability of strong and tightly regulatable promoters. In this study, we investigated the possibility of employing human serum albumin (HSA) as the fusion tag for the secretory expression of heterologous proteins in H. polymorpha. A set of four expression cassettes, which contained the methanol oxidase (MOX) promoter, translational HSA fusion tag, and the terminator of MOX, were constructed. The expression cassettes were also designed to contain sequences for accessory elements including His8-tag, $2{\times}(Gly_4Ser_1)$ linkers, tobacco etch virus protease recognition sites (Tev), multi-cloning sites, and strep-tags. To determine the effects of the size of the HSA fusion tag on the secretory expression of the target protein, each cassette contained the HSA gene fragment truncated at a specific position based on its domain structure. By using the Green fluorescence protein gene as the reporter, the properties of each expression cassette were compared in various conditions. Our results suggest that the translational HSA fusion tag is an efficient tool for the secretory expression of recombinant proteins in H. polymorpha.