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Treerattrakool, Supattra,Eurwilaichitr, Lily,Udomkit, Apinunt,Panyim, Sakol Korean Society for Biochemistry and Molecular Biol 2002 Journal of biochemistry and molecular biology Vol.35 No.5
The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae $\alpha$-factor or Pem-CMG was employed. The results demonstrated that ${\alpha}Pem$-CMG, either with (${\alpha}2EACMG$) or without (${\alpha}CMG$) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either ${\alpha}2EACMG$ or ${\alpha}CMG$ was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both ${\alpha}2EACMG$ and ${\alpha}CMG$ was correctly processed. This produced the mature Pem-CMG peptide.
(Sittiruk Roytrakul),(Lily Eurwilaichitr),(Chittiwat Suprasongsin),(Sakol Panyim) 생화학분자생물학회 2001 BMB Reports Vol.34 No.6
A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N. terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.
(Supattra Treerattrakool),(Lily Eurwilaichitr),(Apinunt Udomkit),(Sakol Panyim) 생화학분자생물학회 2002 BMB Reports Vol.35 No.5
The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae α-factor or Pem-CMG was employed. The results demonstrated that αPem-CMG, either with (α2EACMG) or without (αCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either α2EACMG or αCMG was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both α2EACMG and αCMG was correctly processed. This produced the mature Pem-CMG peptide.
Roytrakul, Sittiruk,Eurwilaichitr, Lily,Suprasongsin, Chittiwat,Panyim, Sakol Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.6
A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.
( Surisa Suwannarangsee ),( Jantima Arnthong ),( Lily Eurwilaichitr ),( Verawat Champreda ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.10
Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is a key step in the conversion of agricultural by-products to biofuels and value-added chemicals. Utilization of a robust microorganism for on-site production of biomass-degrading enzymes has gained increasing interest as an economical approach for supplying enzymes to biorefinery processes. In this study, production of multi-polysaccharide-degrading enzymes from Aspergillus aculeatus BCC199 by solid-state fermentation was improved through the statistical design approach. Among the operational parameters, yeast extract and soybean meal as well as the nonionic surfactant Tween 20 and initial pH were found as key parameters for maximizing production of cellulolytic and hemicellulolytic enzymes. Under the optimized condition, the production of FPase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase was achieved at 23, 663, 88, 1,633, and 90 units/g of dry substrate, respectively. The multi-enzyme extract was highly efficient in the saccharification of alkaline-pretreated rice straw, corn cob, and corn stover. In comparison with commercial cellulase preparations, the BCC199 enzyme mixture was able to produce remarkable yields of glucose and xylose, as it contained higher relative activities of β-glucosidase and core hemicellulases (xylanase and β-xylosidase). These results suggested that the crude enzyme extract from A. aculeatus BCC199 possesses balanced cellulolytic and xylanolytic activities required for the efficient saccharification of lignocellulosic biomass feedstocks, and supplementation of external β-glucosidase or xylanase was dispensable. The work thus demonstrates the high potential of A. aculeatus BCC199 as a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry.
( Asano Krisana ),( Sriprang Rutchadaporn ),( Gobsuk Jarupan ),( Eurwilaichitr Lily ),( Tanapongpipat Sutipa ),( Kirtikara Kanyawim ) 생화학분자생물학회 2005 BMB Reports Vol.38 No.1
During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of 55℃. When tested using xylan from birchwood, it showed K_(m) and V_(max) values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by CuSO₄, EDTA, and by FeSO₄. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-β-xylanase. The full-length gene encoding endo-1,4-β-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-a^-xylanase B previously identified in A. niger.
Krisana, Asano,Rutchadaporng, Sriprang,Jarupan, Gobsuk,Lily, Eurwilaichitr,Sutipa, Tanapongpipat,Kanyawim, Kirtikara Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.1
During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.
( Weerachavangkul Chawannapak ),( Thanaporn Laothanachareon ),( Katewadee Boonyapakron ),( Sarunyou Wongwilaiwalin ),( Thidarat Nimchua ),( Lily Eurwilaichitr ),( Kusol Pootanakit ),( Yasuo Igarashi ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.12
Enzymatic pre-bleaching by modification of pulp fibers with xylanases is an attractive approach to reduce the consumption of toxic bleaching chemicals in the paper industry. In this study, an alkaliphilic endoxylanase gene was isolated from metagenomic DNA of a structurally stable thermophilic lignocellulose-degrading microbial consortium using amplification with conserved glycosyl hydrolase family 10 primers and subsequent genome walking. The full-length xylanase showed 78% sequence identity to an endo-β-1,4-xylanase of Clostridium phytofermentans and was expressed in a mature form with an N-terminal His6 tag fusion in Escherichia coli. The recombinant xylanase Xyn3F was thermotolerant and alkaliphilic, working optimally at 65-70oC with an optimal pH at 9- 10 and retaining >80% activity at pH 9, 60oC for 1 h. Xyn3F showed a Vmax of 2,327 IU/mg and Km of 3.5 mg/ml on birchwood xylan. Pre-bleaching of industrial eucalyptus pulp with no prior pH adjustment (pH 9) using Xyn3F at 50 IU/g dried pulp led to 4.5-5.1% increase in final pulp brightness and 90.4-102.4% increase in whiteness after a single-step hypochlorite bleaching over the untreated pulp, which allowed at least 20% decrease in hypochlorite consumption to achieve the same final bleaching indices. The alkaliphilic xylanase is promising for application in an environmentally friendly bleaching step of kraft and soda pulps with no requirement for pH adjustment, leading to improved economic feasibility of the process.
( Thidarat Nimchua ),( Taksawan Thongaram ),( Tanaporn Uengwetwanit ),( Somchai Pongpattanakitshote ),( Lily Eurwilaichitr ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.4
A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from 50 o C to 55 o C. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.