Papers by Sutipa Tanapongpipat
Lipases and esterases are used as catalysts in synthetic organic chemistry. Most of the industria... more Lipases and esterases are used as catalysts in synthetic organic chemistry. Most of the industrial processes in which the enzymes are employed require high temperatures. Therefore, the hot spring soil sample is an attractive resource for microorganisms producing thermotolerant lipolytic enzymes. In this study, the metagenomic library of Jae Sawn hot spring was constructed and screened for lipolytic activity using both triolein and tributyrin as substrates. Among 36,000 transformants, two colonies showed lipolytic activity by producing clear halo zone around the colony. Blast search results revealed that one clone was found to encode esterase, having 36% amino acid sequence identity to an esterase of Rhodobacter sphaeroides and the other clone was found to encode a phospholipase, having 39% homology to the phospholipase of Carboxydothermus hydrogenoformans
Lipases are used as catalysts in synthetic organic chemistry and various important industries. In... more Lipases are used as catalysts in synthetic organic chemistry and various important industries. In this study, a lipase containing lipolytic activity (capable of using both triolein and tributyrin as substrates and producing clear halo zone around the colony) was isolated from 36,000 transformants of the metagenomic library of Jae-sawn hot spring. The identified lipase has predicted molecular weight of 32 kDa and is encoded by a gene with 876 nucleotides. The enzyme contains 39% homology to the phospholipase of Carboxydothermus hydrogenoformans. This phospholipase
Abstract: Geranylgeraniol (GGOH) is a potent apoptotic inducer in various cancer cells lines such... more Abstract: Geranylgeraniol (GGOH) is a potent apoptotic inducer in various cancer cells lines such as human lung adenocarcinoma cell, colon cancer cell, etc1. It is also only one intermediate of the biosynthetic pathway of plaunotol, an anti ulcer drug from Croton stellatopilosus 2. However low yield and mixture of terpenoids were obtained from recombinant E. coli harboring PDPase alone and chemical synthesized of this compound. To overcome these problems we have used pathway engineering approach for reconstruction of GGOH biosynthetic pathway in E.coli BL21 (DE3) RIPL to increase its GGOH accumulation. Therefore, the recombinant plasmid pETDuet-1 harboring both genes encoding two enzymes involved in GGOH biosynthetic pathway was constructed. The first gene is geranylgeranyl diphosphate synthase (GGPPS) which is responsible for rate determining step of GGPP biosynthesis in E. coli3. The last gene is prenyl diphosphate phosphatase (PDPase) that catalyzes de-phosphorylation of GGPP yie...
Geranylgeraniol (GGOH) is a potent apoptotic inducer in various cancer cells lines and it is also... more Geranylgeraniol (GGOH) is a potent apoptotic inducer in various cancer cells lines and it is also the only one intermediate of plaunotol biosynthesis pathway, an antiulcer drug derived Croton stellatopilosus Ohba (Plau-noi). At present, the production of GGOH by means of chemical synthesis has been in low yield and containing the racemic mixtures. Therefore, the study on gene involved in GGOH biosynthesis pathway has been performed in our group in order to produce GGOH in microorganism systems. From previous studies, cloning and expression of prenyldiphosphate phosphatase (PDPase) from young leaves of C. stellatopilosus was operated in E. coli BL21 (DE3) RIPL yielded very low level of PDPase. Therefore, we changed the expression system into Pichia pastoris. We found that the PDPase still could not express as extracellular proteins. The investigation of mRNA secondary structure formation by using Genebee program showed highly gibbs free energy of their structures (-35.4 to - 46.8 kca...
Journal of Polymers and the Environment, 2014
The high production cost of bio-based plastic polyhydroxyalkanoates (PHAs) limits their use as co... more The high production cost of bio-based plastic polyhydroxyalkanoates (PHAs) limits their use as commercial products. Thus, systems for PHAs production from waste substrates could reduce production costs. Crude glycerol is a by-product of biodiesel fuel production and thus represents an inexpensive, abundant and promising carbon source for production of valorized fermentation products. In this study, industrial crude glycerol by-product from palm oil biodiesel production was used as the carbon source for production of PHAs by recombinant Escherichia coli. Crude glycerol supplemented at 1-5 % (v/v) supported production of poly(3-hydroxybutyrate) (P(3HB)) in E. coli-ABC Ah , which harbors the PHA synthetic genes for b-ketothiolase (PhaA Re ), acetoacetyl-CoA reductase (PhaB Re ) of Ralstonia eutropha and Polyhydroxyalkanoate (PHA) synthase (PhaC Ah ) of Aeromonas hydrophila. The highest P(3HB) content and productivity of 14 wt% of cell dry weight and 0.6 g/L, respectively, were obtained at 1 % (v/v) glycerol concentration. Production of P(3HB-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) was achieved using E. coli-ABC Ah J Ah , harboring genes for PhaA Re , PhaB Re , PhaC Ah , and the (R)-specific enoyl-CoA hydratase (PhaJ Ah ) of A. hydrophila. This led to the copolymer content of 3 wt% of cell dry weight with 1 mol% of 3HHx. Molecular weight and degradation temperature of the polymers were in the range of 110-130 kDa and 295-299°C, respectively. These results indicated that crude glycerol could be an attractive carbon source for economical production of PHAs with properties for industrial application.
Journal of Fluorescence, 2014
Highly water soluble polymer (DD) was prepared and evaluated for its fluorescence response toward... more Highly water soluble polymer (DD) was prepared and evaluated for its fluorescence response towards various amino acids. The polymer consists of dansyl hydrazine unit conjugated into dextran template. The conjugation enhances higher water solubility of dansyl hydrazine moiety. Of screened amino acids, DD exhibited selective fluorescence quenching in the presence of aspartic acid (Asp) and glutamic acid (Glu). A plot of fluorescence intensity change of DD against the concentration of corresponding amino acids gave a good linear relationship in the range of 1 × 10(-4) M to 25 × 10(-3) M. This establishes DD as a potential polymeric sensor for selective sensing of Asp and Glu.
Bioscience, Biotechnology, and Biochemistry, 2013
Biologia, 2009
A thermophilic Streptomyces sp. capable of degrading various aliphatic polyesters was isolated fr... more A thermophilic Streptomyces sp. capable of degrading various aliphatic polyesters was isolated from a landfill site. The isolate, Streptomyces sp. BCC23167, demonstrated rapid aerobic degradation of several polyesters, including polyhydroxyalkanoate copolymers, poly(ε-caprolactone) and polybutylene succinate at 50 • C and neutral pH. The degrading activity was repressed by glucose and cellobiose, but tolerant to repression by other carbon substrates. Degradation of a commercial poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHx) by Streptomyces sp. BCC23167 progressed from surface to bulk as suggested by the slight decrease in polymer molecular weight. Differential scanning calorimetry analysis of PHBHx film degradation by Streptomyces sp. BCC23167 showed that relative crystallinity of the film increased slightly in the early stage of degradation, followed by a marked decrease later on. The surface morphology of degraded films was analyzed by scanning electron microscopy, which showed altered surface structure consistent with the changes in crystallinity. The isolate is thus of potential for application in composting technology for bio-plastic degradation.
Bioscience Biotechnology and Biochemistry, Jun 23, 2012
Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degradi... more Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.
Fems Microbiology Letters, Apr 1, 2003
Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Ent... more Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Enterobacter amnigenus strain An11, a potential host for mosquito control, using in vivo recombination via homologous recombination (An11S4: :6). We now report the successful transfer of two mosquito-larvicidal genes, cry4B from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus, into the host genome. To facilitate the screening procedure, the E. amnigenus derivative, An11S4: :6, was used as a host. The integration of both toxin genes by two successive crossover events interrupted the 6 region yielding two integrants designated An11S4: :cry4B and An11S4: :6: :bin, respectively. Differences in the integration efficiency of these toxin genes were observed. The presence of both genes in the target sites of the host genome was verified by PCR. Cry4B was expressed weakly from An11S4: :cry4B, but no expression of the binary toxin gene could be detected from An11S4: :6: :bin. Nevertheless, these two integrants exhibited mosquitolarvicidal activity against Aedes and Culex, suggesting that both proteins were expressed, but at very low levels.
Applied biochemistry and biotechnology, Jan 30, 2015
The thermotolerant methylotrophic yeast Ogataea thermomethanolica is a host for heterologous prot... more The thermotolerant methylotrophic yeast Ogataea thermomethanolica is a host for heterologous protein expression via secretion to the culture medium. Efficient secretion is a major bottleneck for heterologous protein production in this strain. To improve protein secretion, we explored whether the use of a native signal peptide sequence for directing heterologous protein secretion and overexpression of native ER-resident chaperone genes could improve heterologous protein secretion in O. thermomethanolica. We cloned and characterized genes encoding α-mating factor (Otα-MF) and ER-resident chaperones OtBiP, OtCNE1, and OtPDI. The pre and pre-pro sequences of Otα-MF were shown to promote higher secretion of heterologous endoxylanase comparing with the classical pre-pro sequence of Saccharomyces cerevisiae. However, in the case of heterologous glycosylated phytase, only the Otα-MF pre-pro sequence significantly enhanced protein secretion. The effect of chaperone overexpression on heterolo...
Applied Biochemistry and Biotechnology, 2015
Scheffersomyces stipitis strain BCC15191 is considered as a biotechnologically valuable yeast for... more Scheffersomyces stipitis strain BCC15191 is considered as a biotechnologically valuable yeast for its ability to ferment glucose and xylose, the main sugar components in plant biomass, to ethanol. However, the wild strain lacks of endogenous cellulases and hemicellulases that limited biomass utilization. In order to improve biomass degrading ability of S. stipitis BCC15191, new integrative plasmids harboring constitutive TEF1 promoter and codon-optimized zeocin or hygromycin antibiotic resistance genes were developed. Aspergillus niger endoxylanase and Aspergillus aculeatus endoglucanase activities were demonstrated in transformant cells expressing codon-optimized genes. S. stipitis co-expressing endoxylanase and endoglucanase was able to grow in medium containing xylan and β-glucan as carbon sources and directly produced ethanol with yields of 2.7 g/L. It could also use pretreated corncob as a carbon source for ethanol production. These results suggested that recombinant S. stipilis is possible for consolidated bioprocessing of biomass.
Partial gene encoding a conserved sequence of glycoside hydrolase family 5 was amplified from a m... more Partial gene encoding a conserved sequence of glycoside hydrolase family 5 was amplified from a metagenomic DNA of bovine ruminal bacteria by PCR using a set of degenerate primers. The PCR product of approximate 850 bp was obtained, cloned into the pTZ57R/T vector and transformed into Escherichia coli. Genome walking PCR strategies were applied to amplify the unknown regions of the partial gene. The obtained full-length beta-endoglucanase gene, named cel5, consisted of 1590 bp which encoded a putative protein of 530 amino acids. The amino acid sequence of Cel5 shared 44%, 42%, 41%, 41% and 37% identity with the sequences of the glycoside hydrolase family 5 members; Flavobacterium johnsoniae (YP_001193127), Saccharophagus degradans (YP_525801), Bacteroides uniformis (ZP_2068920), uncultured symbiotic protist of Hodoternopsis sjoestedti (BAF57333) and Saccharophagus degradans (YP_528108) respectively. This result indicated that the isolated beta-endoglucanase gene, cel5, is a new memb...
Biotechnology and Bioprocess Engineering, 2015
ABSTRACT Control of the methanol supply during the production stage is a crucial parameter for ma... more ABSTRACT Control of the methanol supply during the production stage is a crucial parameter for maintaining cell growth and enhancing recombinant protein production by Pichia pastoris. In this study, optimization of the initial specific methanol supply rate was explored for high cell density fed-batch cultivation of recombinant fungal β- glucosidase in a slow methanol utilization strain (MutS), P. pastoris KM71. By varying the methanol feed rates for initiating the induction at a cell concentration of 60 g/L, the optimum initial specific methanol supply rate was determined to be 30.34 ± 0.34 mg/g·h. A methanol feed rate was proposed according to this optimum parameter and applied for the production of recombinant β-glucosidase at the higher cell concentrations of 80 and 100 g/L. The highest recombinant β-glucosidase activity obtained was 2851.7 ± 14.6 U/mL, which was four times higher than that obtained with the reference condition (40 g/L initial cell concentration). The success of this approach suggests that the strategy of optimizing the initial specific methanol supply rate could be adopted and applied for the production of other recombinant proteins in P. pastoris employing a methanol inducible system.
The Biochemical journal, 1998
The eight ccm genes located at minute 47 on the Escherichia coli chromosome, in the order ccmABCD... more The eight ccm genes located at minute 47 on the Escherichia coli chromosome, in the order ccmABCDEFGH, encode homologues of proteins which are essential for cytochrome c assembly in other bacteria. The ccm genes are immediately downstream from the napFDAGHBC genes encoding a periplasmic nitrate reductase. CcmH was previously shown to be essential for cytochrome c assembly. Deletion analysis and a two-plasmid strategy have now been used to demonstrate that CcmA, B, D, E, F and G are also essential for cytochrome c assembly, and hence for cytochrome-c-dependent nitrite reduction. The ccm genes are transcribed from a ccmA promoter located within the adjacent gene, napC, which is the structural gene for a 24 kDa membrane-bound c-type cytochrome, NapC. Transcription from this ccmA promoter is induced approximately 5-fold during anaerobic growth, independently of a functional Fnr protein: it is also not regulated by the ArcB-ArcA two-component regulatory system. The ccmA promoter is an ex...
FEMS Microbiology Letters
This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heter... more This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heterologous expression. Both methanol‐inducible alcohol oxidase (AOX1) and constitutive glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoters from Pichia pastoris were shown to drive efficient gene expression in this host. Recombinant phytase and xylanase were expressed from both promoters as secreted proteins, with the former showing different patterns of N‐glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N‐glycan modification of cell wall mannoproteins. The major glycoprotein oligosaccharide species produced from P. thermomethanolica BCC16875 is Man8‐12GlcNAc2, which is similar to that from other methylotrophs. Moreover, mannosylphosphate and α‐1,6‐ and α‐1,2‐linked mannose modifications of heterologous secreted protein were also detected. The attainably high level of protein production in complement to distinctive th...
Protein Expression and Purification, 2008
Protein Expression and Purification, 2008
A gene encoding a cellobiohydrolase (CBHI) was isolated from Fusicoccum sp. (BCC4124), an endophy... more A gene encoding a cellobiohydrolase (CBHI) was isolated from Fusicoccum sp. (BCC4124), an endophytic fungus belongs in phylum Ascomycota, using 5 0 and 3 0 rapid amplification of cDNA end (RACE) technique. This CBHI gene contains 1395 nucleotides and encodes a 465-amino acid protein with a molecular weight of approximately 50 kDa. The deduced amino acid sequence showed significant similarity to those of other fungal CBHI belonging to family 7 of glycosyl hydrolase. Interestingly, the result from the amino acid alignment revealed that this CBHI does not contain the cellulose binding domain nor the linker region. The CBHI gene was successfully expressed in Pichia pastoris KM71. The purified recombinant CBHI has ability to hydrolyze Avicel, filter paper and 4-methylumbelliferyl b-D-cellobioside (MUC) but not carboxymethylcellulose (CMC). It showed an optimal working condition at 40°C, pH 5 with K m and V max toward MUC of 0.57 mM and 3.086 nmol/min/mg protein, respectively. The purified enzyme was stable at pH range of 3-11. The enzyme retained approximately 50% of its maximal activity after incubating at 70-90°C for 30 min. Due to its stability through wide range of pH, and moderately stable at high temperature, this enzyme has potential in various biotechnology applications.
Protein Expression and Purification, 2010
A mature phytase cDNA, encoding 441 amino acids, from Eupenicillium parvum (BCC17694) was cloned ... more A mature phytase cDNA, encoding 441 amino acids, from Eupenicillium parvum (BCC17694) was cloned into a Pichia pastoris expression vector, pPICZaA, and was successfully expressed as active extracellular glycosylated protein. The recombinant phytase contained the active site RHGXRXP and HD sequence motifs, a large a/b domain and a small a-domain that are typical of histidine acid phosphatase. Glycosylation was found to be important for enzyme activity which is most active at 50°C and pH 5.5. The recombinant phytase displayed broad substrate specificity toward p-nitrophenyl phosphate, sodium-, calcium-, and potassium-phytate. The enzyme lost its activity after incubating at 50°C for 5 min and is 50% inhibited by 5 mM Cu 2+ . However, the enzyme exhibits broad pH stability from 2.5 to 8.0 and is resistant to pepsin. In vitro digestibility test suggested that BCC17694 phytase is at least as effective as another recombinant phytase (r-A170) which is comparable to Natuphos, a commercial phytase, in releasing phosphate from corn-based animal feed, suggesting that BCC17694 phytase is suitable for use as phytase supplement in the animal diet.
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Papers by Sutipa Tanapongpipat