Studies on the extracellular enzymes from thermophilic actinomycete, Thermobifida fusca

• Main Authors: Chao-Hsun Yang, 楊昭順
• Other Authors: Wen-Hsiung Liu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/47147567665882293250

博士 === 國立臺灣大學 === 農業化學研究所 === 91 === Abstract The purpose of this study was to isolate a thermophilic actinomycete that could produce potent extracellular enzymes and to investigate the characteristics of xylanases, esterases and amylase. The results were as follows: From about one hundred compost soil samples collected in Taiwan, seventy strains of thermophilic actinomycetes were isolated by CYC GELRITE plates at 65℃. Among these newly isolated strains, Strain No. 22, which could produce potent extracellular lignocellulolytic enzymes and amylase, was selected. The taxonomical characteristics of Strain No. 22 were examined and it was identified as Thermobifida fusca NTU22. By using bagasse as carbon source, the activity of the lignocellulolytic enzymes that simultaneously accumulated in the culture broth from a 5-L fermentor after 48 h of cultivation at 50℃ were measured as xylanases (4.84 U/mL), cellulases (1.11 U/mL), esterases (0.097 U/mL), and peroxidases (0.093 U/mL). The optimum pH and temperature for xylanases were 6.0-8.0 and 70℃, respectively. Six bands of xylanase activities (pI 5.6 to 9.3) were identified on isoelectric focusing gel. The main product of xylanases from oat-spelt xylan was identified by HPLC as xylobiose. The gene coding for thermostable xylanase (xyl 11) from T. fusca NTU22 has been cloned in E. coli DH5a by pUC19. The xyl 11 gene encompassed 1014 nucleotides and coded for a single polypeptide consisting of 338 amino acids, with a calculated molecular weight of 36 kDa. The base composition of the xyl 11 coding sequence is 66% G+C. The xylanase from E. coli transformant pXYL11 was purified. It was found that the optimal pH and temperature of the E. coli transformant pXYL11 xylanase were similar to those of T. fusca NTU22 xylanases. After 2% of oat-spelt xylan was hydrolyzed by 10 U/mL of the purified xylanase from E. coli transformant pXYL11 at 60℃ and pH 7.0 for 10 h, the yield of xylooligosaccharides was about 30.5% containing 36.4% xylobiose, 33.0% xylotriose and 30.6% xylotetrose. Xylooligosaccharides were recovered by active charcoal chromatography. The recovery of xylooligosaccharides was about 95% and the purity was about 70.2%. The main T. fusca NTU22 esterase in the fermentation broth was purified 51-fold through successive purification steps involving ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified esterase was 14.4%. The molecular weight of purified esterase was estimated to be 28 kDa by Sepharose CL-6B column chromatography and the subunit molecular weight estimated by SDS-PAGE was 30 kDa. It was suggested that the purified esterase from T. fusca NTU22 was a monomer. The pI value of the purified esterase was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimal pH and temperature for the purified esterase was 8.0 and 80℃, respectively. The purified esterase activity was inhibited by Zn2+, Hg2+, PMSF and DIPF. The Km values for p-nitrophenyl acetate and acetylxylan were 1.86mM and 0.15%, respectively. Response surface methodology was used to investigate the optimum medium composition for production of amylase by T. fusca NTU22. After cultured in the optimum medium obtained from response surface methodology which consisted of 20.0 g/L starch, 1.0 g/L yeast extract, 4.0 g/L ammonium sulfate and 13.6 g/L KH2PO4, pH 9.0 by shaken culture at 50℃ for 24 h, the maximum amylase activity produced by T. fusca NTU22 was 6.20 U/mL. The maximum amylase activity of T. fusca NTU22 was 9.40 U/mL after cultured at 50℃ for 60 h under the following fermentation conditions in a 5-L fermentor: working volume 3 L medium, agitation speed 20 rpm, and aeration rate 1 vvm. The amylase from T. fusca NTU22 was purified 7.21-fold through successive purification steps involving ammonium sulfate fractionation, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified amylase was 22.0%. The molecular weight of purified amylase was estimated to be 60 kDa by Sepharose CL-6B column chromatography and the subunit molecular weight estimated by SDS-PAGE was 64 kDa. It was suggested that the amylase from T. fusca NTU22 was a monomer. The pI value of the purified amylase was estimated to be 4.55 by isoelectric focusing gel electrophoresis. The optimal pH and temperature for the purified amylase was 7.0 and 60℃, respectively. The amylase activity was inhibited by Ag+, Hg2+. The Km and Vmax for the purified amylase were determined to be 0.088% and 0.0345 U/mg protein, respectively, using starch as a substrate. The main product from starch was identified by HPLC as maltotriose. The gene coding for amylase (amy 13) from T. fusca NTU22 has been cloned in E. coli DH5a by pUC19. The amy 13 gene encompassed 1815 nucleotides and coded for a single polypeptide consisting of 605 amino acids, with a calculated molecular weight of 65 kDa. The base composition of the amy 13 gene coding sequence is 69% G＋C. The similarities of amylase gene from T. fusca NTU22 were 97.5% to T. curvata CCM3352, 48.0% to S. venezuelae and 46.0% to S. limosus. The amylase from E. coli transformant pAMY13H8 was purified. It was found that the optimal pH and temperature of the E. coli transformant pAMY13H8 amylase were similar to those of T. fusca NTU22 amylase.