Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 92 === The purpose of this study is using the PCR and host-vector system of E. coli DH5?and pUC18 to clone the β-glucosidase gene from thermophilic actinomycete Thermobifida fusca NTU22. After sequencing analysis, the bgl gene encompassed 1,452 nucleotides and code f...

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Main Authors: Yo-Ming Yang, 楊祐銘
Other Authors: Wen-Hsiung Liu
Format: Others
Language:zh-TW
Published: 2004
Online Access:http://ndltd.ncl.edu.tw/handle/64975353090229931015
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spelling ndltd-TW-092NTU053810082016-06-10T04:15:43Z http://ndltd.ncl.edu.tw/handle/64975353090229931015 Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli ThermobifidafuscaNTU22β-葡萄糖苷酶基因在大腸桿菌中之表現 Yo-Ming Yang 楊祐銘 碩士 國立臺灣大學 微生物與生化學研究所 92 The purpose of this study is using the PCR and host-vector system of E. coli DH5?and pUC18 to clone the β-glucosidase gene from thermophilic actinomycete Thermobifida fusca NTU22. After sequencing analysis, the bgl gene encompassed 1,452 nucleotides and code for a polypeptide consisting of 484 amino acid. The base composition of the bgl coding sequence is 69% G+C. The calculated molecular mass of the predicted protein was 53 kDa and with an estimated pI of 4.82 by BLAST Pstd. The derived amino acid sequence of BGL showed 99%, 64% and 60% similarity to that for Thermobifida fusca YX, Strptomyces sp. and Thermotoga maritima, respectively. The bgl gene was overexpression in E. coli BL21(DE3) using the pET32, T7-phage RNA polymerase system with the enzyme activities 2.5 U/mL. The fusion recombinant protein was purified by Ni-affinity gel, and the subunit molecular weight estimated by SDS-PAGE was 79 kDa. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent Km values for pNPG and cellobiose were 0.5 mM and 14.8 mM, Vmax were 0.43 U/μg and 3.7 U/μg, respectively. The enzyme was optimally active at pH 6 and 60 degrees C and was shown to retain 90% activity after incubation for 3 h at 50 degrees C. BGL was inhibited by Ag+, Ba2+, Hg2+, Cu2+, iodoacetate, PCMB and SDS. After isoflavones from methanol extract of soybean meal was hydrolyzed by the purified recombinant BGL at 50 degrees C for 10 min, the molar conversion rate of daidzin and genistin were 56.2% and 94.0%, respectively. Furthermore, the production process of BGL from E. coli BL21 (DE3) was developed and the medium composition was optimized with response surface mtheodology (RSM). The four factors selected on basal medium were glucose, yeast extract, diaminonium hydrogen phosphate and pH. Highest activity yields were obtained in the composition of 1% glucose, 1% yeast extract, 1% diaminonium hydrogen phosphate, and pH 8.79. The optimal medium allowed enzyme yield to be 1.8 folds compared to M9 medium after cultured in Hinton flask for 16 h. The maximum BGL activity (6.0 U/mL) was achieved when a cultivation was carried out with 2 L RSM medium, 1 vvm of aeration volume and 300 rpm of agitation rate in a 5-L fermentor. Wen-Hsiung Liu 劉文雄 2004 學位論文 ; thesis 115 zh-TW
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language zh-TW
format Others
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description 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 92 === The purpose of this study is using the PCR and host-vector system of E. coli DH5?and pUC18 to clone the β-glucosidase gene from thermophilic actinomycete Thermobifida fusca NTU22. After sequencing analysis, the bgl gene encompassed 1,452 nucleotides and code for a polypeptide consisting of 484 amino acid. The base composition of the bgl coding sequence is 69% G+C. The calculated molecular mass of the predicted protein was 53 kDa and with an estimated pI of 4.82 by BLAST Pstd. The derived amino acid sequence of BGL showed 99%, 64% and 60% similarity to that for Thermobifida fusca YX, Strptomyces sp. and Thermotoga maritima, respectively. The bgl gene was overexpression in E. coli BL21(DE3) using the pET32, T7-phage RNA polymerase system with the enzyme activities 2.5 U/mL. The fusion recombinant protein was purified by Ni-affinity gel, and the subunit molecular weight estimated by SDS-PAGE was 79 kDa. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent Km values for pNPG and cellobiose were 0.5 mM and 14.8 mM, Vmax were 0.43 U/μg and 3.7 U/μg, respectively. The enzyme was optimally active at pH 6 and 60 degrees C and was shown to retain 90% activity after incubation for 3 h at 50 degrees C. BGL was inhibited by Ag+, Ba2+, Hg2+, Cu2+, iodoacetate, PCMB and SDS. After isoflavones from methanol extract of soybean meal was hydrolyzed by the purified recombinant BGL at 50 degrees C for 10 min, the molar conversion rate of daidzin and genistin were 56.2% and 94.0%, respectively. Furthermore, the production process of BGL from E. coli BL21 (DE3) was developed and the medium composition was optimized with response surface mtheodology (RSM). The four factors selected on basal medium were glucose, yeast extract, diaminonium hydrogen phosphate and pH. Highest activity yields were obtained in the composition of 1% glucose, 1% yeast extract, 1% diaminonium hydrogen phosphate, and pH 8.79. The optimal medium allowed enzyme yield to be 1.8 folds compared to M9 medium after cultured in Hinton flask for 16 h. The maximum BGL activity (6.0 U/mL) was achieved when a cultivation was carried out with 2 L RSM medium, 1 vvm of aeration volume and 300 rpm of agitation rate in a 5-L fermentor.
author2 Wen-Hsiung Liu
author_facet Wen-Hsiung Liu
Yo-Ming Yang
楊祐銘
author Yo-Ming Yang
楊祐銘
spellingShingle Yo-Ming Yang
楊祐銘
Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli
author_sort Yo-Ming Yang
title Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli
title_short Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli
title_full Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli
title_fullStr Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli
title_full_unstemmed Expression of β-Glucosidase Gene from Thermobifida fusca NTU22 in Escherichia coli
title_sort expression of β-glucosidase gene from thermobifida fusca ntu22 in escherichia coli
publishDate 2004
url http://ndltd.ncl.edu.tw/handle/64975353090229931015
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