毕赤酵母
单宁酶
米曲霉
热稳定性
分子质量
生物化学
重组DNA
酶动力学
化学
酶
凝胶电泳
细胞外
分子克隆
生物
突变体
分子生物学
基因
肽序列
活动站点
抗氧化剂
没食子酸
作者
Kyotaro Ichikawa,Yoshihito Shiono,Tomoko Shintani,Akira Watanabe,Hiroshi Kanzaki,Katsuya Gomi,Takuya Koseki
标识
DOI:10.1016/j.jbiosc.2019.08.002
摘要
A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80–100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-β-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30–35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2–8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.
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