Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter
Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter
复制标题
DOI：
10.1016/j.jbiosc.2019.08.002
复制
发表时间：
2020-02-01
影响因子：
2.8
通讯作者：
Kosekit, Takuya
中科院分区：
文献类型：
--
作者：
Ichikawa, Kyotaro;Shiono, Yoshihito;Kosekit, Takuya
在改良的糖化酶基因启动子PglaA142的控制下，来自米曲霉RIB40的一个编码单宁酶的基因AotanB在源自工业菌株的米曲霉AOK11niaD缺陷型突变体中过表达。这种重组单宁酶被命名为rAoTanBO，作为一种有活性的胞外酶被高效生产。纯化的rAoTanBO在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中显示出分子量约为80 - 100 kDa的弥散条带。经内切 - β - N - 乙酰氨基葡萄糖苷酶H处理后，rAoTanBO的分子量为65 kDa。纯化的rAoTanBO在30 - 35℃和pH 6.0时表现出最大活性。纯化的rAoTanBO对天然和人工底物的单宁酶活性比巴斯德毕赤酵母生产的重组酶（被命名为rAoTanBP）高2 - 8倍。成熟的rAoTanBP的N端比成熟的rAoTanBO的N端多6个氨基酸。动力学分析表明，rAoTanBO比rAoTanBP具有更高的催化效率（kcat / Km）。rAoTanBO在高达60℃时稳定，且比rAoTanBP具有更高的热稳定性。N - 连接寡糖对rAoTanBO和rAoTanBP的活性和稳定性没有影响。（C）2019，日本生物技术学会。保留所有权利。
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-beta-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30-35 degrees 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 (k(cat)/K-m) than rAoTanBP. rAoTanBO was stable up to 60 degrees C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.