A cDNA gene eglA6 encoding β-glucanase was cloned and the recombinant enzyme designated as AnEglA6 showed high thermal stability. The codon of eglA6 was optimized to artificially synthesize a gene. The codon-optimized gene eglA6b was expressed in Pichia pastoris. The average activity of recombinant yeast transformants with eglA6b was 1.8 times higher than those with eglA6. The recombinant AneglA6 was purified and characterized. The molecular weight of AneglA6 was approximately 52 ku. AneglA6 did not showed any activity when the substrate was polysaccharides composed of 1,3-glucoside bond-linked glucoses, such as yeast β-glucan. AneglA6 showed low specific activity of 1 645.7 U/mg when the substrate was polysaccharides containing only β-1,4-glucoside, such as CMC-Na. AneglA6 showed high activities of 12 450.6 U/mg when the substrate was barley glucan. The optimum temperature of AneglA6 was 75 ℃. The half-lives of AneglA6 in 75 ℃ and 70 ℃ were 1.9 h and 4.6 h, respectively. The optimum pH of AneglA was 4.0. Between pH 3.0-5.5, AneglA6 showed more than 50% of its maximum enzymatic activity. In conclusion, AnEglA6 is an endo-β-1,4-glucanase, which showed high activity and stability in high temperature and acid conditions.
HAN Bing
,
WANG Shi-lan
,
DE-qing-mei-duo
,
SHEN Wei
,
CHEN Xian-zhong
,
FAN You
. Gene cloning, expression and characterization analysis of a highly thermal stable β-glucanase gene from Aspergillus niger[J]. Food and Fermentation Industries, 2018
, 44(11)
: 55
-62
.
DOI: 10.13995/j.cnki.11-1802/ts.017330
[1] 熊涛,徐立荣,曾哲灵. β-葡聚糖酶的研究进展[J]. 四川食品与发酵, 2006, 42(6): 1-4.
[2] LI C H, WAN H R, YAN T R. Cloning, purification, and characterization of a heat-and alkaline-stable endoglucanase B from Aspergillus niger BCRC31494[J]. Molecules, 2012, 17(8): 9 774-9 789.
[3] 李永仙,谢焱,朱林江,等. 淀粉液化芽孢杆菌β-1,3-1,4-葡聚糖酶基因的克隆及表达[J]. 生物工程学报, 2009, 25(4): 542-548.
[4] 董自星,李伟国,佟新新,等. 黑曲霉内切β-1,3(4)-葡聚糖酶的基因克隆与酶学特性分析[J]. 食品与发酵工业, 2016, 42(11): 58-64.
[5] 夏许寒,朱成林,李诚. β-1,3-1,4-葡聚糖酶研究进展[J]. 食品科学, 2016, 37(19): 289-295.
[6] PHAM T H, QUYEN D T, NGHIEM N M, et al. Cloning, expression, purification, and properties of an endoglucanase gene (glycosyl hydrolase family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris[J]. Journal of Microbiology and Biotechnology, 2011, 21(10): 1 012-1 020.
[7] ZHAO Shu-miao, HUANG Jun, ZHANG Chang-yi, et al. High-level expression of an Aspergillus niger endo-β-1,4-glucanase in Pichia pastoris through gene codon optimization and synthesis[J]. Journal of Microbiology and Biotechnology, 2010, 20(3): 467-473.
[8] 黄君,张昌毅,赵述淼,等. 黑曲霉内切β-1,4-葡聚糖酶在毕赤酵母中的高效表达[J]. 华中农业大学学报, 2008, 27(5): 611-615.
[9] 韩学易,王春梅,陈惠. 米曲霉giF-10内切葡聚糖酶基因的克隆表达及酶学性质分析[J]. 食品与发酵工业, 2012, 38(10): 17-22.
[10] 郑海英,黄平,蔡少丽,等. 特异腐质霉内切葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质[J]. 微生物学通报, 2012, 39(2): 145-153.
[11] 王瑾,邬敏辰,周晨妍. 内切葡聚糖酶基因在大肠杆菌与毕赤酵母中的表达[J]. 生物技术通报, 2008, 24(3): 110-114.
[12] WANG Kun, LUO Hui-ying, BAI Ying-guo, et al. A thermophilic endo-1,4-β-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials[J]. Appllied Microbiology Biotechnology, 2014, 98(16): 7 051-7 060.
[13] HUA Chen-wei, YAN Qiao-juan, JIANG Zheng-qiang, et al. High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophile in Pichia pastoris[J]. Applied Microbiology and Biotechnology, 2010, 88(2): 509-518.
[14] 李一男,贾会勇,闫巧娟,等. 定向进化提高嗜热拟青霉J18耐热β-1,3-1,4-葡聚糖酶在酸性条件下的催化能力[J]. 生物工程学报, 2011, 27(12): 1 797-1 804.
[15] PEL H J, DE-WINDE J H, ARCHER D B, et al. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88[J]. Nature Biotechnology, 2007, 25 (2): 221-231.
[16] 王兵波,沈微,钱灵紫,等. 一种密码子优化的酸性普鲁兰酶基因在巴斯德毕赤酵母中的高效表达[J]. 食品与发酵工业, 2016, 42(7): 9-15.
[17] YAN Jun-jie, LIU Wei-dong, LI Yu-jie, et al. Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-β-endoglucanase[J]. Biochemical and Biophysical Research Communications, 2016, 475(1): 8-12.
[18] HONG J, TAMAKI H, AKIBA S, et al. Cloning of a gene encoding a highly stable endo-β-1,4-glucanase from Aspergillus niger and its expression in yeast[J]. Journal of Bioscience and Bioengineering, 2001, 92(5): 434-439.
[19] HARA Y, HINOKI Y, SHIMOI H, et al. Cloning and sequence analysis of endoglucanase genes from an industry fungus, Aspergillus kawachii[J]. Bioscience Biotechnology and Biochemistry, 2003, 67(9): 2 010-2 013.
[20] HONG J, WANG Yong-hong, KUMAGAI H, et al. Construction of thermotolerant yeast expressing thermostable cellulose genes[J]. Journal of Biotechnology, 2007, 130(2): 114-123.
[21] MURRAY P G, GRASSICK A, LAFFEY C D, et al. Isolation and characterization of a thermostable endo-β-glucanase active on 1,3-1,4-β-D-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70[J]. Enzyme and Microbial Technology, 2001, 29(1): 90-98.
