通过RT-PCR克隆、鉴定了黑曲霉中编码1种葡聚糖酶基因eglA6,所编码的重组酶蛋白命名为AneglA6。通过密码子优化提高了AneglA6在巴斯德毕赤酵母中的表达水平,对重组酶进行纯化并研究酶学性质,重组酶表观分子量约为52 ku。以大麦葡聚糖为底物时,重组酶比酶活为12 450.6 U/mg;以CMC-Na为底物时,酶活是1 645.7 U/mg;以酵母葡聚糖等β-1,3-葡聚糖为底物时,重组酶未表现出明显的活力。重组酶最适温度为75 ℃,在75℃和70 ℃下半衰期分别为1.9和4.6 h。AneglA6最适pH为4.0,在pH 3.0～5.5范围内具有酶活力的50%以上。AneglA6是一种在酸性和高温条件下有较高活力和稳定性的耐热内切-β-1,4-葡聚糖酶。
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.
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