一株全基因合成耐热甘露聚糖酶的表达及酶学性质分析

Gene Synthesis,Expression and Characterization of a Thermostable Endo-β-1,4-mannanase

根据已知耐热甘露聚糖酶ManAd3氨基酸序列与毕赤酵母密码子使用偏爱性,设计并合成了甘露聚糖酶ManA全基因(Accession No.KJ806637),与表达载体pPIC9k重组后,转化毕赤酵母GS115,筛选获得重组菌株ManA-GS115。该重组菌株发酵产物经SDS-PAGE鉴定,其中甘露聚糖酶ManA含量达到电泳纯级别,分子量大小约为30 kDa。其酶学性质检测结果显示该酶最适反应温度为75℃,最适反应pH为6.0,比活力高达3200 IU/mg,并且在75℃下处理30 min仍能维持90%以上相对酶活力。该甘露聚糖酶ManA表达量较高,在偏酸性环境下仍能够维持较高的相对酶活力,且热稳定性显著,可广泛应用于食品、酿造、饲料、纺织和医药等工业领域。

According to the known thermostable mannanase ManAd3 sequence and the codon bias of yeast,the optimized mannanase ManA sequence（ Accession No. KJ806637） was designed and synthesized. The ManA gene was cloned into the pPIC9 k vector,and then expressed in Pichia pastoris GS115 to obtain a recombinant strain ManA-GS115. The fermentation product of mannanase ManA was electrophoresis pure by SDS-PAGE identification with the molecular weight about 30 kDa. The enzymatic properties were shown that the optimal reaction temperature and pH value were 75℃ and pH6. 0 respectively,and the specific activity was up to 3 200 IU /mg. Moreover,the residual activity was above 90% after 30 min treatment under 75℃,pH 6. 0. With high expression,excellent thermostability,and the good thermostable tolerance under acidic condition,the mannanase ManA could be potentially applied in brewing,food,feed,textile and pharmaceuticals fields.