漆酶BmLac的异源表达、酶学特性及棉酚降解的研究

Heterologous Expression,Enzymatic Characterization of Laccase BmLac and Degradation of Gossypol by It

为开发能高效降解游离棉酚的酶蛋白。从土壤中筛选得到一株Bacillus amyloliquefaciens XP-13,从其基因组扩增获得了基因BmLac,进而构建Ppic9k-BmLac重组质粒,将其导入毕赤酵母GS115实现了异源表达,对漆酶BmLac的酶学特性、棉酚降解效果进行研究。结果表明,以ABTS为底物时,该酶的最适反应条件为pH 4,55℃,最适条件下酶活是101.56 U/mL;在90℃下孵育1 h后,相对酶活仍保留70%以上;在pH 3-7孵育1 h,其相对酶活均保留96%以上。1 mmol/L的Cu^(2+)能使该酶酶活提高1.93倍,但高浓度(10 mmol/L)的Fe^(2+)、Fe^(3+)和Al^(3+)完全抑制该酶活性。在55℃下反应2 h后,该酶对棉酚的降解率高达94.57%(无介体)和98.73%(有ABTS)。综上,漆酶BmLac具有良好的热稳定性和广泛的pH适应性,并能有效降解棉酚,该酶为有效降解棉籽粕中的游离棉酚提供基础支撑。

To develop enzymatic proteins for the efficient degradation of gossypol,a strain of B.amyloliquefaciens XP-13 was screened from soil,the gene BmLac was amplified from its genome,then the recombinant plasmid Ppic9k-BmLac was constructed and introduced into Pichia pastoris GS115 for heterologous expression.The characteristics of the laccase BmLac and the degradation effect of gossypol were studied.The results showed that the enzyme activity was 101.56 U/mL at pH 4 and 55℃under the optimal reaction conditions with ABTS as the substrate;the relative enzyme activity was retained over 70%after incubation at 90℃for 1 h;the relative enzyme activity was retained over 96%after incubation at pH 3-7 for 1 h.The enzyme activity increased 1.93 times by adding 1 mmol/L Cu^(2+),but high concentrations(10 mmol/L)of Fe^(2+),Fe^(3+)and Al^(3+)completely inhibited the enzyme activity.After reaction at 55℃for 2 h,the degradation rate of gossyrol by this enzyme reached 94.57%(without mediator)and 98.73%(with ABTS).In conclusion,the laccase BmLac has good thermal stability and wide pH adaptability and can effectively degrade gossypol,and this enzyme provides the basic support for the effective degradation of free gossypol in the cottonseed meal.