海栖热袍菌木聚糖酶B的分子改造、高效表达及其在啤酒中的应用

High-level Expression of an Engineered Thermostable Xylanase B from Thermotoga maritima and Its Application in Beer Brewing

研究海栖热袍菌木聚糖酶B(TmXynB)的定向进化、高效表达和应用。构建突变文库后经两轮筛选,获得一个在酸性高温下具有高比活力的突变体(mTmXynB)。突变体mTmXynB的最适pH值和最适温度为5.5和90℃,较野生型的最适pH值和最适温度分别下降了0.5和10℃,对小麦阿拉伯木聚糖的比活力由529 U/mg提高至1565 U/mg。序列比对表明,mTmXynB中3个氨基酸发生突变,分别为Asn91Ser、Trp129Arg和Arg143Glu。经定点突变实验,Arg143Glu通过改变局部蛋白表面的电荷分布提高了其耐酸性,Trp129Arg通过打破疏水平台降低了酶的最适温度,而Asn91Ser可能通过改变底物结合位点Trp89的构象提高该酶的催化活力。进一步将mTmXynB表达至毕赤酵母,经高密度发酵酶活力达18000 U/mL。突变体mTmXynB和野生型TmXynB分别添加至麦芽糖化过程中,结果表明mTmXynB在150 U/g麦芽添加量时效果最好,可降低45%的过滤时间和8.7%的料液黏度。而TmXynB在600 U/g麦芽添加量时效果最好,分别降低39%和8.1%的过滤速度和料液黏度。因此,突变体mTmXynB在啤酒工业中具有很好的应用前景。

The extremely thermostable xylanase B(TmXynB)from T.maritima was successfully engineered by directed evolution.After two steps of screening,one mutant(mTmXynB)with high specific activity in acidic and high-temperature conditions was obtained.The optimal pH and temperature of mTmXynB were 5.5 and 90℃,respectively,which were 0.5 and 10℃lower than those of the wild-type enzyme,respectively.The specific activity toward wheat arabinoxylan was increased from 529(TmXynB)to 1565 U/mg(mTmXynB).Three amino acid residues were substituted in mTmXynB,namely Asn91Ser,Trp129Arg,and Arg143Glu.Arg143Glu improved the acid tolerance by changing the charge distribution on part of the protein surface.Trp129Arg decreased the optimal temperature by destroying the hydrophobic platform.Asn91Ser improved the enzyme activity by changing the conformation of the substrate-binding site Trp89.mTmXynB was then successfully expressed in Pichia pastoris,and after 180 h high-cell density fermentation,the maximal xylanase activity of 18000 U/mL was obtained.Furthermore,TmXynB and mTmXynB were separately added in the mashing process.Under the standard mashing conditions,the addition of mTmXynB at 150 U/g of malt decreased filtration time by 45%and viscosity by 8.7%.In contrast,the addition of TmXynB at 600 U/g of malt decreased filtration time by 39%and viscosity by 8.1%.Thus,the engineered mTmXynB may be a promising candidate for the brewing industry owing to its favorable properties.