关键词: 木聚糖酶B；海栖热袍菌；定向进化；高效表达；麦芽糖化

Abstract: 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 1 565 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 18 000 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.

Key words: xylanase B; Thermotoga maritima; directed evolution; high-level expression; mashing