After being incubated at 75☌ for 18 h, its residual activity was 96%. Thermomonospora fusca TF xylanase A (TfxA) is one of the most thermostable GH11 xylanases. Therefore, lots of researches confirmed that these strategies mentioned above could be adopted to enhance the catalytic activity and enzymatic properties of xylanase ( Shibuya et al., 2000 Miyazaki et al., 2006 Stephens et al., 2007 Wang et al., 2013 Wahab et al., 2016 Prajapati et al., 2018 Damis et al., 2019). It was reported that numerous strategies were applied to modify enzyme based on the structure-function relationship and included rational, semi-rational, and irrational design (directed evolution) ( Farinas et al., 2001 Wong and Schwaneberg, 2003 Chica et al., 2005 Sarmiento et al., 2015, Cheng et al., 2015). Xylanases with improved properties (i.e., higher activity and stability) are urgently welcomed and needed for environmental, economic and industrial reasons ( Pandey et al., 2016 Bala and Singh, 2019). The most common external factors include temperature, pH, substrate, and inhibitors, while the internal factors include primary structure and crystal structure. Most of the GH11 xylanases lack xylosidase and cellulase activity and they were widely applied in food, feed, and paper pulp bleaching industries ( Juturn and Wu, 2012).Īs a biological catalyst, the catalytic activity and efficiency of xylanases are susceptible to be influenced by both external and internal factors. Generally, the GH10 xylanases typically have high molecular weight and own an (α/β) 8 barrel fold, liking a “salad bowl,” while GH11 xylanases are characterized as low molecular mass and the “right-handed jellyroll” structure ( Paës et al., 2012). The xylanases were classified into glycosyl hydrolase (GH) families 5, 8, 10, 11, 26, 30, and 43 according to the amino acid sequence similarities of catalytic domain ( Henrissat and Davies, 1997). Endo-1,4-beta-xylanase (EC 3.2.1.8, referred hereinafter as xylanase) cleaves internal β-xylosidic glycosidic bonds in the main backbone of xylan, thereby releasing xylose, xylooligosaccharides (XOs), and small polymers from hemicellulose ( Scheller and Ulvskov, 2010). Xylan is the most abundant hemicellulose in nature and is primarily composed of β-D-xylopyranosyl residues linked by β-1,4-glycosidic bonds. The current findings would provide a better and more in-depth understanding of how distal single residue substitution improved the catalytic activity of xylanase at the atomic level. The distal impact rescued the BaxA from native conformation (“closed state”) through weakening interactions between “gate” residues (R112, N35 in DS241 and DS428 W9, P116 in DS153) and active site residues (E78, E172, Y69, and Y80), favoring conformations with an “open state” and providing improved activity. The tertiary structure of BaxA was not altered under the substitution of distal residues (N29S, S31R, and I51V) it induced slightly changes in active site architecture. Furthermore, molecular dynamics simulations were performed on BaxA and three mutants to explore the precise impact of gain-of-function on xylanase activity. Three mutants have identical hydrolytic function as reBaxA, which released xylobiose–xylopentaose from oat spelt, birchwood, and beechwood xylan. The catalytic efficiency of DS153, DS241, and DS428 enhanced as well, while their sensitivity to recombinant rice xylanase inhibitor (RIXI) was lower than that of reBaxA. The optimum pH for DS153, DS241, and DS428 was 6.0, 7.0, and 6.0, respectively. The optimum temperature of the three mutants was 50☌. The specific activity of DS153, DS241, and DS428 in the optimal condition was 4.54, 4.35, and 3.9 times compared with the recombinant BaxA (reBaxA), respectively. For these mutants, three residues of BaxA were substituted by the corresponding one of TfxA_CD. Three novel mutants (DS153: N29S, DS241: S31R and DS428: I51V) were identified and characterized in detail. A total of 2,250 colonies were collected and 756 of them were sequenced. The mutant library of GH11 endoxylanase was constructed via DNA shuffling by using the catalytic domain of Bacillus amyloliquefaciens xylanase A (BaxA) and Thermomonospora fusca TF xylanase A (TfxA) as parents. 2State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaĮndoxylanase with high specific activity, thermostability, and broad pH adaptability is in huge demand.1Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou, China.Ming-Qi Liu 1 * Jia-Yi Li 2 Ashfaq Ur Rehman 2 Xin Xu 1 Zhu-Jun Gu 1 Ruo-Chen Wu 1
0 kommentar(er)
