Abstract
Endoxylanase with high specific activity, thermostability, and broad pH adaptability is in huge demand. 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. A total of 2,250 colonies were collected and 756 of them were sequenced. Three novel mutants (DS153: N29S, DS241: S31R and DS428: I51V) were identified and characterized in detail. For these mutants, three residues of BaxA were substituted by the corresponding one of TfxA_CD. 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. The optimum temperature of the three mutants was 50°C. The optimum pH for DS153, DS241, and DS428 was 6.0, 7.0, and 6.0, respectively. 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. Three mutants have identical hydrolytic function as reBaxA, which released xylobiose–xylopentaose from oat spelt, birchwood, and beechwood xylan. Furthermore, molecular dynamics simulations were performed on BaxA and three mutants to explore the precise impact of gain-of-function on xylanase 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. 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 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.
Highlights
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 sequence similarity index results revealed that the catalytic domain of TF xylanase A (TfxA) is nearly 40% identical with that of Bacillus amyloliquefaciens xylanase A (BaxA)
The digested products with a size of
Summary
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 catalytic activity and efficiency of xylanases are susceptible to be influenced by both external and internal factors. 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). The sequence similarity index results revealed that the catalytic domain of TfxA is nearly 40% identical with that of BaxA. The mutant library was constructed via DNA shuffling by using the catalytic domain (CD) of TfxA and BaxA as parents. Exploring effects of distal residue substitution on active site architecture may assist in providing valuable information for better clarifying the mechanism of enhanced activity
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