Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1
December
Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1
Xiaoyu Wang 0 1
Fei Zheng 0 1
Yuan Wang 1
Tao Tu 1
Rui Ma 1
Xiaoyun Su 1
Shuai You 1
Bin Yao 1
Xiangming Xie 0 1
Huiying Luo 1
0 College of Biological Sciences and Biotechnology, Beijing Forestry University , Beijing , People's Republic of China, 2 Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences , Beijing , People's Republic of China
1 Editor: Israel Silman, Weizmann Institute of Science , ISRAEL
Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X), respectively. Xyl10E was most active at 93 to 95ÊC and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80ÊC or 90ÊC for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency.
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Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This work was supported by the National
Natural Science Foundation of China (grant: no.
31472127; received by HL; http://www.nsfc.gov.
cn/) and the National High-Tech Research and
Development Program of China (863 Program,
grant: no.2013AA102803; received by BY; http://
program.most.gov.cn/) and the National Science
Fund for Distinguished Young Scholars of China
(grant: no. 31225026; received by BY; http://www.
Introduction
Xylan is the second most abundant polysaccharide after cellulose. To digest polymerized
xylan, acid, alkaline, and enzymatic methods have been widely used. Enzymatic hydrolysis is
more favorable due to the high efficiency and environmentally friendly characteristics.
Complete hydrolysis of xylan requires the synergetic action of several xylanolytic enzymes,
including β-1,4-xylanases, β-D-xylosidases, α-L-arabinofuranosidases, α-glucuronidases, acetyl xylan
esterases, and feruloyl esterases. Of them, β-1,4-xylanase plays a crucial role in breaking the
backbones of xylan. According to the Carbohydrate-Active enZymes database (CAZy; http://
www.cazy.org/), xylanases are classified into glycoside hydrolase (GH) families 5, 7, 8, 10, 11
nsfc.gov.cn/) and the China Modern Agriculture
Research System (grant: no. CARS-42; received by
BY; http://www.caas.net.cn/zytj/kjpt/xdnycyjztx/
index.html). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
and 43, and those from GH10 and GH11 are most widely studied [
1
]. GH11 xylanases fold
into a β-jelly roll structure, while those of GH10 are TIM-barrel folds.
Xylanases have potential applications in various industries, for instance commercial food
production, animal feed, baking, fruit juice clarification, pulp biobleaching and bioconversion
[2±5]. According to the industrial requirements, the thermophilic and acidophilic enzymes are
specifically used in the bioconversion process, feed and brewing fields [
6
]. For example, the
bioconversion process involves steam explosion and acidic pretreatments prior to enzyme
treatment [
7, 8
]. Since the enzymes cannot be present in steam explosion or strongly acidic
(and high temperature) conditions, the enzymes have to be added at a later process stage.
Xylanase as a feed additive must withstand the pelleting temperature (70±90ÊC) and adapt to
digestive tract pH (4.8) [9]; the brewing industry requires acidic thermostable xylanase for
persistent hydrolysis at 70ÊC [
10
].
Although most thermophilic xylanases of GH10 demonstrate a temperature optimum from
60 to 80ÊC [11±14], a few exceptions have temperature optima up to 105 and 110ÊC [15±16].
For industrial purposes, catalytic efficiency is another important enzyme characteristic for
improvement. Chimeric xylanase CXC-X4,5 of GH10 had (...truncated)