Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei.


Abstract

Xylanases isolated from microorganisms such as the Trichoderma reesei have attracted considerable research interest because of their potential in various industrial applications. However, naturally isolated xylanases cannot withstand harsh conditions such as high temperature and basic pH. In this study, we performed structural analysis of the major T. reesei xylanase (Xyn2), and novel flexible regions of the enzyme were identified based on B-factor, a molecular dynamics (MD) parameter. To improve thermostability of the Xyn2, disulfide bonds were introduced into the unstable flexible region by using site-directed mutagenesis and two recombinant xylanases, XM1 (Xyn2Cys12-52) and XM2 (Xyn2Cys59-149) were successfully expressed in Pichia pastoris. Secreted recombinant Xyn2 was estimated by SDS-PAGE to be 24 kDa. Interestingly, the half-lives of XM1 and XM2 at 60°C were 2.5- and 1.8- fold higher, respectively than those of native Xyn2. The XM1 also exhibited improved pH stability and maintained more than 60% activity over pH values ranging from 2.0 to 10.0. However, the specific activity and catalytic efficiency of XM1 was decreased as compared to those of XM2 and native Xyn2. Our results will assist not only in elucidating of the interactions between protein structure and function, but also in rational target selection for improving the thermostability of enzymes.

Submission Details

ID: fxAJ3y3r

Submitter: Shu-Ching Ou

Submission Date: March 7, 2019, 4:48 p.m.

Version: 1

Publication Details
He J;Tang F;Chen D;Yu B;Luo Y;Zheng P;Mao X;Yu J;Yu F,PLoS One (2019) Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei. PMID:30650138
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4S2H 2015-09-23 Joint X-ray/neutron structure of Trichoderma reesei xylanase II at pH 8.5
4XPV 2015-09-30 Neutron and X-ray structure analysis of xylanase: N44D at pH6
4S2G 2015-09-23 Joint X-ray/neutron structure of Trichoderma reesei xylanase II at pH 5.8
4S2D 2015-09-23 Joint X-ray/neutron structure of Trichoderma reesei xylanase II in complex with MES at pH 5.7
4S2F 2015-09-23 Joint X-ray/neutron structure of Trichoderma reesei xylanase II at pH 4.4
5ZH0 2019-03-13 1.08 Crystal Structures of Endo-beta-1,4-xylanase II
4HKL 2014-01-08 1.1 Crystal Structures of Mutant Endo-beta-1,4-xylanase II Complexed with substrate (1.15 A) and Products (1.6 A)
2DFB 2006-07-11 1.11 Xylanase II from Tricoderma reesei at 100K
4HK8 2014-01-08 1.15 Crystal Structures of Mutant Endo- -1,4-xylanase II Complexed with substrate (1.15 A) and Products (1.6 A)
5ZH9 2019-03-20 1.15 Crystal Structures of Mutant Endo-beta-1,4-xylanase II (Y88F)
2DFC 2006-07-11 1.19 Xylanase II from Tricoderma reesei at 293K
5ZF3 2019-03-06 1.2 Crystal Structures of Endo-beta-1,4-xylanase II Complexed with Xylotriose
4XQ4 2015-09-23 1.25 X-ray structure analysis of xylanase - N44D
5ZIW 2019-03-20 1.3 Crystal Structures of Mutant Endo-beta-1,4-xylanase(Y77F)
5ZKZ 2019-04-10 1.3 Crystal Structures of Mutant Endo-beta-1,4-xylanase II(Y77F) Complexed with Xylotriose
5ZII 2019-03-20 1.3 Crystal Structures of Mutant Endo-beta-1,4-xylanase II (Y88F)Complexed with Xylotriose
4HKO 2014-01-08 1.5 Crystal Structures of Mutant Endo-beta-1,4-xylanase II (E177Q) in the apo form
4XQW 2015-09-23 1.5 X-ray structure analysis of xylanase-N44E with MES at pH6.0
1XYP 1995-08-08 1.5 STRUCTURAL COMPARISON OF TWO MAJOR ENDO-1,4-BETA-XYLANASES FROM TRICHODREMA REESEI
1XYO 1995-08-08 1.5 STRUCTURAL COMPARISON OF TWO MAJOR ENDO-1,4-BETA-XYLANASES FROM TRICHODREMA REESEI
1ENX 1995-08-08 1.5 STRUCTURAL COMPARISON OF TWO MAJOR ENDO-1,4-BETA-XYLANASES FROM TRICHODREMA REESEI
4XQD 2015-09-23 1.5 X-ray structure analysis of xylanase-WT at pH4.0
4HK9 2014-01-08 1.55 Crystal Structures of Mutant Endo-beta-1,4-xylanase II Complexed with substrate (1.15 A) and Products (1.6 A)
1REE 1997-01-11 1.6 ENDO-1,4-BETA-XYLANASE II COMPLEX WITH 3,4-EPOXYBUTYL-BETA-D-XYLOSIDE
1RED 1997-01-11 1.6 ENDO-1,4-BETA-XYLANASE II COMPLEX WITH 4,5-EPOXYPENTYL-BETA-D-XYLOSIDE
3LGR 2011-01-19 1.64 Xylanase II from Trichoderma reesei cocrystallized with tris-dipicolinate europium
4HKW 2014-01-08 1.65 Crystal Structures of Mutant Endo-beta-1,4-xylanase II Complexed with Substrate and Products
5ZO0 2019-05-15 1.65 Neutron structure of xylanase at pD5.4
1REF 1997-01-11 1.8 ENDO-1,4-BETA-XYLANASE II COMPLEX WITH 2,3-EPOXYPROPYL-BETA-D-XYLOSIDE
1XND 1994-12-20 2.0 HIGH-RESOLUTION STRUCTURES OF XYLANASES FROM B. CIRCULANS AND T. HARZIANUM IDENTIFY A NEW FOLDING PATTERN AND IMPLICATIONS FOR THE ATOMIC BASIS OF THE CATALYSIS
2D98 2006-07-11 2.0 Structure of VIL (extra KI/I2 added)-xylanase
2D97 2006-07-11 2.01 Structure of VIL-xylanase
5K7P 2017-04-05 2.3 MicroED structure of xylanase at 2.3 A resolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 Endo-1,4-beta-xylanase G0RUP7 XYN2_HYPJQ
99.5 Endo-1,4-beta-xylanase P36217 XYN2_HYPJR
94.2 Endo-1,4-beta-xylanase B5A7N4 XYN2_TRIHA
93.7 Endo-1,4-beta-xylanase P48793 XYN_TRIHA