PDBsum entry 5gyc

Hydrolase

PDB id: 5gyc

Contents

Protein chain

272 a.a.

Waters ×471

PDB id:

5gyc

Name:

Hydrolase

Title:

Crystal structure of enzbleach xylanase k73r+k185r and t28c+t60c mutant

Structure:

Endo-1,4-beta-xylanase. Chain: a. Engineered: yes

Source:

Termite gut metagenome. Organism_taxid: 433724. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.40Å R-factor: 0.173 R-free: 0.203

Authors:

P.Chitnumsub,A.Jaruwat,K.Boonyapakorn

Key ref:

K.Boonyapakron et al. (2017). Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching. J Biotechnol, 259, 95. PubMed id: 28774672 DOI: 10.1016/j.jbiotec.2017.07.035

Date:

22-Sep-16 Release date: 30-Aug-17

Protein chain

No UniProt id for this chain

Struc: 272 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.8 - endo-1,4-beta-xylanase.
• Reaction: Endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.

DOI no: 10.1016/j.jbiotec.2017.07.035

J Biotechnol 259:95 (2017)

PubMed id: 28774672

Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching.

K.Boonyapakron, A.Jaruwat, B.Liwnaree, T.Nimchua, V.Champreda, P.Chitnumsub.

ABSTRACT

In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the k_catof Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pK_aand hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry.