Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach

被引:28
作者
Akbarzadeh, Ali [1 ,2 ,3 ]
Pourzardosht, Navid [4 ]
Dehnavi, Ehsan [2 ]
Siadat, Seyed Omid Ranaei [3 ]
Zamani, Mohammad Reza [1 ]
Motallebi, Mostafa [1 ]
Jamnani, Farnaz Nikzad [3 ]
Aghaeepoor, Mojtaba [2 ]
Tashnizi, Mohammad Barshan [5 ]
机构
[1] NIGEB, Dept Plant Biotechnol, Tehran, Iran
[2] Shahid Beheshti Univ Med Sci, Gene Transfer Pioneers Res Grp, Tehran, Iran
[3] Shahid Beheshti Univ, Biotechnol Prot Res Ctr PRC, GC, Tehran, Iran
[4] Guilan Univ Med Sci, Biochem Dept, Rasht, Iran
[5] Univ Tehran, Dept Life Sci Engn, Fac New Sci & Technol, Tehran, Iran
关键词
Endoglucanase II; Site directed mutagenesis; Disulfide bond; Thermal stability; HIGH-LEVEL EXPRESSION; HUMAN SERUM-ALBUMIN; PICHIA-PASTORIS; MOLECULAR-DYNAMICS; IMPROVED THERMOSTABILITY; SELENOMONAS-RUMINANTIUM; COMPUTATIONAL METHODS; BETA-XYLOSIDASE; CELLULASE; PROTEINS;
D O I
10.1016/j.ijbiomac.2018.09.164
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Endoglucanasell (Cel5A) of Trichoderma reesei is widely used industrially with the high catalytic efficiency, but it is not stable high temperatures. Structural comparison with the closest thermophilic endoglucanase homolog, Cel5A from Thermoascus aurantiacus, demonstrates disulfide bond differences. Replacement of Cysteine99 with Valine and Cysteine323 with Histidine by site directed mutagenesis caused elimination of two disulfide bonds. Recombinant expression in Pichia pastoris showed the catalytic efficiency (kcat/Km) increment toward CMC for single mutant enzymes, C99V and C323H, about 1.87 and 13 folded respectively. This indicates that the elimination of disulfide bond in substrate binding cleft around the catalytic domain of mutant Endoglucanasell may be increased the flexibility of protein, to form a suitable E-S complex. In direct contrast with previous studies suggesting the existence of disulfide bonds increase the protein stability, the results showed mutant endoglucanase enzymes with disulfide bond reduction have higher thermal stability. The thermal stability of C99V and C323H in 80 degrees C were increased 2.4 and 234 folded, respectively. In this project, theoretical data had a good agreement with the experimental results. Because of high enzyme activity and thermal stability, both of C99V and C323H mutant have high potential suitable for different industrial applications. (C) 2018 Published by Elsevier B.V.
引用
收藏
页码:1572 / 1580
页数:9
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