Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei

Abstract

The stability and specific activity of endo-beta-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4-8.8) and thermotolerance (entirely active at 55 degrees C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4-5.2; thermostability, inactive at 55 degrees C for 30 min). k (cat) of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K (m) became twice of that of the wild type.

Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastoris

“…Compared to the kinetic parameters reported by Macarron et al (1993) and Nakazawa et al (2009) for CMCase activity, the K cat value obtained here is higher in comparison to EGIII from T. reesei, with the K m being twice as high. The K m is similar to that found for endoglucanase activity in the crude extract of T. harzianum IOC-3844 (19 g/L) and the endoglucanase from T. harzianum ETS 323 (23 g/L) (de Castro et al, 2010a;Liu et al, 2010).…”

“…Even with a lower degree of thermostability than the EGIII from T. reesei, which displays a high degree of activity until 50°C (Nakazawa et al, 2009), rThEGIII demonstrated stability at temperatures close to the optimal, lasting several days with fairly good activity. Thus, EGIII may be a promising enzyme for the purpose of simultaneous saccharification and fermentation, since this process requires hydrolysis under mild conditions.…”

“…On the other hand, the optimal temperature for rThEGIII is not similar to that for EGIII from other filamentous fungi. The optimal temperature for EGIII from H. schweinitzii and Gliocladium roseum is about 47°C, whereas the optimal temperature for T. reesei, T. koningii and Fusarium javanicum is about 55°C, and finally nearly 70°C is reported for H. grisea (Karlsson et al, 2002;Lynd et al, 2002;Sandgren et al, 2005;Nakazawa et al, 2009).…”

“…This enzyme exhibits a high degree of similarity with endoglucanase III (cel12a) from T. reesei. Endoglucanase III (EGIII) enzymes have a low molecular mass and lack the cellulose binding domain (CBD) (Okada et al, 1998;Henriksson et al, 1999;Nakazawa et al, 2009). These enzymes are also able to degrade amorphous cellulose and have a compact β-jellyroll fold (Okada et al, 1998;Henriksson et al, 1999;Sandgren et al, 2003Sandgren et al, , 2005Nakazawa et al, 2009).…”

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

“…TrCel5A has the least homology to the other T. reesei cellulases and has an N-terminal carbohydrate binding module I (16). TrCel12A is smaller (25 kDa) and has no carbohydrate binding module (20), All three are retaining glycoside hydrolases (21,22).…”