ABSTRACT
Deficient activity of endo-1,4-beta-glucanase II (Cel5A) secreted by Trichoderma reesei is one of the challenges involved in effective cellulase saccharification of cellulosic substrates. Therefore, we expressed Cel5A in Pichia pastoris by constructing a recombinant strain. With the gene optimization based on codon bias, and the construction of expression vector pPIC9K-eg2, the optimized gene was electro-transformed into P. pastoris GS115 to form transformants. Then, a high Cel5A activity producing recombinant, namely P. pastoris GS115-EG Ⅱ, was selected on G-418 resistant plates, followed by shake-flask cultivation. Enzyme characterization showed that the recombinant Cel5A reacted optimally at pH 4.5 and 60 ℃, with 50 kDa of molecular weight, preferentially degrading amorphous cellulose. Recombinant Cel5A was not significantly different from the native T. reesei Cel5A. Moreover, a shake-flask fermentation of the recombinant strain was optimized as below: incubation temperature 28 ℃, initial pH 5.0, inoculum volume 2%, methanol addition (per 24 h) 1.5% (V/V), sorbitol addition (per 24 h) 4 g/L and Tween 80 4 g/L. Under above optimized condition, the recombinant produced 24.0 U/mL of the Cel5A after 192 h fermentation. When incubated in a 5 L fermentation, Cel5A enzyme activity reached 270.9 U/mL at 180 h, with 4.16 g/L of the total protein. The study indicates that the recombinant strain P. pastoris GS115-EG Ⅱ is extremely suitable for heterologous expression of T. reesei cellulase Cel5A. And the recombinant Cel5A can be used as an alternative to the native T. reesei Cel5A in development of a commercially relevant enzyme based biorefinery process.
ABSTRACT
The expensive production of bioethanol is because it has not yet reached the 'THREE-HIGH' (High-titer, high-conversion and high-productivity) technical levels of starchy ethanol production. To cope with it, it is necessary to implement a high-gravity mash bioethanol production (HMBP), in which sugar hydrolysates are thick and fermentation-inhibitive compounds are negligible. In this work, HMBP from an atmospheric glycerol autocatalytic organosolv pretreated wheat straw was carried out with different fermentation strategies. Under an optimized condition (15% substrate concentration, 10 g/L (NH4)2SO4, 30 FPU/g dry matter, 10% (V/V) inoculum ratio), HMBP was at 31.2 g/L with a shaking simultaneous saccharification and fermentation (SSF) at 37 degrees C for 72 h, and achieved with a conversion of 73% and a productivity of 0.43 g/(L x h). Further by a semi-SFF with pre-hydrolysis time of 24 h, HMBP reached 33.7 g/L, the conversion and productivity of which was 79% and 0.47 g/(L x h), respectively. During the SSF and semi-SSF, more than 90% of the cellulose in both substrates were hydrolyzed into fermentable sugars. Finally, a fed-batch semi-SFF was developed with an initial substrate concentration of 15%, in which dried substrate (= the weight of the initial substrate) was divided into three portions and added into the conical flask once each 8 h during the first 24 h. HMBP achieved at 51.2 g/L for 72 h with a high productivity of 0.71 g/(L x h) while a low cellulose conversion of 62%. Interestingly, the fermentation inhibitive compound was mainly acetic acid, less than 3.0 g/L, and there were no other inhibitors detected, commonly furfural and hydroxymethyl furfural existing in the slurry. The data indicate that the lignocellulosic substrate subjected to the atmospheric glycerol autocatalytic organosolv pretreatment is very applicable for HMBP. The fed-batch semi-SFF is effective and desirable to realize an HMBP.