No abstract

BACKGROUND: The deficiency of family 5 endoglucanase (Cel5A) and family 6 cellobiohydrolase (Cel6A) has become a key limiting factor on cellulase enzymatic hydrolysis in bioprocessing of cellulosic biomass. To improve the production of Trichoderma reesei Cel5A / Cel6A, a Vitreoscilla hemoglobin (VHb) gene was tried to co-express extracellularly for the first time with Cel5A / Cel6A in Pichia pastoris GS115.

To address the deficient activity of TrCel5A in naturally secreted cellulase preparation, this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris. A recombinant TrCel5A was screened out after gene optimization, synthesis, and expression. The biochemical and enzymatic properties of the new recombinant were characterized. As a result, optimization of shake-flask fermentation of the recombinant was obtained at 28 °C, 2% inoculum volume, an initial pH of 6.0, as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L, respectively. Under the above-optimized conditions, the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation. To further enhance enzyme production, pilot-scale cultivation was evaluated using 5-L bioreactors. Using high-cell-density fermentation, the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L. In addition, the kinetic factors, including K m and V max values for TrCel5A, were detected to be 5.1 mg/mL and 265.9 μmol/(min . mg), respectively. Thus, TrCel5A was effectively expressed in P.pastoris under the GAP promoter, and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.