According to these results, X3 (sodium acetate), X1 (YE), X1 X2, X2 (CSLP) and X3 are significant model terms.
Based on the statistical analysis, the factors having the greatest impacts on the production of 2,3-BD were identified as X1 (YE, p = 0.010), X2 (CSLP, p = 0.006) and X3 (sodium acetate, p = 0.0050).
Therefore, the fermentation medium was changed to: glucose at 220 g/L, CSLP from 50 to 90 g/L, yeast extract at 10 g/L, deionized water, pH 8.0.
However, when yeast extract (YE), peptone, and beef extract in the fermentation medium were completely replaced with 10.0 g/L CSLP, the concentration of 2,3-BD would decreased from 30.2 ± 0.8 g/L to 19.3 ± 1.2 g/L.
The concentration of 2,3-BD in the medium containing 5.0 g/L YE and 10.0 g/L CSLP was higher than others.
Thus, 5.0 g/L YE and 10.0 g/L CSLP were chosen as the organic nitrogen source of the fermentation medium for future investigations.
To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD).
Phosphate, acetate, ammonium salt, Mg2+, YE and CSLP were used to compose the Plackett Burman design (Table 1 and Table 2).
The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS PAGE to be 22 and 27 kDa, respectively.
The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae (α factor).
As shown in Figure 7, acetoin and 2,3-butanediol gained the highest yields (7.7 g/L of acetoin and 14.5 g/L of 2,3-butanediol) after 2 days of fermentation at the CSLP concentration of 60 g/L.
