通过对6个重组毕氏酵母转化子的产酶试验,筛选出一个优良转化子。在摇瓶条件下对该重组毕氏酵母的发酵过程进行了研究,得到适宜的培养条件为:250 mL三角瓶装液量30 mL,培养基起始pH4.8,此后不加控制,接种量10%,发酵过程中每隔24h补加1%的甲醇对产酶有明显的促进作用,中性纤维素酶活力可达1 144U/mL。研究结果表明:重组毕氏酵母中性纤维素酶的适宜催化条件为:50℃、pH 6.0;Zn2+和Mg2+对该酶有激活作用,Fe2+、Fe3+和Cu2+对该酶有抑制作用。牛仔服水洗试验结果显示:重组毕氏酵母中性纤维素酶可以明显降低靛蓝对牛仔服的反染作用,其工业应用前景良好。

Neutral cellulase has important applications in denim biofinishing.A Pichia pastoris transformant with high activity of neutral cellulase was screened from 6 recombination transformants by fermentation experiment.The research on the neutral cellulase production by the P.pastoris recombinant was conducted under shaking flask condition,and the results were shown as follows: medium volume 30mL in 250mL Erlenmeyer flask,the initial pH 4.8 and uncontrolled during the fermentation process,inoculum volume 10% and methanol addition 1% every 24h which demonstrated an obvious enhancing effect.The above conditions allowed the activity of the neutral cellulase to reach 1144U/mL.The results from the study on enzymatic properties indicate that the appropriate catalysis conditions for the neutral cellulase from the P.pastoris transformant were pH 6.0 and temperature 50℃,and that Zn~2+ and Mg~2+ were activators of the enzyme but Fe~2+,Fe~3+ and Cu~2+ were inhibitors.The outcomes of denim biofinishing test reveal that the neutral cellulase from the P.pastoris recombinant could significantly reduce the backstaining of indigo onto denim and has a promising potential in industrial application.