一株皮革脱毛用工程菌的构建及酶法脱毛应用

Construction of a strain and application in enzyme unhairing

构建一株低胶原蛋白酶活力枯草工程菌,用于皮革脱毛并研究其脱毛特性。以Bacillus subtilis AS1.398中性蛋白酶基因为模板,聚合酶链式反应(PCR)扩增得到中性蛋白酶基因npr,并借助p HT43质粒转化至蛋白酶缺陷型宿主菌WB800N中,培养并经过1mmol/L的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导,发酵液上清经SDS-PAGE电泳证明重组蛋白表达成功,经明胶平板验证重组菌株胶原蛋白酶表达能力显著低于野生菌株AS1.398。扩大培养获得重组中性蛋白酶,对牛皮进行脱毛试验表明:重组中性蛋白酶初始脱毛速率大于野生菌株AS1.398蛋白酶;7h到达脱毛终点,且此时脱毛液中羟脯氨酸值(13μg/m L)较低;经显微及感官鉴定重组中性蛋白酶脱毛后皮块毛孔清晰可见,不损伤皮粒面。综上,重组蛋白酶可以在不损害皮质量的前提下完成脱毛过程,为酶法脱毛制革提供新的技术支撑。

A strain of Bacillus subtilis with low expression level of collagenase was constructed and applied in leather unhairing process,the unhairing properties were investigated. Using the sequence of neutral protease gene of Bacillus subtilis AS1. 398 as the template,neutral protease gene npr was obtained by polymerase chain reaction（ PCR）. The amplified product was transformed to protease deletion engineering Bacillus subtilis WB800 N mediated by an expression plasmid p HT43. The expression was induced by1 m M IPTG,then SDS- PAGE electrophoresis shows neutral protease is successfully expressed,and the recombinant strain is verified by gelatin plate which expresses the collagenase significantly lower than that of AS1. 398. Recombinant enzyme were obtained by expand cultivation,and then applied in leather unhairing process. Studies show that the initial unhairing rate of recombinant enzyme is greater than that of AS1. 398 protease. The process of unhairing is about 7 hours,and the value of hydroxyproline（ 13μg /mL） in depilatory is low. The microstructure and sensory evaluation after unhairing show that after recombinant enzyme unhairing the hide pore is clearly visible,and with little damage of leather grain. In summary recombinant enzyme is suitable for unhairing without damage to the hide quality.