Abstract: As an important glycoside hydrolases, cellulases can be widely used in biofuel, feed, pulp and paper, laundry and textile. The GH45 family endo-cellulase gene ncGH45 from N. crassa contained 935 nucleotides (including a 53-bp intron) coding a 272 amino acids peptide. The putative protein consisted of a N-terminal catalytic motif and 1 C-terminal carbohydrate binding motif. This gene was expressed successfully in P. pastoris and the recombinant enzyme rNcGH45 showed a single band in the culture supernatant with enzyme activity of 20 U/mL. The recombinant rNcGH45 displayed maximum activity at pH 4.5~5.0 and 60~65℃, the relative activity were over 75% at pH 4.0~8.0 and at 30~70℃. Almost no activity was lost after being treated for 2 h at 37℃ and 50℃, and pH range was 3.0~11.0. The recombinant enzyme could resist high temperature, which remained 89.6% and 77.7% residual activities after pretreated at 70℃ and 80℃ for 2 h and even could remain 10% residual activity after boiled for 10 min. Besides, the purified enzyme had the highest activity against lichenin (1 116.0 U/mg, 438.8%), followed by barley β-glucan (754.2 U/mg, 296.6%) and CMC-Na (254.6 U/mg, 100%). The kinetic parameter of Km and Vmax against barley β-glucan and CMC-Na were 6.26 mg/mL and 997.4 μmol/mg·min, 19.96 mg/mL and 722.1 μmol/mg·min, respectively. The predominant hydrolysis products of rNcGH45 against the polysaccharides were cellopentose, and the minimum substrate was cellotriose against cello-oligosaccharides. Furthermore, the defibrillation activity on dyed cotton of rNcGH45 was investigated, when 10 U rNcGH45 was added in, the weight-loss ratio reached 1.55%. In conclusion, the highly expression strain of rNcGH45 was successfully constructed and the recombinant protein showed excellent enzymatic property, which made the rNcGH45 have great potential application in textile and other industrial fields.

Key words: Neurospora crassa, GH45 endoglucanase, defibrillation, textile, Pichia pastoris

摘要： 纤维素酶作为一种重要的糖苷水解酶，在生物质能源生产、饲料、造纸、洗涤和纺织领域都有着非常广泛的应用。粗糙脉孢菌来源的GH45家族内切纤维素酶基因 （ncGH45）全长935 bp，包含一个53 bp的内含子（339-391），编码272个氨基酸，包括一个N-端的GH45家族催化结构域和C-端的1家族碳水化合物结合结构域。该基因在毕赤酵母中得到了分泌表达，且表达蛋白条带单一，酶活性达到20 U/mL。重组酶rNcGH45的最适pH为4.5~5.0，最适温度为60~65℃，并且在较宽的pH范围（pH 4.0~8.0）和温度范围（30~70℃）都能维持75%以上的酶活。在37℃和50℃，pH 3.0~11.0都有着非常好的稳定性；该酶的热稳定性非常好，在70℃和80℃处理2 h还能剩余89.6%和77.7%的酶活，即使在沸水中煮10 min还能剩余10%的酶活。rNcGH45的最适底物是地衣多糖（1 116.0 U/mg），其次是大麦葡聚糖（754.2 U/mg）和CMC-Na（254.6 U/mg），对大麦葡聚糖和CMC-Na的动力学常数Km和Vmax分别为6.26 mg/mL和997.4 μmol/mg·min, 19.96 mg/mL和722.1 μmol/mg·min。水解产物分析结果表明，rNcGH45对多糖的水解产物主要是纤维五糖，对寡糖的最小作用底物是纤维三糖，而对纤维二糖没有作用。对纺织品脱毛实验的结果表明，酶的添加量为10 U时，减量率为1.55%。上述结果表明成功构建了rNcGH45高效表达工程菌株，表达的重组蛋白性质优越，有着良好的工业应用前景。

关键词: 粗糙脉胞菌, GH45内切纤维素酶, 脱毛, 纺织, 毕赤酵母