中文摘要：

以里氏木霉（Trichoderma reesei）RNA为模板，采用RT—PCR扩增的方法获得不带自身信号肽man1基因的cDNA片段。构建了重组表达载体pPIC9K-man1，重组质粒SacⅠ线性化后用PEG（聚乙二醇）法导入毕赤酵母（Pichia pastoris）菌株GS115中，通过PCR和表型鉴定表明，man1基因已经整合到毕赤酵母染色体上。经大量筛选，获得高效分泌表达甘露聚糖酶的毕赤酵母工程菌株RMAN23。将此菌株在5L发酵罐中进行高密度发酵，测定酶活最高达470IU／mL，同时对重组甘露聚糖酶的性质进行了初步研究，重组酶的最适反应pH4．5，最适反应温度60℃，并且在小于75℃，pH4．0-45．0范围内有较好的稳定性。

英文摘要：

The manl gene encoding mannanase without the signal peptide was cloned by RT-PCR using RNA of the Trichoderma reesei as template. The secreted expression plasmid pPIC9K-man1 of Pichia pastoris was constructed and digested with Sac Ⅰ and transformed into Pichia pastoris GS115 by PEG （polyethylene glycol）. PCR and phenotype anaijets showed man1 has been integrated into P.pastoris genome. Alter screening, the recombinant P. pastoris strain RMAN23 was obtained and fermented in large scale 5 L fermenter for 120 h. The recombinant mannanase activity could reach to 470 IU/mL .The properties of the recombinant mannanase were characterized. Optimum pH and temperature for the recombinant enzyme were 4.5 and 60℃, respectively. The enzyme was stable below 75℃ between pH 4.0-6.0