外切-β-葡聚糖酶基因重组里氏木霉的筛选及其产酶性能

外切-b-葡聚糖酶是纤维素酶的重要组分之一，提高该组分的活力是增强纤维素酶协同降解性能、降低纤维素水解成本的关键。分别采用微晶纤维素琼脂平板法和滤纸崩解法，对已有的基因重组转化子进行筛选试验，获得了6个优良转化子，其滤纸崩解速率和微晶纤维素琼脂平板上的生长速率都较大。进一步在摇瓶条件下进行复筛试验，获得了外切-β-葡聚糖酶（C₁）高产转化子Trichoderma reesei ZU-101，液体培养48 h，其C₁酶活力可达18.24 U·ml^-1，是出发菌株的2.16倍；分析结果表明：重组转化子的纤维素酶体系中内切-b-葡聚糖酶和纤维二糖酶的活力与出发菌株相比变化不大，但由于外切-b-葡聚糖酶活力得到了大幅度提高，纤维素酶的总活力（滤纸酶活力FPA）也提高了61.9%。采用纤维素酶对碱预处理玉米秸秆进行酶解试验，当酶用量为20 FPIU·（g底物）^-1，水解48 h，重组转化子T.reesei ZU-101纤维素酶的酶解得率高达94.4%。本文的研究结果在可再生纤维素资源的生物转化与利用方面具有广阔的应用前景。

Screening and cellulase production of recombinant Trichoderma reesei with high activity exo-β-glucanases

Increasing the activities of exo-β-glucanases, one of the important components of cellulase, is a key to strengthening the synergistic degradation of cellulase and reducing the cost of cellulolytic hydrolysis. The methods of MCC-agar plate screening and liquid culture medium of filter paper were used in this work respectively to screen Trichodermareesei transformants, obtaining 6 superior transformants with higher filter paper collapsing rate and growing rate on MCC-agar plates. Further screening experiment was conducted under shaking flask condition, obtaining the highly exo-b-glucanase (C₁)-producing transformant T.reesei ZU-101 whose C₁ activity was18.24 U·ml^-1 after 48 h liquid culture, 2.16-fold higher than the original strain. Analysis results demonstrated that filter paper activity (FPA) of the cellulase system of the transformant, representing total activity, increased by 61.9% as exo-β-glucanase activity ascended significantly, although endo-b-glucanase and cellobiase activities varied insignificantly from the original strain. The enzymatic hydrolysis yield of T.reesei ZU-101's cellulase in the hydrolysis of alkali pretreated corn stover was 94.4% when the enzyme dosage and hydrolysis time were 20 FPIU·(g substrate)^-1 and 48 h, respectively. The results of the present work have promising application prospects in the bioconversion and utilization of renewable cellulosic materials.