成果简介

本技术可用于工业化生产不含纤维素酶的木聚糖酶。木聚糖酶是一类可广泛用于纤维素乙醇生产、饲料添加剂的生产、以及生物制浆等工业过程的重要酶制剂。一些真菌如里氏木霉、黑曲霉等被认为是重要的木聚糖酶产生菌。如里氏木霉在富含阿拉伯糖培养基的诱导下,可产生1350IU/ml的木聚糖酶。然而,在诱导的情况下,里氏木霉除木聚糖酶外还产生纤维素酶,含有纤维素酶的木聚糖酶的应用在某些行业会受到限制,如造纸行业。为解决这一问题,本技术通过RT-qPCR方法筛选了里氏木霉的强组成型启动子,并通过强组成型启动子pdc构建了新型高效的生产技术,用于生产不含纤维素酶的木聚糖酶。在改进的Mandels培养基中,可以获得高达9266IU/ml的木聚糖酶产量,为迄今报道的最高水平。通过SDS-PAGE分析证明,使用本技术生产重组木聚糖酶时,发酵液中的杂蛋白极少。且通过酶活性分析表明,发酵液中几乎没有纤维素酶活性,在造纸行业的应用前景十分广阔。

This technology is ready for efficient production of cellulase-free recombinant xylanase in industrial scale. Xylanase is an enzyme that is widely applied in various industrial processes, such as cellulosic ethanol production, feedstock additives, and biobleaching, etc. T. reesei is regarded as an important producer of xylanase. Induced by arabinose-rich plant hydrolysates and lactose in fed-batch cultures, the mutant strain T. reesei Rut C-30 produces up to 1350 IU/ml of xylanase. However, under induced conditions, T. reesei produces a large amount of cellulase aside from xylanase, and the cellulase side product is problematic for the application of xylanase in some industrial processes, such as biobleaching. To resolve this problem, we screened strong constitutive promoters of T. reesei through q RT-PCR, and developed a novel approach to produce cellulase-free xylanase by using the promoters of the pdc gene. The T. reesei pxyn2 recombinant strain, in which the pdc promoter were used to conduct the xyn2 gene expression, produced 9266 IU/ml of activity in a modified Mandels medium. Moreover, under constitutive production with high glucose concentrations, the recombinant strains produce little non-objective proteins as revealed by the SDS-PAGE image, and only a trace of cellulase activity is detected, with 0.29 IU/ml of CMCase activity and 0.03 FPIU/ml of filter paper activity. As xyanlases are a group of industrial enzymes with applications in feed and food, pulp and paper, textiles, etc., a number of production systems for these enzymes have been developed, including native production of xylanases in T. reesei, and heterologous production of xylanase in bacteria or fungi. To date, the highest native production of obtained in T. reesei is 1800 IU/ml with a fed-batch cultivation process, and the highest heterologous production of xylanase obtained is 3676 IU/ml with a fed-batch cultivation of Pchia pastoris expressing A. niger xylB . We used strong constitutive promoters in the xylanase production in T. reesei, and obtained recombinant productivity of 9266 IU/ml for the pdc promoter and 8866 IU/ml for the eno promoter in batch cultivation, which are much higher than the best native and heterologous xylanase production levels to date.

技术创新

    构建具有自主知识产权的高效组成型表达系统,获得了迄今报道最高的木聚糖酶生产水平。

专利情况

    中国发明专利:里氏木霉组成型表达盒、表达载体、重组菌株及其应用,申请号:201110196079.6,实质审查。

市场前景及应用领域

 酶制剂行业、造纸行业、饲料行业

合作方式

   (技术转让,合作开发等)

 

 

项目单位:深圳大学生命科学学院  成果负责人:刘刚

联系方式:深圳大学科学技术部 孙老师 0755-26536623

电子邮箱:sunni@szu.edu.cn