N-糖基化对在毕赤酵母中表达的瑞氏木霉外切纤维素酶酶活力的影响

【作者】 魏联果；

【导师】 曲音波； 庄国强；

【作者基本信息】 山东大学 ， 微生物学， 2006， 硕士

【摘要】 当前，新能源、新食品资源的开发是世界各国都在研究的重大课题，其中纤维素科学、纤维素的降解成为课题的主要组成部分。纤维素降解的方法有很多，常用的酸或高温处理的方法虽然也能降解纤维素，但同时会产生很多难以接受的副产物，污染环境，并使葡萄糖受到破坏。于是人们把目光逐渐转移到纤维素的酶解，但目前纤维素酶对纤维素的降解效率太低，成为大规模推广使用的制约因素。人们试图利用蛋白质工程的手段对纤维素酶系的主要组分（如CBHI）和其它丝状真菌的纤维素酶进行改造，以改善酶的性质，用于工业生产。这样就需要一个能高效表达丝状真菌纤维素酶的表达系统。实践证明纤维素酶在大肠杆菌中不能成功大量表达。丝状真菌虽然能大量分泌纤维素酶且适合工业化生产，但作为蛋白质工程的表达系统转化效率非常低，并且转化子的筛选费时又费力，不适合高通量筛选。毕赤酵母（Pichia pastoris）表达系能表达单一的酶，且自身分泌的蛋白非常少，利于下游蛋白的纯化，而成为人们关注的焦点。 毕赤酵母与高等真核细胞非常接近，可对表达的蛋白质进行翻译后的修饰，如信号序列的加工、蛋白质的折叠、二硫键形成以及糖基化等，使表达的外源蛋白具有天然蛋白所应有的生物学活性。但有些蛋白在毕赤酵母中分泌表达时会发生过度糖基化，又是毕赤酵母表达系统的缺点。 目前纤维素酶在毕赤酵母中表达的研究报道并不多见。1999年，Shubhada Godbole等报道了其在毕赤酵母中表达瑞氏木霉外切纤维素酶CBHI的结果，外泌的CBHI的V_max是瑞氏木霉中自然分泌的CBHI的1％，通过western分析发现，表达出来的CBHI被高度糖基化，Shubhada Godbole等分析认为在两种宿主中糖基化程度不同可能是造成CBHI在酵母中表达后酶活低的主要原因。基于上述分析，我们希望通过对CBHI的糖基化位点进行改造，研究过度糖基化对CBHI在酵母中表达酶活力的影响，为构建丝状真菌纤维素酶的酵母表达系并开展纤维素酶的蛋白质工程工作做准备。 本研究通过点突变的方法，对CBHI的四个N-糖基化位点进行改造，将其45、64、270和384位的天冬酰胺突变为丝氨酸，构建重组质粒，并在毕赤酵母中进行表达，以研究过度糖基化对酵母中表达的CBHI酶活力的影响。更多还原

【Abstract】 Converting cellulose to ethanol has become an important task in the world. Hydrolysing cellulose to glucose is key step for this conversion. Until now, there are many methods to hydrolyse cellulose, and acid or high temperature is in common use, which may result in unwilling byproducts and polluting environments and even damaging glucose. More and more emphasis has been put on enzymatic hydrolysis. But low efficiency of cellulase astrict its extensive application at present. To solve the problem, protein engineering technology is practiced to modify CBHI or other cellulases from filamentous fungi. A high efficiency system is needed to express cellulase. However, T. reesei and E.coli were proved to be unfit expression systems. P. pastoris is evoluted from 80’s, which can express single enzyme and secrete little self proteins, which could be used as a perfect cellulase expression system.Nowadays, it is seldom reported that cellulase was expressed in P. pastoris. Shubhada Godbole reported in 1999 that the V_max of cellobiohydrolase I expressed in P. pastoris was one percent than that of the natural CBHI. He found that the expressed CBHI in P. pastoris had been hyperglycosylated and the activity was significantly lower than that of T. reesei CBH I also. Shubhada Godbole believed that hyperglycosylation of rCBHI in P. pastoris is the reason for its low activity.To understand the effect hyperglycosylation on recombinant CBH I from T. reesei in P. pastoris, asparagines of the four N—glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of CBH I were replaced by serines using site-directed mutagenesis. These four mutants and the cbh1 gene from T. reesei were transformed into P. pastoris’, then the enzymes expressed were purified and analyzed. Data showed that the activity of mutant enzyme N64S was higher than that of rCBHI expressed in P. pastoris. It indicated that the Asn64 glycosylation has an effect on the activity of the CBHI expressed in P. pastoris. Comparing the activities of CBHI from different mutants and native CBH I, experimental data showed that hyperglycosylation is only a limited factor for the low activity of recombinant CBHI in P. pastoris.更多还原