Functional analysis of substrate binding domain in cellulase and hemicellulase

(1)提高裂殖酵母分泌里氏木霉内切葡聚糖酶III（EG III）和阿拉伯呋喃糖苷酶（AF）的能力研究EG III与纤维素结合域嵌合酶的酶学性质，并对AF C端木聚糖结合域的功能进行分析。酵母中EG III和AF的表达得到改善。在EG III信号肽取代的其他纤维素酶和木聚糖酶，CBH II信号肽导致3倍增加的EG III生产比天然信号肽。此外，在S.粟酒裂殖酵母的信号肽的100倍。(2)除了EG III之外的纤维素结合结构域（CBD）对于纤维素水解的作用为了阐明CBD对于酶促纤维素水解的作用，嵌合体EG III由EG III构建，EG III由不含CBD的单个催化结构域组成，其连接到来自其他纤维素酶的CBD。两种嵌合体酶，T. reesei CBH I或CBH II连接到EG III，在S.与CBH II信号肽的pombe。虽然由于过度糖基化，嵌合体的分子量增加，但它们的比活性在添加CBD之前和之后没有变化。酶与纤维素的吸附实验表明嵌合体中的CBD是功能性的。无论是EG III与CBD的细菌微晶纤维素的水解活性增加，并在协同酶促反应与CBH结晶纤维素。

(1) Improvement of secretion in Schizosaccharomyces pombe of Trichoderma reesei endoglucanase III (EG III) and arabinofuranosidase (AF)To study on enzymatic characterization of the chimera enzyme of EG III with a cellulose binding domain and on functional analysis of C-terminal xylan binding domain of AF, heterologous protein secretion system in S. pombe was improved for EG III and AF expression. Among the EG III signal peptide substituted to those of other cellulases and a xylanase, the CBH II signal peptide led to 3-fold increase the EG III production than the native signal peptide. Furthermore AF substituted with CBH II signal peptide was secreted in S. pombe to 100-fold compared to its own signal peptide.(2) Effect of cellulose binding domain (CBD) in addition to EG III for cellulose hydrolysisTo clarify the role of CBD for enzymatic cellulose hydrolysis, the chimera EG IIIs were constructed by the EG III, which consists of single catalytic domain without CBD, joined to the CBDs from other cellulases. Two chimera enzymes, the CBD of T. reesei CBH I or CBH II attached to the EG III, were expressed in S. pombe with the CBH II signal peptide. Although the chimeras increased in molecular mass because of over-glycosylation, their specific activity was unchanged before and after the addition of the CBD. Adsorption experiment of the enzymes with cellulose suggested the CBD in chimera to be functional. Either EG III with CBD increased in the hydrolysis activity for bacterial microcrystalline cellulose and in synergistic enzymatic reaction with CBH for crystalline cellulose.