Functional analysis of substrate binding domain in cellulase and hemicellulase

(1)里氏木霉内切葡聚糖酶III(EG III)和阿拉伯呋喃诺苷酶(AF)分泌功能的改进为了研究EG III与纤维素结合域嵌合酶的酶学性质，并对AF的C-末端木聚糖结合域进行功能分析，改进了裂殖酵母异源蛋白分泌系统，使其能够表达EG III和AF。在被其他纤维素酶和木聚糖酶取代的EG III信号肽中，CBH II信号肽使EG III的产量比天然信号肽增加3倍。此外，被CBH II信号肽取代的AF在S.pombe中的分泌是其自身信号肽的100倍。(2)纤维素结合结构域(CBD)和EG III对纤维素酶的作用为了阐明CBD在纤维素酶水解中的作用，将EG III与其他纤维素酶的CBD连接，构建了嵌合体EG III。在带有CBH II信号肽的庞氏链霉菌中表达了两种嵌合体酶，即附着在EG III上的里氏锥虫CBH I或CBH II嵌合酶。虽然嵌合体的分子质量因过度糖基化而增加，但其比活性在加入CBD前后没有变化。酶与纤维素的吸附实验表明嵌合体中的CBD具有功能。无论是EG III和CBD都能提高细菌微晶纤维素的水解酶活性，以及与CBH对结晶纤维素的协同酶促反应。

(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.