厭氣性真菌Neocallimastix frontails 木聚醣酶基因之選殖、表現與重組蛋白酵素特性分析

Abstract

木聚醣

Xylanases are widely used in varies industries, majored in pulp and paper, animal feed and food. Cost required for enzymes is concerned in its industry application and xylanases with high specific activity are asked for its enzyme market. Anaerobic fungi harbored in rumen, a competitive ecosystem has evolved to produce highly specific-activity xylanases or other polysaccharidases and become to one of the potential xylanases producing species. The absolutely anaerobic condition of anaerobic fungi limited them to ferment in large scale easily. Since that, an expression system with benefits of easy-handled and large-scale fermentation is more suitable for production of anaerobic fungal xylanase by introduced its related genes into. In this study, a xylanase cDNA library of anaerobic fungus Neocallimastix frontalis was constructed by mRNA substraction and screened a lot of xylanase genes. These selected genes were deduced into two kinds of modular structure of family 11 xylanases. One has one catalytic domain and two dockerin domains in one polypeptide; another is two catalytic domains and two dockerin domains. Among these genes, the xylanase gene (xyn11C) with one catalytic domain and two dockerin domains discovered was first reported in genus of Neocallimastix. Xylanase is one of anaerobic fungal cellulosomal enzymes, its dockerin domains were used to anchor them to its cellulosome. Does removal of dockerin domains have effects on the enzyme-activity exhibition of the related catalytic domains? Xyn11A and Xyn11B represented the anaerobic fungal xylanases with one or two catalytic domains were investigated. After deletion of dockerin domains of Xyn11A and Xyn11B, both of them had significantly increment of the optimal temperatures as attributed to the lower stability of the second structure in full length xylanase than that in the truncated one. The removal of dockerin domains increase specific activity as reflected by the values of kcat/Km. According to these results, the existence of dockerin domains had side effects on the enzyme-activity exhibition of the related catalytic domains. For large-scale expression of anaerobic fungal xylanase, Xyn11B’ exhibited the highest specific activity in studies of effect of dockerin domains on enzyme activity was selected to express in methyltrophic yeast Pichia methanolica. In P. methanolica expression of Xyn11B’, high copy-number integration of xyn11B’ resulted by heterologous recombination was identified. Transformant 34 with the highest xylanase expression level in culture supernatant reached 635 U/ml after 120 h by induction of 1 % methanol per 24 h. No significant signals of Xyn11B’ in cytoplasm and cell lysate of P. methanolica revealed the efficient secretion of Xyn11B’ by host The Xyn11B’ expressed by P. methanolica was truncated at C-terminal and resulted in smaller protein size than prediction To realizing the reasons of xyn11B’ truncated by P. methanolica, the completeness of xyn11B’ transcript and proteases-sensitive test of Xyn11B’ were conducted. The full-length of xyn11B’ transcript was amplified by 3’-RACE. Xyn11B’ expressed by E. coli was truncated at N-terminal by serine, cystein and aspartic proteases of P. methanolica. There was no effect on eliminating proteolysis of Xyn11B’ by protease-deficient strain P. methanolica PMAD16.