"嗜鹽菌Bacillus spp. NTU-06 β-1,4-內切型聚木醣水解酶基因之選殖與表現"

Abstract

半纖維素 (Hemicellulose) 為自然界中含量僅次於纖維素的多醣，此為一由多種多醣所組成的無結晶型異質多醣，其中又以聚木醣 (xylan) 為最主要的成分，聚木醣主要是木糖六元環 (xylopyranose) 以 β-1,4鍵結而成的多醣，若要完整降解聚木醣成單醣，則需要多種類型之聚木醣水解酶的參與，包含β-1,4-內切型聚木醣水解酶 (β-1,4-endoxylanase, EC 3.2.1.8)。本次實驗自嗜鹽菌Bacillus spp. NTU-06選殖出一642 bp之基因片段，此核酸序列與Bacillus licheniformis MS5-14 之內切型聚木醣水解酶基因 xyn11 (Accession no. EU591524) 相似度達99%，經推導後可轉譯出213個胺基酸，且與Bacillus licheniformis之內切型聚木醣水解酶基因 (Accession no. ACF05486) 具99%之相似度，屬於醣苷水解酶第11家族β-1,4-內切型聚木醣水解酶之成員，並命名為xyn6。XYN6之N端約30個胺基酸為訊息肽鏈 (signal peptide)，此訊息肽鏈可將前驅蛋白質引導至細胞膜上，經訊息胜肽酶 (signal peptidase) 切解作用，將具催化能力之成熟酵素分泌至細胞膜外。成熟之重組XYN6分子量大小為21.13 kDa，而重組之β-1,4-內切型聚木醣水解酶XYN6之最適作用環境溫度為60℃，並可穩定存在於40℃之環境達24小時，而其最適作用環境pH值為 pH 6.0，最適作用環境鹽度則為15-20% NaCl，且於30% NaCl的環境下仍能維持其一定之活性。以樺木聚木醣 (birchwood xylan) 做為基質進行酵素動力學分析，其Km值為10.48 mg mL-1、Vmax值則為217.39 IU mL-1，比活性則為869.27 IU mg-1。重組之XY6屬於嗜高溫與高嗜鹽性之β-1,4-內切型聚木醣水解酶，具有發展成為處理含鹽之農作廢棄物或木質纖維生物質之酵素單元，以改良或開發新型纖維質酒精之製程。

Hemicellulose is the second major component of lignocellulose biomass in nature. Complete degradation of this substrate requires several different enzymes, including β-1,4-endoxylanase (EC 3.2.1.8). A gene encoding a β-1,4-endoxylanase, named xyn6, was cloned from Bacillus sp. NTU-06 and was heteroexpressed in Escherichia coli BL21 (DE3). The gene, xyn6, consisted of 642 bp, and the translated protein encoded 213 amino acid residues. The sequence of xyn6 showed 99% similarity to β-1,4-endoxylanase xyn11 (Accession no. EU591524) from Bacillus licheniformis MS5-14, and the deduced amino acid sequence of xyn6 showed highest identity of 99% with an endoxylanase from Bacillus licheniformis (Accession no. ACF05486). According to the amino acid sequence similarities, this enzyme was assigned as a member of glycosyl hydrolases family 11. The 30 residue N-terminal sequence of the XYN6 was a putative signal peptide, and the mature recombinant xyn6 protein posses molecular mass of 21.13 kDa. The recombinant XYN6 showed an maximal activity at 60℃, pH 6.0. It also showed an optimal activity at 15-20% NaCl, and substantial activity remained at 30% NaCl. Using birchwood xylan as substrate, the enzyme displayed Km of 11.05 mg mL-1 and Vmax of 227.27 IU mL-1. The specific activity of purified XYN6 toward birchwood xylan was 869.27 IU mg-1. The xylanase XYN6 showed high activity under saline conditions, and shown to be industrial potential for various industrial processes such as wastewater treatment and bioethanol production.