Vibrio alginolyticus KL-10之褐藻膠裂解酶基因的選殖、特性分析及異源表現

Abstract

Global warming and energy crisis are major drivers for a shift from the use of fossil fuels to renewable energy, and then, brown macroalgae exhibit several key features of an ideal feedstock for production of biofuels, such as requiring no arable land, fertilizer, or fresh water resources. Alginate is the most abundant sugars in brown macroalgae, therefore when microorganism fermentation combine alginate degradation technology, it will be a new strategy for bioethanol production. This study has isolated a gram-negative bacterium, which could degrade alginate and use as the sole carbon source for its growth, and it was taxonomically identified as Vibrio alginolyticus KL-10 by 16S rRNA sequencing. By referring to the homologus sequence of alginate lyase gene of Vibrio alginolyticus 40B which has been finished whole genome sequencing to design the PCR primers, the alginate lyase gene of Vibrio alginolyticus KL-10 was amplified from genomic DNA and sequenced. The alginate lyase gene was 1566 base pairs in length and could code for a protein product of 57.6 kDa including signal peptide. Cloning this gene with expression vector pET30c into the E.coli BL21 (DE3) and inducing with 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) at 16℃ for 18 hour, the alginate lyase activity of 69.82 Umg-1 was obtained from the cell curde extract, but the activity increased to 135.82 Umg-1 after purifing by nickel column. SDS-PAGE and zymogram analysis displayed that the fusion protein of alginate lyase and 6X His-tag had a molecular mass of 70 kDa, its optimum temperature and pH for enzyme activity were 30℃ and pH 8, respectively. In order to heterologously express the alginate lyase gene in ethanol-producing microorganisms, four shuttle vectors: pKT230 of Zymomonas mobilis/E.coli, pIMP1 of the Clostridium spp./E. coli, and pRS423 and pRS426 of Saccharomyces cerevisiae/E. coli were used to construct recombinant plasmids pKT230-alg, pIMP1-alg ,pRS423-alg, pRS426-alg. However, pKT230-alg and pIMP1-alg can not be transformed into Z. mobilis and C. xylanolyticum Ter3, respectively, by electroporation transformation. On the other hand, only pRS426-alg was successfully introduced into S. cerevisiae BY4741 by LiAc/PEG transformation, but for unknown reason the transformant did not express alginate lyase activity.