Cloning of a novel cellulase and lipase gene from bagasse compost metagenome and characterizations of their gene products

• Main Authors: Yi-Yun Hsieh, 謝依芸
• Other Authors: Ming-Chung Chang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/76368304651873622281

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 97 === The global petroleum crisis awakes human to concern about an exhaustive problem that energy will soon be insufficient. Hence the development of substitute energy becomes a newly anxious and worldwide subject. In all kinds of substitute energy, to use biomass derivatives as source for energy production is more environmental friendly than others. The biomass mainly takes the crops and the agricultural residues as raw materials and exhibits lower environment impacts. Biofuel composes two major types: bioethanol and biodiesel. We focused on key enzymes involved in bioethanol and biodiesel production, such as cellulases and lipases. In this study, we established microbial metagenomic library from bagasse compost and isolated two clones containing a novel glycosyl hydrolase family 6 (GHF 6) homologous partial sequence using colony hybridization. Subclone this gene revealed that both two clones composed of the same 1.5 kb DNA fragment, in which encoded a protein with cellulase activity (named as YYH-1). Amino acid sequences alignment showed YYH-1 contained a GHF 6 conserved domain sharing 56% similarity to those of cellulases belonged to this family. The molecular weight of YYH-1 was about 44.7kDa and its optimal enzyme activities reached under pH 6.5 and 45-55°C which is similar to the condition in bagasse compost. In substrate specificities of YYH-1, a wide range of polysaccharides including β-1,4-, β-1,4/β-1,3- or β-1,3/β-1,6-linked polysaccharides could be hydrolyzed into sugars. In addition, we found that YYH-1 can be secreted into cultured medium of E. coli and also exhibited its cellulase activity. In signal peptide prediction, there were two putative cleavage sites at 28th or 31th amino acid of YYH-1. To study the extracellularly secreted mechanism of YYH-1 was undertaken. As for another section, we also isolated a clone with lipase activity that contained a lipase gene (named as lip2). The molecular weight of Lip2 is 32.3 kDa and contained an esterase-lipase conserved domain and a GXSXG motif. Expression and purification of recombinant Lip2 in E. coli using pET expression vector was performed. We determined the Lip2 optimum activity occurred at pH 8.0 and 45-65°C. Additionally, Lip2 could hydrolyze preferentially carbonated p-nitrophenyl esters, especially the C2, C4, C6, C8 and C14 ones. For all, it is expected that YYH-1 and Lip2 could be utilized in the fiber digestion in compost of rice and corn stalks and transesterification of middle or long chain lipid. It is anticipated that the research would contribute to bioehtanol and biodiesel production and recycle of agriculture waste and various lipid source.