嗜鹽菌Vibrionaceae sp. NTU 05纖維水解酶基因之選殖與表現

Abstract

Cellulase plays an important role in the degradation and recycle of fiber wastes. Conversion of cellulose has great potential in biomass application such as energy. This study has investigated cellulase gene from a halophilic strain Vibrionaceae sp. NTU 05, isolated from saline area in southern Taiwan. Cellulase gene from the bacteria has been cloned and heterogeneous expressed for application in simultaneous hydrolysis and fermentation. This study has amplified cellulase gene from Vibrionaceae sp. NTU05 genomic DNA containing 1,232 bp by PCR using CELCF / CELCR primer, which was suitable in the amplification of celC in Bacillus licheniformis. The target gene contains a complete opening reading frame composes of 807 nucleotide that translates 269 amino acid residues that showed 96 % homology to Bacillus licheniformis celC (AM183792.1). The celC was ligated with pGEM®-T easy vector and further transformed in E. coli DH5α. The celC was subsequently subcloned into the pET30a（+）expression vector, and expressed it in Escherichia coli BL21 (DE3). The transformant T1 was cultivated on the LB-CMC plate and detected by Congo red test. Hydrolysis halo is clearly been observed around the colony. The activity was determined at 63.7 mU / mL in intracellular, and no activity was detected in extracellular. The enzyme is specific for the substrate of β-1,4 -linkages of CMC and β-1,3、β-1,4 –linkages of β-glucan. The optimal temperature and pH value for activity of enzyme are detected at 40℃ and pH 7.0, respectively. The activity remained at 80 % when 5 % salinity was given, and retained higher than 70% in 10 % salinity environment for more than 1 hour. The protein was analyzed by fast protein liquid chromatography (FPLC). The purified products were analyzed by native-PAGE, which showed a molecular weight of 29.5 kDa, as expected. This study has successfully established the cloning and heterogeneous expression system of the celC from Vibrionaceae sp. NTU 05.