Cloning of Partial β-Mannanase Gene from Indonesia Marine Bacteria Bacillus Subtilis LBF-005

Abstract

The strain LBF-005 from marine bacteria have been already isolated and screened for mannanase degrading enzyme in submerged fermentation process. This strain was further identified by using 16S rRNA identification and showed that bacterium is belong to Bacillus subtilis that could produce mannanase with activity around 9.5 U/mL. The optimum pH and temperature for the activity of crude enzyme for mannanase were 6.0 and 50 o C, respectively. Cloning of mannanase gene from B. subtilis was conducted using six primer / set designed based on the homology analysis conserved region of several mannanases from bacteria (Bacillus sp.) glycosyl hydrolase (GH) family 26. Optimization of PCR conditions was performed by gradient PCR to obtain PCR product of -mannanase gene. The PCR product was obtained by the third primer combination and was estimated to be around 972-bp. Analysis of the nucleotide sequence indicated that the sequence has similarity with mananase gene from other Bacillus sp., such as the B. subtilis strain W LY-12, B. subtilis strain W L-8, B. subtilis strain CICC 10260, B.subtilis strain CD-25, B.subtilis strain G1, and Bacillus sp. SW U60 about 98%, 98%, 98%, 98%, 97% and 95%, respectively.