Enzyme Characterization and Cloning of Mannanase Gene from Bacillus subtilis Isolated from Tempe

Abstract

Nutrition value and characteristics of tempe are primarily derived from enzymatic activities derived from complex microorganisms community during its fermentation. As a rich source of bacterial diversity, tempe could be explored for its potential to yield enzyme, such as mannanase. This study was intended to isolate mannanase producing bacteria derived from a number of tempe samples from Java (Bogor, Surabaya, Malang, Kediri, Lamongan, and Wonogiri); characterize its enzyme; and clone its mannanase gene into E. coli. Crude enzyme from each isolate was prepared for characterization. Showed the highest specific activity (0,41 U/mg protein), isolate N mannanase was further characterized. Nmannanase crude enzyme showed optimum activity at 60oC and pH 5.0. This enzyme has better activity in palm kernel meal (PKM) as its substrate with average activity 1.13 U/ml compared to soybean meal (SBM) (0.43 U/ml) and copra meal (CM) (0.48 U/ml). This enzyme was also stable at 60oC for 4 hours, in wide range of pH (4-10) for 1 hour, and various metal ions. SDS-PAGE and Zymogram analysis showed that this mannanase has molecular weight approximately 39.8 kilodalton. Isolate N was identified having 98% similarity with Bacillus subtilis (Genebank access JN644507) employing 16S rRNA sequence analysis. The B. subtilis N-mannanase gene was successfully isolated and cloned into E.coli. Recombinant’s mannanase activity was detected in E. coli intra- (8.82 U/ml) and extracellular (6.39 U/ml) extracts.