Purified PCR products were sequenced and sequence search similarities were conducted using BLAST.4 and 15 Phylogenetic analysis of sequence data of bacteria under study was aligned with reference sequence homology from the NCBI database using the multiple sequence alignment of MEGA 5.0 Program.16 Scale up studies were carried out in a 5 L glass fermentor (Model: Bio Spin-05A, Bio-Age) with a working volume of 3.5 L containing [Sago starch – 10 g, Yeast Extract – 20 g, KH2PO4 – 0.05 g, MnCl2·4H2O – 0.015 g, MgSO4·7H2O – 0.25 g, CaCl2·2H2O CHIR99021 – 0.05 g, FeSO4·7H2O – 0.01 g, Cysteine 1 g (g/L)] at pH – 7.0. Fermentor

glass vessel containing 3.5 L of fermentation medium was sterilized in an autoclave for 20 min at 15 lbs pressure (at 121 °C) and cooled to room temperature. 350 ml of 10% inoculum was transferred to the fermentor vessel through a port at the top plate under aseptic conditions. The incubation temperature was click here 32 °C, while the aeration and agitation rates were maintained at 0.8 L/L/min (DO) and 95 rpm respectively throughout the fermentation period. The air to be supplied was sterilized by passing through Millipore membrane filters (0.2 μm pore size). Sterilized

solution of 1 N HCl/NaOH was used for pH adjustment. Sterilized polypropylene glycol (0.01% (v/v) of 50%) was used to control foam, formed during the heptaminol fermentation process. After incubation, the fermented broth was filtered. The filtrate was used for the estimation of alpha amylase.17 Sago industrial waste soil samples were used for isolation

of amylase producing bacteria on SAM. Totally 30 different soil samples were collected from sago starch industry waste sites. Among that 22 isolates showed amylase activity upon primary screening using SAM supplemented with cassava starch as a carbon source. Only two out of 22 isolates showed high amylase activity. One potential isolate (SSII2) was identified by standard morphological and biochemical characterization and it was confirmed to be Bacillus sp. The maximum amount of amylase production was observed with 42 h incubation. The high protein content of 2.99 U/mg and the maximum enzyme activity of 456 U/ml was observed at 24 h (Fig. 1a). The main advantage of enzyme production by Bacillus sp. is a shorter incubation period which will reduce cost as well as autolysis of the enzyme created by protease itself during the fermentation process. 18 Previously amylase activity had been reported in B. subtilis (22.92 U/ml) after 72 h and Bacillus amyloliquefaciens after 72 h. 6 Maximum yield of 550 U/ml of enzyme and protein content 3.43 U/mg was observed at 32 °C ( Fig. 1b). A decrease in enzyme yield was observed with further increases in temperature.