Sequence analysis of starch degrading enzymes from anoxybacillus species

Abstract

The thermophilic Anoxybacillus sp. SK3-4 was formerly isolated from Sungai Klah hot spring, Malaysia. In a recent work, the genome of this bacterium was sequenced and the presence of several amylolytic enzymes was identified. Starch degrading enzymes have been greatly involved in several types of industrial applications typically for degradation and convertion to value-added sugars. Present work aimed to clone pullulanase type I, a-glucosidase, and pullulanase type II (amylopullulanase, Apu) and analyze the primary sequences through bioinformatic tools. The full length for pullulanase type I from Anoxybacillus was 2121bp (706 amino acids) with theoretical molecular weight of 82kDa while the a-glucosidase gene was 2343bp (780 amino acids, 90.5kDa). The Apu was 6102bp (2033 residues, 224.8kDa) and appeared to be huge in the size. Based on the analysis using SAPS software, the proteins were mainly constituted of hydrophobic amino acids. Pullulanase type I and a-glucosidase are most likely expressed inside the cell while Apu is an extracellular protein due to the presence of signal peptide. According to similarity search using Blastp, pullulanase and a- glucosidase shared higher similarities with counterpairs from Geobacillus and relative lower to Bacillus species. Most of these closest match sequences are deduced proteins obtained from genome sequencing data; hence the related information is thus limited. From conserved domain search, a-glucosidase is a member of Glycosyl Hydrolase (GH) 31 family, and was further proved by the evolutionary tree. The Apu exhibited a single active site for hydrolyzing a-1,4 and a-1,6 linkages in starch and is different to amylopullulanase from Bacillus sp. KSM1378 and Bifidobacterium breve UCC2003 that had dual catalytic properties. The Anoxybacillus Apu also has a surface-layer homology (SLH) domain at C-terminal which functions as an anchor that binds to the cell wall. In conclusion, based on the bioinformatic analysis, it is shown that the studied enzymes from Anoxybacillus sp. SK3-4 are novel and unexplored and future work should be done to study the biochemical properties of these enzymes.