A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization

Abstract

An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.

Morphological, enzymatic screening, and phylogenetic analysis of thermophilic bacilli isolated from five hot springs of Myagdi, Nepal

“…Amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was detected in cell-bound fraction [44]. The emerging picture is that association of fibrolytic enzymes within cell differs from one enzyme to another and with the age of the cell.…”

Characterization of the starch-acting MaAmyB enzyme from Microbacterium aurum B8.A representing the novel subfamily GH13_42 with an unusual, multi-domain organization

“…The role of FNIII domains in carbohydrate acting enzymes in general has not been studied in detail. Studies that addressed the roles of FNIII domains in such enzymes are inconclusive25262728293031323334353637. FNIII domains have been suggested to act as flexible linkers252936 which corresponds to our findings for MaAmyA.…”

The evolutionary origin and possible functional roles of FNIII domains in two Microbacterium aurum B8.A granular starch degrading enzymes, and in other carbohydrate acting enzymes

“…In these studies, removal of only the CBM usually affects activity on insoluble substrates [15,16,[34][35][36][37][38][39][40], while the additional removal of the FNIII domains showed no further effects. In a study of the chitinase A1 from Bacillus circulans WL-12a, the single CBM present was re-attached after removal of one or two intermediate FNIII domains, allowing a direct test for the FNIII function [14].…”