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.

Figure 1

(a) The protein relationship tree of Apu from Anoxybacillus, Bacillus, Geobacillus, Thermoanaerobacter, Thermoanaerobacterium, Bifidobacterium, and Lactobacillus. (b) Schematic representation of conserved domains identified by motif search for the respective Apus.

Figure 2

Biochemical characterizations of ApuASK. (a) Effects of pH on activity (●) and stability (○) of ApuASK; (b) Effects of temperature on activity (●) and stability (○) of ApuASK; (c) Thermostability of ApuASK at 60 °C (■), 65 °C (□), and 70 °C (▼). Values are the mean ± standard error of triplicate analyses.

Figure 3

Analysis of reaction products using HPLC. (a) Production of glucose (black), maltose (light grey), and maltotriose (grey) by ApuASK on pullulan at different time intervals; (b) Production of glucose (black), maltose (light grey), and maltotriose (grey) by ApuASK on individual substrate of pullulan, soluble starch, amylose, amylopectin, glycogen, and dextrin.

Figure 4

Illustration of carbohydrate (i.e., starch) utilization in Anoxybacillus sp. SK3-4. Several types of putative transporters and enzymes that are involved in carbohydrate utilization in Anoxybacillus sp. SK3-4 were identified through the analyzed sequence data from genome sequencing. The localization of the enzymes was predicted using PSORTb 3.0. ApuASK and α-amylase from Anoxybacillus sp. SK3-4, ASKA [33] are cell-bound proteins whiles other enzymes are expressed intracellularly.