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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 ?-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 ?-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.

SUBMITTER: Kahar UM

PROVIDER: S-EPMC3709733 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A high molecular-mass Anoxybacillus sp. SK3-4 amylopullulanase: characterization and its relationship in carbohydrate utilization.

Kahar Ummirul Mukminin UM Chan Kok-Gan KG Salleh Madihah Md MM Hii Siew Mee SM Goh Kian Mau KM

International journal of molecular sciences 20130528 6

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 ...[more]

PMID: 23759984

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Project description:Polysaccharides comprising plant biomass are potential resources for conversion to fuels and chemicals. These polysaccharides include xylans derived from the hemicellulose of hardwoods and grasses, soluble ?-glucans from cereals and starch as the primary form of energy storage in plants. Paenibacillus sp. JDR-2 (Pjdr2) has evolved a system for bioprocessing xylans. The central component of this xylan utilization system is a multimodular glycoside hydrolase family 10 (GH10) endoxylanase with carbohydrate binding modules (CBM) for binding xylans and surface layer homology (SLH) domains for cell surface anchoring. These attributes allow efficient utilization of xylans by generating oligosaccharides proximal to the cell surface for rapid assimilation. Coordinate expression of genes in response to growth on xylans has identified regulons contributing to depolymerization, importation of oligosaccharides and intracellular processing to generate xylose as well as arabinose and methylglucuronate. The genome of Pjdr2 encodes several other putative surface anchored multimodular enzymes including those for utilization of ?-1,3/1,4 mixed linkage soluble glucan and starch.To further define polysaccharide utilization systems in Pjdr2, its transcriptome has been determined by RNA sequencing following growth on barley-derived soluble ?-glucan, starch, cellobiose, maltose, glucose, xylose and arabinose. The putative function of genes encoding transcriptional regulators, ABC transporters, and glycoside hydrolases belonging to the corresponding substrate responsive regulon were deduced by their coordinate expression and locations in the genome. These results are compared to observations from the previously defined xylan utilization systems in Pjdr2. The findings from this study show that Pjdr2 efficiently utilizes these glucans in a manner similar to xylans. From transcriptomic and genomic analyses we infer a common strategy evolved by Pjdr2 for efficient bioprocessing of polysaccharides.The barley ?-glucan and starch utilization systems in Pjdr2 include extracellular glycoside hydrolases bearing CBM and SLH domains for depolymerization of these polysaccharides. Overlapping regulation observed during growth on these polysaccharides suggests they are preferentially utilized in the order of starch before xylan before barley ?-glucan. These systems defined in Pjdr2 may serve as a paradigm for developing biocatalysts for efficient bioprocessing of plant biomass to targeted biofuels and chemicals.

Dataset Information

A high molecular-mass Anoxybacillus sp. SK3-4 amylopullulanase: characterization and its relationship in carbohydrate utilization.

Publications

A high molecular-mass Anoxybacillus sp. SK3-4 amylopullulanase: characterization and its relationship in carbohydrate utilization.

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