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Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis

Kahar, U. M. and Ng, C. L. and Chan, K. G. and Goh, K. M. (2016) Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis. Applied Microbiology and Biotechnology, 100 (14). pp. 6291-6307. ISSN 0175-7598

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Abstract

Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0–6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26–72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362–370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.

Item Type:Article
Uncontrolled Keywords:Efficiency, Hydrolysis, Oligosaccharides, Starch, Sugars, Anoxybacillus, Glycoside hydrolases, Isoamylase, Pullulanase, Pullulans, Thermostable enzymes, Enzymes, amino acid, amylose, carbohydrate, dextrin, glycoside, maltotriose, oligosaccharide, pullulan, pullulanase, starch, type I pullulanase, unclassified drug, bacterial protein, culture medium, glucan, glycosidase, maltopentaose, maltose, maltotetraose, pullulanase, recombinant protein, trisaccharide, adsorption, bacterium, digestibility, enzyme activity, hydrolysis, polysaccharide, starch, substrate, temperature effect, Anoxybacillus, Article, bacterial gene, bioinformatics, controlled study, digestion, enzyme activity, enzyme specificity, hydrolysis, molecular weight, nonhuman, pH, protein expression, temperature, amino acid sequence, analogs and derivatives, Anoxybacillus, biology, carbohydrate metabolism, chemistry, culture medium, enzymology, gene expression regulation, genetics, hydrolysis, metabolism, protein conformation, sequence alignment, Anoxybacillus, Amino Acid Sequence, Anoxybacillus, Bacterial Proteins, Carbohydrate Metabolism, Computational Biology, Culture Media, Gene Expression Regulation, Enzymologic, Glucans, Glycoside Hydrolases, Hydrogen-Ion Concentration, Hydrolysis, Maltose, Molecular Weight, Oligosaccharides, Protein Conformation, Recombinant Proteins, Sequence Alignment, Starch, Substrate Specificity, Temperature, Trisaccharides
Subjects:Q Science > QH Natural history
Divisions:Biosciences and Medical Engineering
ID Code:72363
Deposited By: Narimah Nawil
Deposited On:20 Nov 2017 16:23
Last Modified:20 Nov 2017 16:23

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