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

• Published in: Applied microbiology and biotechnology 2016-07, Vol.100 (14), p.6291-6307
• Main Authors: Kahar, Ummirul Mukminin, Ng, Chyan Leong, Chan, Kok-Gan, Goh, Kian Mau
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analysis; Anoxybacillus - enzymology; Bacillus (Bacteria); Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bioinformatics; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Carbohydrate Metabolism; Computational Biology; Culture Media - chemistry; Enzymes; Gene Expression Regulation, Enzymologic; Genes; Genomes; Glucans - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Health aspects; Hydrogen-Ion Concentration; Hydrolysis; Life Sciences; Maltose - analogs & derivatives; Maltose - chemistry; Microbial Genetics and Genomics; Microbiology; Molecular Weight; Observations; Oligosaccharides - chemistry; Protein Conformation; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharides; Sequence Alignment; Servers; Starch; Starch - chemistry; Studies; Substrate Specificity; Sugar; Temperature; Trisaccharides - chemistry
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-016-7451-6

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.