title: Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis.
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.
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors.forename: Ummirul Mukminin
authors.initials: UM
authors.lastname: Kahar
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
authors.forename: Chyan Leong
authors.initials: CL
authors.lastname: Ng
authors.affiliation: Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia.
authors.forename: Kok-Gan
authors.initials: KG
authors.lastname: Chan
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. gohkianmau@utm.my.
authors.forename: Kian Mau
authors.initials: KM
authors.lastname: Goh
mesh_terms: D000595:Amino Acid Sequence; D058677:Anoxybacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D019295:Computational Biology; D003470:Culture Media / Q000737:chemistry*; D015971:Gene Expression Regulation, Enzymologic; D005936:Glucans / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013213:Starch / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D014312:Trisaccharides / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D005936:Glucans; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides; C019193:maltopentaose; C008317:maltotriose; D008320:Maltose; C009109:pullulan; D013213:Starch; C009819:maltotetraose; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: Anoxybacillus; Glycoside hydrolase 13; Isoamylase; Pullulan; Pullulanase; Thermostable enzyme