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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.

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Acknowledgments

This work was supported by the University of Malaya via High Impact Research Grants (UM.C/625/1/HIR/MOHE/CHAN/01 [Grant No. A-000001-50001] and UM.C/625/1/HIR/MOHE/CHAN/14/1 [Grant No. H-50001-A000027]) awarded to Kok-Gan Chan. Kian Mau Goh is grateful for funding received from Universiti Teknologi Malaysia GUP (Grant 09H98).

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Kahar, U.M., Ng, C.L., Chan, KG. et al. Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis. Appl Microbiol Biotechnol 100, 6291–6307 (2016). https://doi.org/10.1007/s00253-016-7451-6

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  • DOI: https://doi.org/10.1007/s00253-016-7451-6

Keywords

  • Anoxybacillus
  • Glycoside hydrolase 13
  • Isoamylase
  • Pullulan
  • Pullulanase
  • Thermostable enzyme