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

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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.

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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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Authors and Affiliations

  1. Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

    Ummirul Mukminin Kahar & Kian Mau Goh

  2. Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

    Chyan Leong Ng

  3. Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia

    Kok-Gan Chan

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  1. Ummirul Mukminin Kahar
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Correspondence to Kian Mau Goh.

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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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