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

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 80Â kDa) was stable at pHÂ 5.0–6.0 and was most active at pHÂ 6.0. Th...

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Main Authors: Kahar, U. M., Ng, C. L., Chan, K. G., Goh, K. M.
Format: Article
Published: Springer Verlag 2016
Subjects:
QH Natural history
Online Access:http://eprints.utm.my/id/eprint/72363/
https://www.scopus.com/inward/record.uri?eid=2-s2.0-84961806498&doi=10.1007%2fs00253-016-7451-6&partnerID=40&md5=14cd6e31e1ec4d5fe20914288469a594
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spelling my.utm.723632017-11-20T08:23:44Z http://eprints.utm.my/id/eprint/72363/ Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis Kahar, U. M. Ng, C. L. Chan, K. G. Goh, K. M. QH Natural history 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. Springer Verlag 2016 Article PeerReviewed 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 https://www.scopus.com/inward/record.uri?eid=2-s2.0-84961806498&doi=10.1007%2fs00253-016-7451-6&partnerID=40&md5=14cd6e31e1ec4d5fe20914288469a594
institution Universiti Teknologi Malaysia
building UTM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Teknologi Malaysia
content_source UTM Institutional Repository
url_provider http://eprints.utm.my/
topic QH Natural history
spellingShingle QH Natural history
Kahar, U. M.
Ng, C. L.
Chan, K. G.
Goh, K. M.
Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis
description 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.
format Article
author Kahar, U. M.
Ng, C. L.
Chan, K. G.
Goh, K. M.
author_facet Kahar, U. M.
Ng, C. L.
Chan, K. G.
Goh, K. M.
author_sort Kahar, U. M.
title Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis
title_short Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis
title_full Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis
title_fullStr Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis
title_full_unstemmed Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis
title_sort characterization of a type i pullulanase from anoxybacillus sp. sk3-4 reveals an unusual substrate hydrolysis
publisher Springer Verlag
publishDate 2016
url http://eprints.utm.my/id/eprint/72363/
https://www.scopus.com/inward/record.uri?eid=2-s2.0-84961806498&doi=10.1007%2fs00253-016-7451-6&partnerID=40&md5=14cd6e31e1ec4d5fe20914288469a594
_version_ 1643656420409737216
score 13.211869

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