Transglycosylation Activity of Catalytic Domain Mutant of Endo-1,3-β-glucanase from Cellulosimicrobium cellulans

Author(s): Yoshiji Hantani, Shoko Motoki, Atsushi Miyagawa, Hatsuo Yamamura, Masayuki Oda*.

Journal Name: Protein & Peptide Letters

Volume 25 , Issue 8 , 2018

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Background: Oligosaccharides are of great value in drug discovery programs which address a wide range of therapeutic strategies in medical specialties. However, owing to difficulties in oligosaccharide synthesis by conventional methods, oligosaccharide assembly using enzymes has been explored. The transglycosylases have been demonstrated to be effective for the oligosaccharide synthesis. Further studies are required to improve the specificity and activity of transglycosylases. There is an additional approach to use mutated glycosidase which transforms into glycosyltransferase with a decreased hydrolytic activity. The substitution of catalytic residue in glycosidase results in the loss of hydrolytic activity. During the reaction with glucanase, reaction of water with the substrate - enzyme intermediate results in the production of a hydrolyzed sugar. When the water molecule is replaced by a competing sugar, a new glycoside linkage is formed as a result of transglycosylation.

Objective: In this article, we evaluated the transglycosylation activity of endo-1,3-β-glucanase mutant, E119G, toward laminarioligosaccharides under various pH and temperature conditions, in comparison with those of the wild-type enzyme. We also analyzed the effect of glucose and laminaribiose on the transglycosylation activity.

Method: In this article, we generated the E119G mutant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1. The residue, Glu119, would act as a nucleophile in the reaction and affect the balance between hydrolysis and transglycosylation. The enzymatic activities of wild-type and E119G were estimated by detecting the products obtained from laminarioligosaccharides as substrates. We also analyzed the effect of reaction conditions such as temperature and pH on the enzymatic activity of E119G toward laminaritriose. We further analyzed the enzymatic activity of E119G toward laminaritriose in the presence of glucose or laminaribiose to investigate whether these additional molecules could accelerate the transglycosylation activity.

Results: The purified E119G mutant of endo-1,3-β-glucanase was properly folded, and exhibited the secondary structure, similar to that of wild-type. The E119G mutant exhibited enhanced transglycosylation activity and decreased hydrolytic activity, relative to the wild-type. The hydrolytic as well as transglycosylation activities of E119G decreased with the decrease in temperature, however, the ratio of transglycosylation products increased. The temperature-dependent degree of reduction in hydrolytic activity was higher than that in the transglycosylation activity. The enzymatic activities were similar within the range of pH 4.0 - 7.4, while those at pH 8.0 and 8.5 were slightly decreased. The enzymatic activity of E119G toward laminaritriose in the presence of glucose was ineffective, while the addition of laminaribiose evidently increased the transglycosylation products such as laminaritetraose and laminaripentaose.

Conclusion: A mutation of catalytic residue, Glu119 to Gly, in endo-1,3-β-glucanase from Cellulosimicrobium cellulans exhibited transglycosylation activity on laminarioligosaccharides. The combination of laminaribiose and laminaritriose as a substrate enhanced the transglycosylation activity. According to the structural information previously reported, laminaritriose mainly binds to the enzyme at the subsites from −1 to −3 and forms a link with laminaribiose, which transiently binds to the subsites +1 and +2. To increase the amount of transglycosylation product, the reaction was found to be effective at low temperature.

Keywords: Endo-1, 3-β-glucanase, laminarioligosaccharide, mutation, structure - function relationship, substrate specificity, transglycosylation.

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Article Details

Year: 2018
Page: [734 - 739]
Pages: 6
DOI: 10.2174/0929866525666180704094109
Price: $65

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