Dataset Information

Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin.

ABSTRACT: The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10(8) to 10(7) Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying alpha-amylase, bacterial liquefying alpha-amylase, beta-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.

SUBMITTER: Kamasaka H

PROVIDER: S-EPMC123897 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin.

Kamasaka Hiroshi H Sugimoto Kazuhisa K Takata Hiroki H Nishimura Takahisa T Kuriki Takashi T

Applied and environmental microbiology 20020401 4

The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10(8) to 10(7) Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrol ...[more]

PMID: 11916682

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Project description:Increasing the ratio of amylose in the diet can increase the quantity of starch that flows to the large intestine for microbial fermentation. This leads to the alteration of microbiota and metabolite of the hindgut, where the underlying mechanism is not clearly understood. The present study used a combination of 16S amplicon sequencing technology and metabolomics technique to reveal the effects of increasing ratios of amylose/amylopectin on cecal mucosa- and digesta-associated microbiota and their metabolites in young goats. Twenty-seven Xiangdong black female goats with average body weights (9.00 ± 1.12 kg) were used in this study. The goats were randomly allocated to one of the three diets containing starch with 0% amylose corn (T1), 50% high amylose corn (T2), and 100% high amylose corn (T3) for 35 days. Results showed that cecal valerate concentration was higher (P < 0.05) in the T2 group than those in the T1 and T3 groups. The levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were decreased (P < 0.05) in cecal tissue while IL-10 was increased (P < 0.05) in the T2 group when compared with T1 or T3 groups. At the phylum level, the proportion of mucosa-associated Spirochaetes was increased (P < 0.05), while Proteobacteria was deceased by feeding high amylose ratios (P < 0.05). The abundance of Verrucomicrobia was decreased (P < 0.05) in the T3 group compared with the T1 and T2 groups. The abundance of digesta-associated Firmicutes was increased (P < 0.05) while Verrucomicrobia and Tenericutes were deceased (P < 0.05) with the increment of amylose/amylopectin ratios. The LEfSe analysis showed that a diet with 50% high amylose enriched the abundance of beneficial bacteria such as Faecalibacterium and Lactobacillus in the digesta and Akkermansia in the mucosa compared with the T1 diet. The metabolomics results revealed that feeding a diet containing 50% high amylose decreased the concentration of fatty acyls-related metabolites, including dodecanedioic acid, heptadecanoic acid, and stearidonic acid ethyl ester compared with the T1 diet. The results suggested that a diet consisting of 50% high amylose could maintain a better cecal microbiota composition and host immune function.

Project description:The disproportionation activity of cyclodextrin glucosyltransferase (CGTase; EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. In this study, Leu277 of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu253, was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced Km for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased kcat/Km (1,175 s-1 mM-1 versus 686.1 s-1 mM-1), compared with those of the wild-type enzyme. The disproportionation-to-hydrolysis ratio of L277M was 2.4-fold greater than that of the wild type. Existing structural data were combined with a multiple-sequence alignment and Gly282 mutations to examine the mechanism behind the effects of the Leu277mutations. The Gly282 mutations were included to aid a molecular dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu253 and the hydrophobicity of the +2 subsite combine to produce the observed effects. IMPORTANCE In this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation-to-hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.

Dataset Information

Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin.

Publications

Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin.

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