Dataset Information

Crystal structure of a raw-starch-degrading bacterial ?-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13.

ABSTRACT: Subfamily 37 of the glycoside hydrolase family GH13 was recently established on the basis of the discovery of a novel ?-amylase, designated AmyP, from a marine metagenomic library. AmyP exhibits raw-starch-degrading activity and consists of an N-terminal catalytic domain and a C-terminal starch-binding domain. To understand this newest subfamily, we determined the crystal structure of the catalytic domain of AmyP, named AmyP?SBD, complexed with maltose, and the crystal structure of the E221Q mutant AmyP?SBD complexed with maltotriose. Glu221 is one of the three conserved catalytic residues, and AmyP is inactivated by the E221Q mutation. Domain B of AmyP?SBD forms a loop that protrudes from domain A, stabilizes the conformation of the active site and increases the thermostability of the enzyme. A new calcium ion is situated adjacent to the -3 subsite binding loop and may be responsible for the increased thermostability of the enzyme after the addition of calcium. Moreover, Tyr36 participates in both stacking and hydrogen bonding interactions with the sugar motif at subsite -3. This work provides the first insights into the structure of ?-amylases belonging to subfamily 37 of GH13 and may contribute to the rational design of ?-amylase mutants with enhanced performance in biotechnological applications.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5355875 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Crystal structure of a raw-starch-degrading bacterial α-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13.

Liu Yanhong Y Yu Jigang J Li Fudong F Peng Hui H Zhang Xuecheng X Xiao Yazhong Y He Chao C

Scientific reports 20170317

Subfamily 37 of the glycoside hydrolase family GH13 was recently established on the basis of the discovery of a novel α-amylase, designated AmyP, from a marine metagenomic library. AmyP exhibits raw-starch-degrading activity and consists of an N-terminal catalytic domain and a C-terminal starch-binding domain. To understand this newest subfamily, we determined the crystal structure of the catalytic domain of AmyP, named AmyP<sub>ΔSBD</sub>, complexed with maltose, and the crystal structure of th ...[more]

PMID: 28303907

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Project description:BackgroundRaw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value.ResultsIn this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production.ConclusionsThis study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Project description:Hemicelluloses, such as xyloglucan, xylan and mannans, consist of a heterogeneous array of plant-derived polysaccharides that form the plant cell wall. These polysaccharides differ from each other in their structure and physiochemical properties, but they share a β-(1,4)-linked sugar backbone. Hemicelluloses can be hydrolyzed by plant-cell-wall-degrading enzymes (PCWDEs), which are widely distributed in phytopathogenic microbes. Recently, it has become apparent that phytophagous beetles also produce their own PCWDEs. Our previous work identified genes encoding putative mannanases belonging to the subfamily 10 of glycoside hydrolase (GH) family 5 (GH5_10) in the genomes of the leaf beetle, Gastrophysa viridula (Chrysomelidae, Chrysomelinae; one gene), and of the bean beetle, Callosobruchus maculatus (Chrysomelidae, Bruchinae; four genes). In contrast to proteins from other GH5 subfamilies, GH5_10 proteins are patchily distributed within the tree of life and have so far hardly been investigated. We addressed the following questions: Are beetle-derived GH5_10s active PCWDEs? How did they evolve? What is their physiological function? Using heterologous protein expression and enzymatic assays, we show that the G. viridula GH5_10 protein is an endo-β-1,4-mannanase. We also demonstrate that only one out of four C. maculatus GH5_10 proteins is an endo-β-1,4-mannanase, which has additional activity on carboxymethyl cellulose. Unexpectedly, another C. maculatus GH5_10 protein has evolved to use xylan instead of mannans as a substrate. RNAi experiments in G. viridula indicate (i) that the sole GH5_10 protein is responsible for breaking down mannans in the gut and (ii) that this breakdown may rather be accessory and may facilitate access to plant cell content, which is rich in nitrogen and simple sugars. Phylogenetic analyses indicate that coleopteran-derived GH5_10 proteins cluster together with Chelicerata-derived ones. Interestingly, other insect-derived GH5_10 proteins cluster elsewhere, suggesting insects have several independent evolutionary origins.

Dataset Information

Crystal structure of a raw-starch-degrading bacterial ?-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13.

Publications

Crystal structure of a raw-starch-degrading bacterial α-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13.

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