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Unravelling the diversity of glycoside hydrolase family 13 ?-amylases from Lactobacillus plantarum WCFS1.

ABSTRACT: BACKGROUND:?-Amylases specifically catalyse the hydrolysis of the internal ?-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main ?-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming ?-amylase (Lp_0179) and maltogenic ?-amylase (Lp_2757) were included. RESULTS:In this study, Lp_0179 and Lp_2757 L. plantarum ?-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-?-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards ?-1,4-linkages typical of maltogenic amylases. CONCLUSIONS:The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic ?-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of ?-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

SUBMITTER: Plaza-Vinuesa L

PROVIDER: S-EPMC6815381 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1.

Plaza-Vinuesa Laura L Hernandez-Hernandez Oswaldo O Moreno F Javier FJ de Las Rivas Blanca B Muñoz Rosario R

Microbial cell factories 20191026 1

<h4>Background</h4>α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included.<h4>Results</h4>In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases we ...[more]

PMID: 31655584

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Project description:BACKGROUND: Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality. RESULTS: We have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA. CONCLUSION: This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

Dataset Information

Unravelling the diversity of glycoside hydrolase family 13 ?-amylases from Lactobacillus plantarum WCFS1.

Publications

Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1.

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