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Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.

ABSTRACT: A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0-9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30-32.5?°C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20?°C, respectively, and the Ea, ?G(*) (30?°C), Km (30?°C) and kcat (30?°C) values for hydrolysis of sucrose by rInvHJ14 was 47.6?kJ mol(-1), 57.6?kJ mol(-1), 62.9?mM and 746.2?s(-1), respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0?mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged.

SUBMITTER: Zhou J

PROVIDER: S-EPMC4995436 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.

Zhou Junpei J He Limei L Gao Yajie Y Han Nanyu N Zhang Rui R Wu Qian Q Li Junjun J Tang Xianghua X Xu Bo B Ding Junmei J Huang Zunxi Z

Scientific reports 20160824

A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0-9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30-32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG(* ...[more]

PMID: 27553125

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Project description:BackgroundSucrose invertase is an important catalytic enzyme that is widely distributed in plants and can irreversibly hydrolyze sucrose into fructose and glucose. Daylily is an important perennial flower worldwide and a traditional vegetable in East Asia. Previous studies have suggested that sucrose invertase is involved in the aging of daylily flowers. However, knowledge about the number, physicochemical properties, and expression patterns of daylily sucrose invertases is still lacking. Identifying the daylily sucrose invertase family genes in the genome is highly important for understanding phylogenetic evolution and determining the genetic function of sucrose invertase.MethodsTo obtain basic knowledge about the number, classification, sequence composition, and physicochemical properties of sucrose invertases in daylily, bioinformatics software was used to analyze the genome of Hemerocallis citrina (H. citrina), and the basic properties of sucrose invertase genes and proteins were obtained. Then, combined with transcriptome data from flower organs at different developmental stages, the expression patterns of each gene were clarified. Finally, the reliability of the transcriptome data was verified by quantitative real-time polymerase chain reaction (PCR).ResultsThrough software analysis, 35 sucrose invertases were identified from the H. citrina genome and named HcINV1-HcINV35; these enzymes belong to three subfamilies: cell wall invertases, vacuolar invertases, and chloroplast invertases. The amino acid composition, motif types, promoter composition, gene structure, protein physicochemical properties, gene chromosomal localization, and evolutionary adaptability of daylily invertases were determined; these results provided a comprehensive understanding of daylily invertases. The transcriptome expression profile combined with fluorescence quantitative reverse transcription-polymerase chain reaction (RT‒PCR) analysis suggested that almost all daylily invertase genes were expressed in flower organs, but even genes belonging to the same subfamily did not exhibit the same expression pattern at different developmental stages, suggesting that there may be redundancy or dissimilation in the function of daylily sucrose invertases.

Dataset Information

Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.

Publications

Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.

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