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Functional study of C-terminal domain of the thermoacidophilic raw starch-hydrolyzing α-amylase Gt-amy.

ABSTRACT: Since the thermoacidophilic raw-starch hydrolyzing α-amylase Gt-amy can effectively hydrolyze corn starch under starch liquefaction conditions, it has potential for many industrial applications. To identify the raw starch-binding domain of Gt-amy, a C-terminal domain (CTD)-truncated mutant (Gt-amy-T) was constructed, and its enzymatic properties were compared with Gt-amy. In comparison to CTD of Gt-amy, which could effectively bind corn starch, the Gt-amy-T could not bind to and hydrolyze corn starch under similar conditions. In addition, Gt-amy-T showed significantly lower thermal activity and thermal stability. Using soluble starch as the substrate, the k cat of Gt-amy-T at 80 °C was approximately 77.9% of that of Gt-amy. The half-life of Gt-amy at 80 °C was 3 h, while that of Gt-amy-T was 2 h. These results reveal that the CTD plays a vital role in raw starch binding and degradation by Gt-amy and helps Gt-amy maintain thermal activity and stability.

SUBMITTER: Zeng J

PROVIDER: S-EPMC7105561 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Functional study of C-terminal domain of the thermoacidophilic raw starch-hydrolyzing α-amylase Gt-amy.

Zeng Jing J Guo Jianjun J Tu Yikun Y Yuan Lin L

Food science and biotechnology 20190826 3

Since the thermoacidophilic raw-starch hydrolyzing <i>α</i>-amylase Gt-amy can effectively hydrolyze corn starch under starch liquefaction conditions, it has potential for many industrial applications. To identify the raw starch-binding domain of Gt-amy, a C-terminal domain (CTD)-truncated mutant (Gt-amy-T) was constructed, and its enzymatic properties were compared with Gt-amy. In comparison to CTD of Gt-amy, which could effectively bind corn starch, the Gt-amy-T could not bind to and hydrolyze ...[more]

PMID: 32257525

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Project description:The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

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.

Dataset Information

Functional study of C-terminal domain of the thermoacidophilic raw starch-hydrolyzing α-amylase Gt-amy.

Publications

Functional study of C-terminal domain of the thermoacidophilic raw starch-hydrolyzing α-amylase Gt-amy.

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