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Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain.

ABSTRACT: Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5-10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

SUBMITTER: Safary A

PROVIDER: S-EPMC6378094 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain.

Safary Azam A Moniri Rezvan R Hamzeh-Mivehroud Maryam M Dastmalchi Siavoush S

BioImpacts : BI 20180913 1

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, O ...[more]

PMID: 30788256

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Project description:Background2,3-Butanediol (2,3-BD), a valuable compound used for chemicals, cosmetics, pesticides and pharmaceuticals, has been produced by various microbes. However, no high-temperature fermentation of the compound at high productivity has been reported.MethodsThermotolerant xylose-utilizing microbes were isolated from 6 different districts in Laos and screened for a low accumulation of xylitol in a xylose medium at 37 ˚C. One isolate was found to produce 2,3-BD and identified by 16S rDNA sequencing. The 2,3-BD fermentation capacity was investigated at different temperatures using xylose and glucose as carbon sources, and the fermentation parameters were determined by a high-performance liquid chromatography system.ResultsBy screening for a low accumulation of xylitol in a xylose medium, one isolate that accumulated almost no xylitol was obtained. Further analyses revealed that the isolate is Cronobacter sakazakii and that it has the ability to produce 2,3-BD at high temperatures. When xylose and glucose were used, this strain, named C. sakazakii OX-25, accumulated 2,3-BD in a short period before the complete consumption of these sugars and then appeared to convert 2,3-BD to acetoin. The optimum temperature of the 2,3-BD fermentation was 42 ˚C to 45 ˚C, and the maximum yield of 2,3-BD was 0.3 g/g at 12 h in 20 g/l xylose medium and 0.4 g/g at 6 h in 20 g/l glucose medium at 42 ˚C. The 2,3-BD productivity of the strain was higher than the 2,3-BD productivities of other non-genetically engineered microorganisms reported previously, and the highest productivity was 0.6 g/l·h and 1.2 g/l·h for xylose and glucose, respectively.ConclusionsAmong thermotolerant microbes isolated in Laos, we discovered a strain, C. sakazakii OX-25, that can convert xylose and glucose to 2,3-BD with high efficiency and high productivity at high temperatures, suggesting that C. sakazakii OX-25 has the potential for industrial application to produce 2,3-BD as an important platform chemical.

Dataset Information

Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain.

Publications

Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant <i>Bacillus</i> strain.

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