An Alternative Bacterial Expression System Using Bacillus pumilus SG2 Chitinase Promoter

Solid phase fermentation of wheat bran coupled with powdered fish scales could yield chitinase using a novel Citrobacter strain. The fermentation conditions are optimized conventionally (84.14 U/gds) and statistically (94.3 U/gds). The partially purified chitinase had a specific activity of 53.41 U/mg with optimal activity at pH 8.5 and 50 °C, it was stable at 7.5-10 pH range for 24 h with more than 80% stability at 45-60 °C, and the Km value of chitinase with swollen chitin (substrate) is 8.11 mg/ml with a V max of 2.43 mmol h −1 ml −1 . The partially purified enzyme was halotolerant showing maximum activity and stability up to 10% of Nacl, also possessing potential anticoagulant activity. This is the first report to date elucidating the production of halotolerant chitinase from wheat bran supplemented with fish scales using Citrobacter freundii haritD11 and its application as an anticoagulant agent and notable tolerance to heavy metal ions.

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Expression and Secretion of Cyan Fluorescent Protein (CFP) in B. subtilis using the Chitinase Promoter from Bacillus pumilus SG2

Shali

Rigi²,

Pornour

et al. 2017

IBJ

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Background:Improved cyan fluorescent protein (ICFP) is a monochromic, green fluorescent protein (GFP) derivative produced by Aequorea macrodactyla in a process similar to GFP. This protein has strong absorption spectra at wavelengths 426-446 nm. ICFP can be used in cell, organelle or intracellular protein labeling, investigating the protein-protein interactions as well as assessing the promoter activities.Methods:In our previous study, the promoters of two chitinases (ChiS and ChiL) from Bacillus pumilus SG2 were assessed in B. subtilis and their regulatory elements were characterized. In the present study, icfp was cloned downstream of several truncated promoters obtained in the former study, and ICFP expression was evaluated in B. subtilis.Results:Extracellular expression and secretion of ICFP were analyzed under the control of different truncated versions of ChiSL promoters grown on different media. Results from SDS-PAGE and fluorimetric analyses showed that there were different expression rates of CFP; however, the UPChi-ICFP3 construct exhibited a higher level of expression and secretion in the culture medium.Conclusion:Our presented results revealed that inserting this truncated form of Chi promoter upstream of the ICFP, as a reporter gene, in B. subtilis led to an approximately ten fold increase in ICFP expression.

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An Alternative Bacterial Expression System Using Bacillus pumilus SG2 Chitinase Promoter

Abstract: The chitinase promoter developed in this study, has potential to be used in an expression vector that could be induced by chitin. In addition, compared to the other inducers like IPTG and lactose, chitin is definitely cheaper and more available as an inducer.

Cited by 3 publications

References 36 publications

A Contemporary Appraisal on Impending Industrial and Agricultural Applications of Thermophilic-Recombinant Chitinolytic Enzymes from Microbial Sources

A Contemporary Appraisal on Impending Industrial and Agricultural Applications of Thermophilic-Recombinant Chitinolytic Enzymes from Microbial Sources

Anticoagulant activity of partially purified chitinase produced by Citrobacter freundii str. nov. haritD11 by fermentation of wheat bran coupled with fish scales

Expression and Secretion of Cyan Fluorescent Protein (CFP) in B. subtilis using the Chitinase Promoter from Bacillus pumilus SG2

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