A method for screening 1-deoxyxylulose-5 phosphate racemase inhibitors from marine microbial fermentation broth

Abstract

The invention discloses a method of screening a 1-deoxyxylulose-5-phosphate racemase inhibitor from a marine microorganism fermentation broth. The method includes: 1) a step of preparing a marine microorganism fermentation broth sample, namely, a step of inoculating a fungus, fermenting, extracting metabolites, concentrating and eluting; 2) a step of designing a primer and performing PCR amplification by adopting the whole genome of vibrio vulnificus as a template, cloning a vector, converting escherichia coli, inducing expression of a target protein, and subjecting the target protein to elution, dialysis, ultrafiltration and desalting to obtain His-DXR; 3) a step of reacting a bacterial suspension, a resazurin solution and the marine microorganism fermentation broth sample obtained by the step 1), measuring the fluorescence value, calculating the inhibition rate through an equation, and screening the fraction the inhibition rate of which is higher than 80%; and 4) reacting the fraction screened in the step 3) and the His-DXR obtained in the step 2), calculating the inhibition rate, and screening the fraction the inhibition rate of which is not less than 50%. The method is simple and accurate, and lays a foundation for development of novel antibacterial medicines adopting Dxr as a therapeutic target or development of leading compounds.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for screening 1-deoxyxylulose-5 phosphate racemase inhibitors from marine microbial fermentation broth. Background technique [0002] Since Fleming discovered penicillin from fungi in 1929, microbial active metabolites have become an important resource for drug discovery. The application of penicillin marked the beginning of the era of antibiotics, and more and more terrestrial microbial metabolites were developed into natural medicines. In the past 70 years, about 30,000-50,000 kinds of natural products have been discovered from microorganisms, of which more than 10,000 kinds have biological activity, and more than 8,000 kinds are antibacterial and antitumor agents. According to incomplete statistics, more than 100 kinds have been produced by The pesticides, antineoplastic agents and antibiotics produced by microorganisms and used in clinics, the annual global antibiotic p...

AI Technical Summary

Problems solved by technology

In the study of the DXR gene of Arabidopsis, Rohdich et al. (2006) expressed the recombinant in Escherichia coli (Escherichia coli) after removing the N-terminal transit peptide sequence, and the purified protein obtained by affinity chromatography could be catalyzed by DXP to form MEP. The catalytic rate is 5.6molmin-1mg-1 (protein), this reaction requires the participation of NADPH and the divalent metal ion Mn 2+ and Mg 2+ , the optimal pH value is 8.0, although NADPH can be replaced by NADH, but the reaction rate after replacement drops to 14% of the original

Images