A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

Abstract

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3 , the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples.

Figure 1

SDS-PAGE analysis of the ADA expression. E. coli BL21(DE3) cells containing pET-28a-add were grown and induced with 1 mM IPTG. The cells were sonicated and then centrifuged to divide into two fractions, soluble and insoluble fractions. Soluble fractions were then purified using Ni-NTA agarose. Lane 1, size markers; Lane 2, total proteins of the uninduced cells; Lane 3, total proteins of the IPTG-induced cells; Lane 4, purified protein of ADA.

Figure 2

Adenosine standard curves using ADA assay in H₂O (A and B), modified M9 medium (C and D), LB medium (E) and 10-fold diluted LB medium (F and G), respectively. Each plot represents the average of three samples. Absorbance was measured using a micro-plate reader.

Figure 3

A. Adenosine concentration in the fermentation flask determined using the CDA assay and HPLC.B. Correlation between the enzymatic determination of adenosine and adenosine added concentration in fermentation broth.Each experiment run in triplicate.

Figure 4

Screening high adenosine-producing strains using the ADA assay.A. Production of adenosine in a 96-well culture plate from randomly picked mutation strains. A1 represented for adenosine concentration of parent strain.B. Top four samples (B8, B9, B12 and A3) determined using the ADA assay were chosen to detect adenosine by HPLC. Each experiment run in triplicate.

Figure 5

Scheme of the enzymatic assay for adenosine detection at 697 nm.