Recombinant Expression and Functional Assessment of Uricase from a Pertinent Origin of the Enzyme, Streptomyces sp. Strain 17-1

Abstract

Background: Uricase or urate oxidase, as a therapeutic enzyme, is extensively applied to oxidize accumulated uric acid in the body to soluble form to treat related illnesses.

Objectives: This study was conducted with the aim of searching for potential sources of uricase-producing Streptomyces from Eshtehard salt desert in Alborz province, Iran and heterologous expression, purification and functional assay of the enzyme.

Materials and methods: Main screening was conducted by cultivation of the strains on a medium enriched with 0.3 percent (w/v) uric acid. The uricase gene from the most potent strain was then recombinantly expressed in E. coli BL21 (DL3).

Results: Out of the tested strains, only seven showed uricase activity. The highest level of native uricase activity (11.5735 U.mL^-1) belonged to strain 17-1, which had the closest similarity to Streptomyces nigra. A recombinant uricase with a molecular mass of approximately 38 kDa was produced. The purified uricase exhibited a specific activity of about 28.29±0.59 U.mg^-1, which is among the highest level of uricase activity reported by other studies.

Conclusions: This enzyme is a promising candidate for further applicable investigations and large-scale production in terms of its large volume of soluble expression and selective competitive activity.

Keywords: Actinobacteria; Uricase; pET28a+; Streptomyces.

Figure 1

Primary screening of the strains: A) Screening of strain 17-1 for uricase activity using plate method (formation of clear zones around the colonies indicated the presence of uricase activity); B) Negative Control

Figure 2

Characteristics of the selected strain: Morphological A-F) and microscopical G) characteristics of strain 17-1. Morphological features are studied in six culture media, which indicates the good growth of strain in all culture environments, but the colors of mycelium and pigments are different. complete explanations are given in the table1.

Figure 3

Relationships between strain 17-1 and related species of the genus Streptomyces; Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences was conducted in MEGA7 software with bootstrap degree of 10000. GenBank sequence accession numbers are indicated in parentheses after the strain names.

Figure 4

Recombinant uricase purification steps: A) Fractions collected after nickel2+-nitrilotriacetic acid (Ni-NTA) chromatography were visualized on 12% SDS-PAGE. Lane S, soluble purified protein; Lane FT, flow-through; Lane M, protein Marker(SM0431); Lane W1-W3, washing step (20mM imidazole); Lane E1-E3, purified uricase after elution with 800mM imidazole; Lane P, precipitate. B) Western blot detection of recombinant uricase using anti-histidine tag monoclonal antibody. lane 1: protein ladder; lane 2 and 3: purified uricase, 1 and 2 μL, respectively.