Selection of uric acid oxidizing- Lactobacillus plantarum isolates based on their genetic determinant and uricase kinetics

Hyperuricemia is a condition characterized by abnormally elevated levels of uric acid in the blood. It has been a leading morbidity disease. Microbial uricase can be used to oxidize uric acid into allantoin and hydrogen peroxide in the presence of oxygen and therefore has the potential to play an essential role in reducing uric acid in the people suffering from degenerative disease of hyperuricemia. The present study aims to select uric acid oxidizing- Lactobacillus plantarum isolates based on their genetic determinant and uricase kinetics. A collection of Lactobacillus plantarum isolates were grown on a selective differ-ential medium followed by measuring their uricase activity spectrophotomet-rically. Speci(cid:977)ic primers for detection of uricase gene were designed. The uricase coding gene (uox) was then detected in all of the selected isolates by using a qPCR method employing the designed speci(cid:977)ic primers. The uricase kinetics was determined by the Lineweaver-Burk method. Results showed that all isolates had uricase activity and 4 potential isolates were selected based on their superior uricase activity. The uox gene was detected in all of the selected isolates. The kinetics analysis, however, revealed that only the L. plantarum K-Mar-A2 show strongest substrate af(cid:977)inity and was considered a potential candidate to be developed as a source of therapeutic agent for hyperuricemia.


INTRODUCTION
Hyperuricemia is a condition characterized by abnormally elevated levels of serum uric acid (Benn et al., 2018). Hyperuricemia has been a leading morbidity disease. It has been shown to be a risk fac-tor for gout (Fang et al., 2020;Zhang et al., 2020) the most common form of in lammatory arthritis, arises from the subsequent deposition of urate crystals when concentrations become saturated (Benn et al., 2018). Uric acid (2,6,3 trihydroxypurine) is the insoluble end product of exogenous and endogenous purine catabolism. The relationship between diet and serum uric acid seems to be more complex than simple purine intake, as beer and high fructose soft drinks have been shown to in luence serum uric acid levels irrispective of their purine content (Benn et al., 2018).
The biosynthesis of uric acid is catalyzed by the enzyme xanthine oxidase also known as xanthine oxidoreductase. The enzyme is widely distributed throughout various organs including the liver, gut, lung, kidney, heart, and brain as well as the plasma and plays roles in two stages of uric acid production: conversion of hypoxanthine to xanthine and subsequently xanthine to uric acid (Benn et al., 2018).
In most mammalian species uric acid is further metabolized by the enzyme uricase to the more soluble metabolite which is subsequently excreted in the urine (Benn et al., 2018). Human and certain primates, however, lost their uric acid oxidizing enzyme, the uricase, consequently uric acid is the inal product of purine catabolism and can not be metabolized to the soluble allantoin, urea, or ammonia for excretion from the body (Kratzer et al., 2014;Iswantini et al., 2014;Tan et al., 2016).
Notably, an isolate of probiotic bacteria of Lactobacillus plantarum has been reported to show an uric acid oxidizing activity (Iswantini et al., 2014). L. plantarum is commonly found in plantderived foods such as fruits, pickles, wine, and bean sauces (Li et al., 2014). It is essential then to explore, select and characterize its uricase in term of both genetic and kinetic properties which is important for developing a potentially probiotic for hyperuricemia.

MATERIALS AND METHODS
Forty six L. plantarum isolates were collected from the fruit of mangosteen, eggplant, mango, and passion fruit. The culture medium was a selective differential GYP (1% glucose 1% yeast 0.5% peptone b/v) liquid and agar medium suplemented with 0.3% b/v uric acid. Uricase activity in the supernatant fraction was measured spectrophotometrically.

Culture and selection of L. plantarum
All L. plantarum isolates were grown on GYP agar medium with CaCO 3 for 24 hour at incubation 37 • C. The uric acid (0.3% b/v) was supplemented to GYP broth in the selection process. The amount of uric acid oxidized was measured spectrometricaly at λ 595 nm and reported as relative the control medium without bacterial treatment.

Primer Design for the uox gene
Both forward and reverse primers of uox gene were derived from the uox sequence alignmented of Bacillus subtilis subsp. subtilis str. 168, Bacillus subtilis subsp. spizizenii TU-B-10, Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Amy-colatopsis vancoresmycina strain NRRL B-24208, and Streptomyces venezuelae ATCC 10712. The Primer-BLAST and Clustal Omega program were used in the primer design. The primers were synthesized by IDT (Integrated DNA Technologies) of Singapore.

Detection of uox gene
The bacterial DNA was isolated through a heatshock technique of at 95 • C for 15 minutes, then cooled down at -20ºC for 5 minutes. The q-PCR reaction of 20 µL was performed in a Bio-Rad CFX of 40 cycles : 3 min pra-denaturation at 95 • C, 10 sec denaturation at 95 • C, and 30 sec annealing at 55 • C.

Uricase Assay
The selected L. plantarum isolates were grown on Glucose Yeast Peptone (GYP) broth for 24 hour at incubation 37 • C. Crude extract of uricase were harvested by centrifugation (10000 rpm, 14 min). The crude extract of uricase activity was measured spectrophotometrically at λ 293 nm.

Characterization of the selected L. plantarum isolates
The selected L. plantarum isolates were subjected to measurement of activity of crude extract uricase at pH and temperature range: pH 6; 7; and 8, and temperature 25; 30; and 37ºC. Michaelis-Menten constant (K M ) and maximum velocity (V max ) of crude extract uricase based on various substrate 2-7 mg/dL at the best pH and temperature, then plotted into a Lineweaver-Burk curve.

Uric acid oxidizing activity of L. plantarum
In this study, we report uric acid oxidizing activity in strains of L. plantarum, isolated from tropical fruits in Indonesia. The fruit's sweet and acidic condition would support the presence of lactic acid bacteria. The mannose-rich fruit mesocarp would be the best fruit part to ind the mannose speci ic adhesin (MSA)-producing L. plantarum. The MSA plays important roles in the probiotic interaction with host intestinal tract, immunologic re ponses and health related bene its (Gross et al., 2010;Pretzer et al., 2005;Nieuwboer et al., 2016).
All of the 46 L. plantarum isolates were be able to oxidize the uric acid down to 3.6 to 82.1 percent. The top ten uric acid oxidizing isolates where presented in Figure 1. The four best isolates, K-Mar-A2, Mar-A18, Mgs-Bst-3, and Mgs-Psmb-3 were selected and then subjected to uox gene identi ication and uricase enzyme kinetics analysis (Figure 1). The four selected isolates were tested for the presence of the uox gene. The best designed primers were a forward primer: TGATCGCTTCGGCTTTCCTT and a reverse primer: TCCGGGATTTGCTTCACTCC. The uox gene was detected in all of the four selected isolates and their qPCR pro iles were presented in Figure 2. The K-Mar-A2 isolate showed better qPCR signal than the other isolates. Speci ic primers were created to identify uox gene by qPCR. The best designed primers succesfully ampli ied the uox gene in the four isolates, and the K-Mar-A2 isolate showed the best qPCR signal compared with the other selected isolates.

Detection of uox gene
Our data con irmed the presence of uox gene in every isolate tested which encodes the uricase enzyme with detected activity in each isolate. The detected uricase genes of the four selected isolates can potentially be used to produce recombinant uricase by cloning and expressing the genes in host cells. Production of recombinant microbial uricase has been reported (Cheng et al., 2012).
Recombinant Aspergillus lavus uricase (Rasburicase) has been used for the treatment of gout and hyperuricemia occurring in tumor lysis syndrome. Compared to allopurinol, a competitive inhibitor of xanthine oxidase, a common agent for blocking the conversion of hypoxanthine and xanthine to uric acid, thereby, reduces uric acid production,  the use of Rasburicase has advantages, in that, it reduces pre-existing hyperuricemia and does not cause accumulation of xanthine or hypoxanthine which occurs after allopurinol uptake (Imani and Shahmohamadnejad, 2017).

Kinetics of L. plantarum uricase
Our results show that the uricase works better at pH 6 and 25 • C ( Figure 3). As expected from genetic analysis, the K-Mar-A2 isolate showed a better catalytic rate and substrate af inity as revealed by the Lineweaver-Burk curves (Figure 4). The urate oxidase expressed by K-Mar-A2 isolate exhibited uric acid oxidase activity, with crude enzyme activity was 0.4028 U/mL with K M and V max were 0.0137 mg/mL and 0.1311 µmol mL −1 min −1 .
The Lactobacillus plantarum uricase of our study showed different optimum temperature and pH from microbial uricase previously reported. Characterization of bacterial uricase from Sphingobacterium thalpophilum (VITPCB5) showed that the optimum temperature of the enzyme activity was between 25 and 45 • C, and the optimum pH was 8.0 (Ravichandran et al., 2015). Studies of puriied uricase of Pseudomonas aeruginosa showed optimum temperature of 30 o C and optimum pH of 9.0 (Saeed et al., 2004). The kinetic data presented in the present study were obtained from crude enzymes. Futher experiments are required to determine the kinetics of pure Lactobacillus plantarum uricase.