Enzymatic Conversion For Preparation Of L-citrulline And Characteristics Analysis Of Arginine Deiminases

Arginine deiminase (EC3.5.3.6; ADI), accompanying with ornithine transcarbamylase (EC2.1.3.3; OTC) and carbamate kinase (EC2.7.2.2; CK), combines the arginine deiminase pathway, which is widely distributed in the microorganisms that are able to utilize arginine as an energy source. The ADI protein hydrolyzes L-arginine to L-citrulline and releases ammonia. ADI is considered to be a useful producer in the industrial production of L-citrulline. In addition, it is also proved to be a potential anti-tumor agent for its efficient inhibition effects on the growth of arginine-auxotrophic tumor cells via depleting extracellular supply of L-arginine, especially on hepatocellular carcinomas and melanomas. Hence, many ADI genes from different sources, including Mycoplasma, Pseudomonas plecoglossicida, Lactococcus lactis, Streptococcus sanguis and Giardia intestinalis, have been cloned and expressed in E. coli cells. However, the recombinant ADI proteins were mainly existed in inclusion body forms, which is the major bottleneck of its research and development.This paper selected three different ADI genes, named TADI, PADI, LADI respectively, which were extracted from Thermoanaerobacter tengcongensis, Pseudomonas putida and Listeria welshimeri with significantly different isolation environment. Low identity of their amino acid sequences make it possible to explore the novel characteristics and their potential to produce L-citrulline and antitumor agents.In E. coli system, we constructed a series of different types of expression vectors. With optimizing the culture conditions, all of the ADIs still exist in inclusion bodies. Fortunatly, We found that on the basis of co-expression of GroEL-GroES, combined addition of L-arginine and D-glucose in Luria-Bertani medium markedly increased the solubility of rADI. Accordingly, the total activity of rPADI and rLADI were increased to15-fold and7-fold. However, many strategies including truncated expression, refolding in vitro, co-expression with the chaperon teams and addition of substrate didn’t make rTADI soluble.After purified using Ni affinity chromatography, the enzymatic characteristics of rPADI and rLADI were further studied. The optimum temperature and pH of rPADI were40℃and6.0, respectively. The specific activity of rPADI was determined to be76±0.03U/mg, which located in a upper level according to the literatures reported. The optimum temperature and pH of rLADI were45℃and6.0, respectively. Its specific activity was3.12±0.01U/mg. Cr3+, Co2+, Ni2+, Cu2+, Hg2+, SDS and DTT strongly inhibited the activity of rLADI. Slight changes of temperature and pH, or presence of metal ions and reagents showed strong influences on its catalytic ability. Given the advantages of rPADI on solubility (45-50mg/L)and catalytic ability in this paper, crude enzymes of rPADI were directly used to convert L-arginine to L-citrulline. The crude enzymes from25ml cultures could completely convert20g L-arginine into L-citrulline in22h, which indicated its potential industrial application.To explore new rADI with great solubility and catalytic ability was the key to produce L-citrulline and its anti-tumor research. In this paper, we explored a potential rPADI with good solubility and catalytic ability, and found an efficient method to promote the yield of soluble rADI, which suggested a new strategy to increase the solubilization of other over-expressed recombinant proteins in E. coli cells.