Studies On Enzymes Degrading Creatine From Bacillus Sp. BSD-8

In clinical diagnosis, the concentration of creatinine in serum is an important parameter for the evaluation of kidney function.The enzymatic determination methodhas characteristic of high specificity and sensitivity. In previous work, a Bacillus sp. BSD-8 that produced thermostable sarcosine oxidase (SOX) was isolated in our laboratory and this enzyme had been purified and systematically studied. It was shown that the enzyme had a relatively high thermostability and may have advantage in clinical application for measurement of creatinine, so it is worth mading thorough study. In additon, it was found that the strain could grow under the condition of creatine as only carbon source, so we guess it probably could produce creatininase and creatinase. In this study, the Bacillus sp. BSD-8 sarcosine oxidase gene was coloned and expressioned; the thermostability of sarcosine oxidase was enchanced by directed evolution and screening with developed high-throughput screening method; the Bacillus sp. BSD-8 creatinase gene was coloned and expressioned and the enzyme characterization were also described; in addition, the Bacillus sp. BSD-8 creatininase gene was cloned and expressioned. The results of this experiment are summarized as follows.1 Cloning, sequencing and expression of SOX gene from Bacillus sp. BSD-8:The complete SOX gene from BSD-8 strain was cloned by chromosome walking method. The gene was composed of 1164 bp that encoded 387 amino acids. The alignment result of amino acids sequences of SOX showed that the SOX gene sequence from BSD-8 strain had high similarity with those from Genbank. Two highly effective expression systems were successfully constructed: 1 highly effective expression vector pET-15b(-) and BL21(DE3) expression system. The protein was effectively expressed with inducer IPTG, the expressed protein SOX comprised more than 30% of the total cell protein ; 2 Cloning vector pBluscript KS(+)II and E.coli DH-5αexpression system. The vector has blue/white blot, positive clones display white spots and are easy to be identified. SOX was expressed with no need to add inducer IPTG and the expression level was about half of that of above, which could be used for directed evolution screening.2 Purification and characteristics of SOX: The expressed SOX in E. coli was purified by Ni-IDA column, The purity of the enzyme was examined by SDS-PAGE and a clear single band corresponding to the 51 kDa protein was observed .The specific activity of enzyme was 28.6 U/mg. The purified recombinant SOX shared almost similar properties with that wild type SOX protein, the optimum pH for SOX was 8.0 and the optimal temperature was 60℃. Between pH 7.0-10.0 the SOX was stable below 60℃. The Km and the Kcat value of SOX for sarcosine were 3.1mM and 24/s, respectively. However, they were different in flavin binding mode: recombinant SOX presumably contained covalently bound flavin and SOX from BSD-8 contained noncovalently bound flavin.3 Directed evolution of thermostable sarcosine oxidase: In order to improve the thermostability of sarcosine oxidase, directed evolution method was used.The evolution strategy involves error-prone PCR and site-directed mutation. The enzyme activity was determined by microplate with wall-breaking of surfactant, which could effectively be used for screen thermostable sarcosine oxidase.Through mutation and screening, the mutant soxM2 had three amino acid substitutions,these substitutions resulted in an increase of thermostability by 5℃(from 60℃increase to 65℃).4 Expression and characterization of creatinase from Bacillus sp. BSD-8 in Escherichia coli: The complete creatinase gene from BSD-8 strain was cloned by chromosome walking method. The gene was composed of 1236 bp that encoded 411 amino acids. Two highly effective expression systems were successfully constructed: pET-28a-CRE and BL21(DE3), pET-28a-CRE and BL21(DE3)pLysS. Result showed that the expression level in BL21(DE3)pLysS was 1.46 times more than in BL21(DE3). The expressed creatinase in BL21(DE3)pLysS was purified by ammonium sulfate precipitation and Ni-IDA column. The purity of the enzyme was examined by SDS-PAGE and a clear single band corresponding to the 54 kDa protein was observed .The specific activityof enzyme was 79.4 U/mg. 5 Cloning and sequencing of creatininase gene from Bacillus sp. BSD-8: Thecomplete creatininase gene from BSD-8 strain was cloned by DNA WalkingSpeedUp^TM Premix Kit II. The gene was composed of 747 bp that encoded 248 amino acids. The alignment result of amino acids sequences of creatininase showed that the creatininase gene sequence from BSD-8 strain had relatively low similarity with those from Genebank.