High Expression And Characteristic Of Tool Enzymes For The Creatinine Measurement

Creatinine is generated from phosphocreatine in muscle that an irreversible process.Under normal conditions of kidney,the concentration of creatinine can maintain a relatively low level,however.If something wrong with kidney,it can lead to the accumulation of creatinine in the blood,so the concentration of creatinine is an important indicator of kidney function.The most widely used method for creatinine test is alkaline picrate method,but it has poor specificity and low sensitivity in the reaction that can be interfered by many interfering substances,it gradually be replaced by the enzymatic method with creatininase coupled sarcosine oxidase that consist of three enzymatic-reactions and need three important enzymes?creatininase,EC 3.5.2.10;creatinase,EC 3.5.3.3;sarcosine oxidase,EC 1.5.3.1?.creatininase hydrolyzed creatinine to creatine reversibly,creatininase catalyze creatine to urea and sarcosine,sarcosine oxidase decompose sarcosine into methanol,glycine and H2O2 that can be coupled with Trinder reaction,finally,we can calculate the content of creatinine after colorimetric assay of rinder reaction.Enzymatic method for creatinine test have so many advantages,such as strong resistance to interference,high specificity and high sensitivity,that be used in most commercial assay kit,it got more and more use in the clinic.In this study,I found the amino acid sequence of creatininase?Genebank no.1Q3K_A?that from Pseudomonas sp in the Genebank and synthesized the whole codon optimized gene.I took use of E.coli and P.pastoris as expression host and realized the expression of creatininase successfully.The gene of creatinase?Genebank no.KM027338?was cloned from genome of Arthrobacter nicotianae 23710 and the gene of sarcosine oxidase?Genebank no.AY626822?that from Bacillus sp was synthesized,I took use of E.coli as expression host and realized the expression of creatinase and sarcosine oxidase.The main contents and results are as follows:The creatininase gene from Pseudomonas sp was synthesized base on the codon preference of E.coli and was named cine-c.The synthesized cine-c was amplified by PCR.The successfully constructed recombinant plasmids pET28a/cine-c were transformed into E.coli BL21?DE3?.The positive transformants B/pET28a-cine-c was screened andfermented in TB medium.The IPTG was added into the TB to induce the production of enzyme.The specific activity of Cine-c is 489.2U/mg,the optimal temperature and pH is60? and 7.0.When incubated at 60? for 3 h,about 80% activity is maintained,it was stable at pH 7.0-9.0.Cu²⁺ can inhibit some enzyme activity,Mn²⁺?Zn²⁺ and Co²⁺ can improve the enzyme activity,NaN₃ have no influence on enzyme activity.The creatininase gene from Pseudomonas sp was synthesized base on the codon preference of P.pastoris and was named cine-p.Gene cine-p was cloned to vector pHKA,the recombinant plasmid pHKA/cine-p was were transformed into P.pastoris GS115.After screened the positive transformant,the recombinant yeasts were cultured in BMMY with methanol was added into the BMMY to induce the production of enzyme.The enzyme activity is 14.0U/mL in flask fermentation,the specific activity of Cine-p is 649.2 U/mg,the optimal temperature and pH is 70? and 8.0.it was stable at pH 7.0-9.0 and 50?.Cu²⁺ can inhibit enzyme activity seriously,Mn²⁺ and Mg²⁺ can improve the enzyme activity,NaN₃ have no influence on enzyme activity.The creatinase gene cre was cloned from genome of Arthrobacter nicotianae 23710,cre was constructed recombinant plasmid pET22b/cre that were transformed into E.coli BL21?DE3?.The positive transformants was screened and fermented in TB medium.The IPTG was added into the TB to induce the production of enzyme.The molecular weight of protein subunit almost 46.4kDa,the specific activity of CRE is 19.6U/mg,the optimal temperature and pH is 35? and 8.0.CRE was stable at pH 6.0-8.0 and temperature blow 35?.Cu²⁺ can lead CRE lost enzyme activity,Zn²⁺ can inhibit the enzyme activity seriously,NaN₃ have no influence on enzyme activity.The sarcosine oxidase gene from Bacillus sp was synthesized base on the codon preference of E.coli that was named sox.Gene sox was were transformed into E.coli BL21?DE3?.The positive transformants was screened and fermented in TB medium.The IPTG was added into the TB to induce the production of enzyme.The molecular weight of protein subunit almost 43 kDa,the specific activity of SOX is 15.9U/mg,the optimal temperature and pH is 60? and 8.0.CRE was stable at pH 7.0-8.0.Cu²⁺ no influence on enzyme activity,Fe²⁺and EDTA can inhibit the enzyme activity slightly,NaN₃ have no influence on enzyme activity.