Search for Microbial Enzymes Useful for Creatinine Assay

恶臭假单胞菌77和许多其他微生物中发现了一条以N-甲基海因、N-氨甲酰肌氨酸和肌氨酸为中间产物降解肌酸的新代谢途径。从恶臭假单胞菌细胞中分离纯化了参与该途径的酶，并对其进行了鉴定。第一步，肌酐的脱亚胺是由胞嘧啶脱氨酶/肌氨酸脱氨酶催化的。N-甲基海因开环和N-氨甲酰肌氨酸脱氨基两步反应分别由N-甲基海因氨基水解酶和N-氨甲酰肌氨基水解酶催化。前一种酶需要三磷酸腺苷，水解和反应过程如下：N-甲基海因-&gt；N-氨甲酰肌氨酸ADP-PI。后者催化以下反应：N-氨甲酰肌氨酸H_20-&GT；肌氨酸NH。在恶臭假单胞菌77中，肌氨酸脱氢酶被发现是肌氨酸氧化为甘氨酸的负责酶，但在几种微生物中也发现肌氨酸氧化酶参与了肌氨酸的氧化。这些酶被发现对肌酐的测定很有用。

A novel metabolic pathway for the degradation of creatinine with N-methylhydantoin, N-carbamoylsarcosine and sarcosine as successive intermediates was found to operate in Pseudomonas putida 77 and many other microorganisms. Enzymes involved in this pathway were purified from cells of P. Putida and characterized. The first step, deimination of creatinine, is catalyzed by cytosine deaminase/creatinine deiminase. The following two steps, ring-openinig of N-methylhydantoin and decarbamoylation of N-carbamoylsarcosine, are catalyzed by new enzymes, N-methylgydantoin amidohydrolase and N-carbamoylsarcosine amidohydrolase, respectively. The former enzyme requires ATP, Mg^<2+>, and for the hydrolysis and the reaction proceeds as follows : N-methylhydantoin ---> N-carbamoylsarcosine + ADP + Pi. The latter catalyzes the following reaction ; N-carbamoylsarcosine + H_20 ---> sarcosine + NH. Sarcosine dehydrogenase was found to be the responsible enzyme for the oxidation of sarcosine to glycine in P. Putida 77, but sarcosine oxidase was also found to be involved in this oxidation in several microorganisms. These enzymes were found to be useful for determination of creatinine.