Oksalat dekarboksilaz enziminin rekombinant DNA teknolojisi ile üretimi ve karakterizasyonu / Characterization and production via recombinant dna technology of oxalate decarboxylase enzyme

Abstract

Oxalate stones are calcium and oxalic acid formed structures that accumulate in urinary system which result in oxalate stone accumulate and kidney stones. Early diagnosis of kidney stones requires measurement of urinere oxalate stone level. Currently there are two different method for measuring oxalate level namely oxalate oxidase and oxalate decarboxylase assay. While, in oxalate method the H2O2 and CO2 produced by oxalate is being measured, oxalate decarboxylase method reveals NADH+ level released from digestion of formate by formate dehydrogenase enzyme so that the level of oxalate stones can be determine. In this study it has been aimed to produce oxalate decarboxylase enzyme by recombinant DNA technology. This produced recombinant oxalate decarboxylase enzyme can be used with commercially available formate dehydrogenase enzyme for measuring oxalate stones as a new method. For this purpose the yvrK gene from Basillus subtilis M168 strain cloned and expressed in One Shot® Mach1™-T1R Escherichia coli and BL21 (DE3) One Shot® Escherichia coli microorganism via pET-SUMO TA Cloning vector system. Thus 6xHis-SUMO-yvrK fussion protein was produced successfully. Requisite cofactor, optimum pH and temperature were provided and characterization was specified. In this way recombinant oxalate decarboxylase enzyme can easily produced and, a method by higher enzymatic activity has been developed to measure oxalate level.

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