Development Of An Efficient Procedure For Galactose Oxidase Of Gibberella ZEAE PH-1

The enzyme galactose oxidase(EC 1.1.3.9, Gao), catalyzes the oxidation of primary alcohols to corresponding aldehydes with the concomitant reduction of O2 to H2O2. Recently, galactose oxidase received increasing attention for its potential various applications in diagnostics, biosensing and industrial biocatalyst. For example, sensors incorporating Gao have been used to detect D-galactose, lactose and other related substrates. Gao was also applied in biodegradation or biosynthesis of glycolipids, glycoproteins and polysaccharides to produce desirable products, such as sweeteners, flavorants and paper strength additives. Compared with conventional chemical systems, Gao-mediated biocatalysis has several advantages in industrial applications due to its high stereospecificity, mild operation conditions and environmental-friendliness.Copper is essential for the posttranslational activation of metalloenzyme galactose oxidase. In this study, the Gibberella zeae PH-1 gene Gao encoding the galactose oxidase Gao was successfully cloned and overexpressed in Escherichia coli. Furthermore, Gao was overexpressed in E. coli as soluble protein and the preparation technology for Gao was systematically studied. The main results are as follows:1. The gene Gao encoding the galactose oxidase Gao was successfully amplified from the first strand cDNA of Gibberella zeae PH-1. The insert DNA Gao was ligated with pET32Xa/LIC vector to give the recombinant plasmid pET32-Gao, which adds the N-ternimal His6-tag to the protein. The recombinant pET32-Gao plasmid was transformed to TrxB (DE3). Recombinant Gao was expressed in the cytoplasm as soluble protein.2. The effect of Cu2+-addition on E. coli growth was studied. The results showed that presence of 0.5 mmol/L CuSO4 during cell growth at incubation or induction time exhibited no significant effect on cell growth.3. Various preparation procedures of Gao were systematically and compared in this work, In conclusion, by optimization ofpreparation technology, it is able to significantly increase the yield of active Gao, from the original 5 U/mg to nearly 25 U/mg, which could be useful in industrial application.