Enzymatic Properties and Isozyme Electrophoresis of Spinach Glycolate Oxidase

doi:10.7668/hbnxb.201750895

Abstract

Abstract: The enzymatic properties of Spinacia oleracea glycolate oxidase (SpGLO) were studied in the paper. Firstly, the RNA of spinach was extracted and reversely transcribed into cDNA. Based on the mRNA sequence information of spinach GLO gene provided in NCBI database, the specific primers were designed, and the target sequences were cloned and connected to the pMD19-T vector for sequence identification. Then the primer was designed again and His label was added to the upstream primer, and SpGLO gene was cloned to build pYES2 vector. The SpGLO was heterologously expressed in Saccharomyces cerevisiae and purified by immobilized metal-affinity chromatography, and the enzyme activities were detected at different induction time. The results showed that the transformed S.cerevisiae strain presented the highest GLO activity after 20 hours of induction. The catalytic activities of SpGLO at different pHs and temperatures were determined by using glycolate as substrate, and the optimum pH was 8.0 and the optimum temperature was 39℃. The enzymatic properties of SpGLO were analyzed with glycolate, glyoxylate and glycine as substrates respectively. Which showed that SpGLO displayed the highest affinity and activity with glycolate as substrate, and the Km of SpGLO for glycolate was 0.41 mmol/L, and the Vm was 45.92 μmol/(min·mg). Oxalate could more strongly inhibit the glyoxylate-oxidizing activity of SpGLO, and the Ki values were 4.61, 2.09 mmol/L, respectively. The purified SpGLO isozyme zymogram analysis was performed with a Caps-ammonium Clear Native-PAGE system, and two isozyme bands appeared after staining, indicating that there may be two GLO isozymes existed in spinach leaves. It provided a good foundation for future research on the biochemical difference between GLO isozyme in plant and the analysis of its physiological function.

Key words: Glycolate oxidase, Enzymatic characteristics, Isozyme electrophoresis, Spinach

CLC Number:

S636.01

LUO Sha, HUANG Luyao, YIN Qin, ZHANG Yuying, ZHAO Yan, ZHANG Zhisheng. Enzymatic Properties and Isozyme Electrophoresis of Spinach Glycolate Oxidase[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(1): 156-164. doi: 10.7668/hbnxb.201750895.

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