Purification and identification of galloyltransferase involved in catechin metabolism in‘Cabernet Sauvignon’grape

Abstract:【Objectives】The aim of this paper focuses on identification and purification of the galloyl transferase involved in catalyzing EGC and βG to generate EGCG in‘Cabernet’grape, named ECGT.【Methods】The berries of‘Cabernet Sauvignon’grape were picked from the Experimental Grape Gar- den of Xiao County for identifying ECGT and its gene expression. Firstly, the fine precipitated powder was collected by vacuum filtration based on acetone precipitation, then it was dissolved in Tris-HCl buf-fer for ECGT crude enzyme extraction after centrifuging at 12 000 r · min- 1 for 15 min at 4 °C; ECGT protein was then purified by ammonium sulfate fraction, affinity column chromatography, hydroxyapa- tite chromatography, and Superdex 200 gel filtration chromatography based on its enzyme activity ana- lyzed by HPLC. Secondly, the protein strips (55 ku, 28 ku) were achieved by SDS-Polyacrylamide Gele- lectrophoresis (SDS-PAGE) and identified by HPLC-ESI-MS/MS, and subsequently, the functional an- notations of proteins were conducted by Mascot search engine (Matrix Science, London, UK; version 2.3.02) and Blast2GO program against the database of NR (the non-redundant protein database), KEGG (http: //www. genome. jp/kegg/) and COG (http: //www. ncbi. nlm. nih. gov/COG/). Thirdly, qRT-PCR was used to detect the expression level of genes including ECGT and 3 VvGT1-3 in fruits at different de- velopmental stages.【Results】The specific activity of ECGT in the crude enzyme extraction was 0.32 U·mg-1, and compared with crude enzymes, the specific activity was 3.14, 11.78, 31.80 and 88.82 U·mg-1, respectively, via ammonium sulfate precipitation, ConA-affinity chromatography, hydroxyapatite chro- matography and a Superdex 200 column. The purification-folds were 9.81-fold, 36.81-fold, 99.38-fold and 277.56-fold; the enzyme activity yields were 83.33%, 4.64%, 0.96% and 0.13%. According protein score, a protein SCPL acyltransferase (XP_002272116.1) was the highest; the expressions of SCPL and 3 GT1- 3 were all negatively correlated with fruit development.【Conclusion】The ECGT activity was detected in‘Cabernet Sauvignon’grape, and its gene belonged to SCPL family. In addition, its function may be performed as gene cluster.