Expression and activity analysis of sucrose:sucrose 1-fructosyltransferase from onion.
This study was designed to express the onion fructosyltransferase by Escherichia coli DH5alpha, and obtain the optimal conditions of FST-1 activity. Thereby, fructosyltransferase gene was obtained by RT-PCR from onion in this experiment, and named FST-1. The expressed proteins were analyzed by SDS-PAGE. FST-1 activity was identified by the high performance liquid chromatography (HPLC). The optimal conditions of FST-1 were analyzed by the dinonylnaphthalene sulfonic acid (DNS) and orthogonal test. Results revealed that FST-1 was identified to 98% similarity with fructosyltransferase mRNA of onion (accession number: AJ006066). FST-1 was successfully expressed in E. coli DH5alpha. HPLC results indicated that the expressed protein from FST-1 had a good transferring activity for fructose. The optimal conditions of FST-1 in catalyzing reaction were the pH 5.0, 45 degrees C and 60% sucrose substrate. The results in this experiment would lay the foundation for the large-scale of kestose by bio-catalysis method.
Han
Yawei
Y
Zhengzhou University of Light Industry, Henan Province, 450002, China.
Chen
Liping
L
Mao
Duobin
D
Tang
Lijun
L
Guan
Lihong
L
D002851:Chromatography, High Pressure Liquid; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D019697:Onions / Q000201:enzymology* / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D001426:Bacterial Proteins
D006602:Hexosyltransferases; C021451:inulosucrase; D001426:Bacterial Proteins