Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production.

Kai Song,Jianhua Hao,Wei Wang,Jingjing Sun

doi:10.3389/fmicb.2021.664339

Abstract

In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G.

Highlights

Cyclodextrin glycosyltransferase (CGTase), called 4-α-D-(1,4-α-D-glucanol)-transferase (EC 2.4.1.19), is a member of the α-amylase family (Han et al, 2014)
Toyoda used the method of chemical synthesis to produce AA-2G, but the results identified the synthesis process as complicated and associated with high cost; making enzymatic production unattractive (Toyoda et al, 2004)
The optimal isopropyl β-D-L-thiogalactopyranoside (IPTG) concentration was 0.4 mM, and both too low and too high IPTG concentration [which may be caused by cell death and protein degradation (Chu et al, 2018)] decreased the level of CGTase expression (Figure 1B)

Summary

Introduction

Cyclodextrin glycosyltransferase (CGTase), called 4-α-D-(1,4-α-D-glucanol)-transferase (EC 2.4.1.19), is a member of the α-amylase family (glycosyl hydrolase family 13) (Han et al, 2014). CGTase catalyzes four types of reactions, including hydrolysis reaction and three types of transglycosylation reactions (i.e., disproportionation, cyclization, and coupling). The cyclization reaction, which is the characteristic reaction of CGTase, is used to produce. Α-CD, β-CD, and γ-CD are the major structures of cyclodextrins (CD) (Leemhuis et al, 2010; Tao et al, 2020). Disproportionation and coupling reactions format glycosylated derivatives by transferring maltooligosaccharides to a variety of receptors (Fenelon et al, 2015). CGTase can produce high value-added products, such as CD and 2-O-α-D-glucopyranosylL-ascorbic acid (AA-2G). An increasing number of researchers focus on CGTase, and its demand is gradually increasing

Methods

Results

Conclusion