Optimization of Media, Expression Conditions and Extraction of SrUGT76G1 Glucosyltransferase and Sucrose Synthase for Glycoside Production
DOI:
https://doi.org/10.48048/tis.2025.9272Keywords:
Sucrose synthase, SrUGT76G, Glycosyltransferase, Box-Behnken design, Recombinant protein expression, MicrofluidizationAbstract
The objective of this work was to produce the soybean sucrose synthase and SrUGT76G1 glucosyltransferase for glycoside production. Media, expression conditions, fermentation, and extraction were screened to gain high amounts of soluble enzymes from E. coli BL21(DE3). Lysogeny broth (LB) was identified as the best of 5 media tested. Then, Box-Behnken design was used to determine the suitable expression conditions, including temperature, lactose concentration, and time. Application of the expression conditions identified in flask cultures to the fermenter successfully increased the soluble enzymes from 7.3 to 14.1 mg/L for sucrose synthase, and from 14 to 17.7 mg/L for SrUGT76G1. To improve scalability, microfluidization was used for extraction and the condition of 25 g/L cell concentration and 69 MPa pressure gave 86 % disruption efficiency in a single pass, after which cell fragmentation was observed by scanning electron microscopy. The yields of sucrose synthase and SrUGT76G1 were 12.7 ± 1.2 and 18.4 ± 1.2 mg/L, respectively. This work demonstrates the benefit of surface response methodology in optimizing enzyme production for glycosylation reactions.
HIGHLIGHTS
- Optimal conditions for SuSy and SrUGT76G1 were identified by Box-Behnken design.
- The optimal conditions could be scaled up to 5-L and 50-L fermenters with higher yields.
- Microfluidization provided efficient enzyme extraction for economical scalability.
GRAPHICAL ABSTRACT
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