Exploring the in Vitro Operating Window of Glycosyltransferase PtUGT1 from Polygonum Tinctorium for a Biocatalytic Route to Indigo Dye

Abstract

The eobiotic compound indican lends itself to a compelling biocatalytic dyeing strategy for denim, in which the formation of corrosive by-products is avoided. However, the efficient and scalable production of indican remains a key bottleneck. This work focuses on the in vitro characterization of PtUGT1, a glycosyltransferase from Polygonum tinctorium that catalyzes the formation of indican via the glycosylation of indoxyl. Here, the buffer composition and enzyme concentration were identified as key parameters for enzyme activity and stability. The short lifetime of the enzyme under reaction conditions initiated an immobilization study. As a consequence, an amino-functionalized methacrylate resin was identified as a highly functional option for efficient immobilization of PtUGT1, allowing immobilization yields of > 98% for enzyme loadings up to 7.6 w-%. We further report a stabilization factor of 47 and significantly improved overall biocatalytic productivity. The straightforward handling and reuse of the described heterogeneous biocatalyst is demonstrated.

Chemoenzymatic indican for sustainable light-driven denim dyeing

“…PtUGT1 is a soluble UDP-dependent glycosyltransferase (UGT) 14 found in the indigo-producing plant Polygonum tinctorium. Its limited stability in operating conditions was previously partly alleviated by immobilization but with severe activity loss 9 . Instead, we used a combination of consensus mutagenesis, loop grafting, and disulfide bridge engineering, to rationally introduce stabilizing mutations outside the active site of PtUGT1 (Fig.…”

“…Indigo dyeing has significant negative environmental and social implications, including pollution of waterways and soil as well as human toxicity [4][5][6][7] . Indican, the O-glycoside of the indigo precursor indoxyl, has been suggested as an environmentally mild blue denim dyeing agent, since indican dyeing would eliminate reducing agents while still ending as indigo in the yarn [8][9][10][11][12] . To efficiently leverage indican in denim dyeing, two processes must be developed that have environmental benefits over the conventional method while still being affordable: a) a production process (Fig.…”

“…1D). We envisage the use of PtUGT1 stable and SuSy as lysates in solution, since previous attempts to immobilize these enzymes resulted in dramatic losses of activities 9,20 . With these data and projections, the raw material price reaches 9.9 USD/kg (Extended Data Fig.…”