Immobilization of the Highly Active UDP-Glucose Pyrophosphorylase From Thermocrispum agreste Provides a Highly Efficient Biocatalyst for the Production of UDP-Glucose.

Antje Kumpf,Dirk Tischler,André Pollender,Aleksandra Łochowicz,Katarzyna Szymańska,Isabel Bento,Andrzej Jarzȩbski,Maria Mehnert,Daria Kowalczykiewicz

doi:10.3389/fbioe.2020.00740

Abstract

Biocatalysis that produces economically interesting compounds can be carried out by using free enzymes or microbial cells. However, often the cell metabolism does not allow the overproduction or secretion of activated sugars and thus downstream processing of these sugars is complicated. Here enzyme immobilization comes into focus in order to stabilize the enzyme as well as to make the overall process economically feasible. Besides a robust immobilization method, a highly active and stable enzyme is needed to efficiently produce the product of choice. Herein, we report on the identification, gene expression, biochemical characterization as well as immobilization of the uridine-5′-diphosphate-glucose (UDP-glucose) pyrophosphorylase originating from the thermostable soil actinobacterium Thermocrispum agreste DSM 44070 (TaGalU). The enzyme immobilization was performed on organically modified mesostructured cellular foams (MCF) via epoxy and amino group to provide a stable and active biocatalyst. The soluble and highly active TaGalU revealed a Vmax of 1698 U mg–1 (uridine-5′-triphosphate, UTP) and a Km of 0.15 mM (UTP). The optimum reaction temperature was determined to be 50°C. TaGalU was stable at this temperature for up to 30 min with a maximum loss of activity of 65%. Interestingly, immobilized TaGalU was stable at 50°C for at least 120 min without a significant loss of activity, which makes this enzyme an interesting biocatalyst for the production of UDP-glucose.

Highlights

Uridine-5 -diphosphate-glucose (UDP-glucose) is a fundamentally important molecule in biology, food, biopharmaceuticals and cosmetic chemistry
It consisted of a backbone (5740 bp) coding for a resistance against Ampicillin and the codon usage optimized UDPglucose pyrophosphorylase gene from Thermocrispum agreste DSM 44070 as insert (911 bp, NCBI accession of the protein sequence: WP_028847555; GenBank accession of the codon usage optimized nucleotide sequence: MT321102; see Supplementary Material) under the control of the lactose or isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible lacpromoter
After successful transformation of E. coli BL21(DE3) pLysS cells with the pET16bP-TagalU plasmid, the recombinant enzyme TaGalU was produced with a maximum yield of 73 mg of purified protein per liter of broth

Summary

Introduction

Uridine-5 -diphosphate-glucose (UDP-glucose) is a fundamentally important molecule in biology, food, biopharmaceuticals and cosmetic chemistry. Increases in the stereoselectivity of more than 90% have been observed for one of the stereoisomer, but under harsh reaction conditions (e.g., temperatures of −78◦C or pressures of 3 atm) and with harmful chemicals (e.g., dichloromethane, dioxane, DBU or tert-butyldimethylsilyl chloride) (Guo and Ye, 2010) In contrast to this type of chemical synthesis, enzymatic sugar coupling offers several advantages. Product degradation and/or the synthesis of unnecessary by-products, which are the result of the presence of other enzymes of the cell’s metabolic shock can constitute an even bigger problem (Park et al, 2020) This can be the case during UDP-glucose biosynthesis by means of a whole cell system, as several pathways will utilize it and limit the amount of product for a subsequent down-stream processing. The down-stream processing needs to be carried out under mild conditions as UDP-glucose is not very stable and decomposes for example at non-neutral pH conditions or in the presence of reactive solvents (Hill et al, 2017)

Objectives

Methods

Results

Discussion

Conclusion