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

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 ( Ta GalU). 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 Ta GalU revealed a V max of 1698 U mg –1 (uridine-5′-triphosphate, UTP) and a K m of 0.15 mM (UTP). The optimum reaction temperature was determined to be 50°C. Ta GalU was stable at this temperature for up to 30 min with a maximum loss of activity of 65%. Interestingly, immobilized Ta GalU 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.

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

“…The synthesis of UDP- d -Glc using immobilized pyrophosphorylase from Thermocrispum agreste ( Ta GalU) was also reported recently . This thermostable enzyme was active at 50 °C for 30 min, losing up to 65% of its activity over this time.…”

“…Recently,119 the entire cascade illustrated in Figure5(including PmHAS for HA synthesis) was immobilized onto magnetically recoverable beads, and the system reused up to five times 120. These modules have also been compartmentalized and transferred into continuous flow reactors to permit automated glycan (HA) synthesis,121 although no information with regard to scale was disclosed in this most recent report.The synthesis of UDP-D-Glc using immobilized pyrophosphorylase from Thermocrispum agreste (TaGalU) was also reported recently 122. This thermostable enzyme was active at 50 °C for 30 min, losing up to 65% of its activity over this time.…”

“…However, Ta GalU immobilized on amino-functionalized mesostructured cellular foams (MCFs) demonstrated retained activity up to 80 °C (40% activity after 5 h) and for greater lengths of time (up to 96 h). These reactions were carried out on a preparative (10 mL) scale at 2 mM substrate concentration, and the immobilized enzyme could be reused up to five times …”

“…These reactions were carried out on a preparative (10 mL) scale at 2 mM substrate concentration, and the immobilized enzyme could be reused up to five times. 122 The immobilization of three enzymes required for the synthesis of CMP-Neu5Ac within hydrogels as a method to avoid CTP cross-inhibition of N-acyl-D-glucosamine 2epimerase (AGE) and N-acetylneuraminate lyase (NAL) has been described. 123 Utilizing a confinement and compartmentalization strategy enabled higher substrate concentrations to be used.…”

“…However, TaGalU immobilized on amino-functionalized mesostructured cellular foams (MCFs) demonstrated retained activity up to 80 °C (40% activity after 5 h) and for greater lengths of time (up to 96 h). These reactions were carried out on a preparative (10 mL) scale at 2 mM substrate concentration, and the immobilized enzyme could be reused up to five times 122. The immobilization of three enzymes required for the synthesis of CMP-Neu5Ac within hydrogels as a method to avoid CTP cross-inhibition of N-acyl-D-glucosamine 2epimerase (AGE) and N-acetylneuraminate lyase (NAL) has been described 123.…”