A Novel β-Glucuronidase from Talaromyces pinophilus Li-93 Precisely Hydrolyzes Glycyrrhizin into Glycyrrhetinic Acid 3- O -Mono-β- d -Glucuronide

Abstract

Glycyrrhetinic acid 3--mono-β-d-glucuronide (GAMG), which possesses a higher sweetness and stronger pharmacological activity than those of glycyrrhizin (GL), can be obtained by removal of the distal glucuronic acid (GlcA) from GL. In this study, we isolated a -glucuronidase (TpGUS79A) from the filamentous fungus Li-93 that can specifically and precisely convert GL to GAMG without the formation of the by-product glycyrrhetinic acid (GA) from the further hydrolysis of GAMG. First, TpGUS79A was purified and identified through matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF-TOF MS) and deglycosylation, indicating that TpGUS79A is a highly -glycosylated monomeric protein with a molecular mass of around 85 kDa, including around 25 kDa of glycan moiety. The gene for TpGUS79A was then cloned and verified by heterologous expression in TpGUS79A belonged to glycoside hydrolase family 79 (GH79) but shared low amino acid sequence identity (<35%) with the available GH79 GUS enzymes. TpGUS79A had strict specificity toward the glycan moiety but poor specificity toward the aglycone moiety. Interestingly, TpGUS79A recognized and hydrolyzed the distal glucuronic bond of GL but could not cleave the glucuronic bond in GAMG. TpGUS79A showed a much higher catalytic efficiency on GL (/ of 11.14 mM s) than on the artificial substrate NP-glucopyranosiduronic acid (/ of 0.01 mM s), which is different from the case for most GUSs. Homology modeling, substrate docking, and sequence alignment were employed to identify the key residues for substrate recognition. Finally, a fed-batch fermentation in a 150-liter fermentor was established to prepare GAMG through GL hydrolysis by Li-93. Therefore, TpGUS79A is potentially a powerful biocatalyst for environmentally friendly and cost-effective production of GAMG. Compared to chemical methods, the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3--mono--d-glucuronide (GAMG), which has a higher sweetness and stronger pharmacological activity than those of GL, via catalysis by -glucuronidase is an environmentally friendly approach due to the mild reaction conditions and the high yield of GAMG. However, currently available GUSs show low substrate specificity toward GL and further hydrolyze GAMG to glycyrrhetinic acid (GA) as a by-product, increasing the difficulty of subsequent separation and purification. In the present study, we succeeded in isolating a novel-glucuronidase (named TpGUS79A) from Li-93 that specifically hydrolyzes GL to GAMG without the formation of GA. TpGUS79A also shows higher activity on GL than those of the previously characterized GUSs. Moreover, the gene for TpGUS79A was cloned and its function verified by heterologous expression in Therefore, TpGUS79A can serve as a powerful biocatalyst for the cost-effective production of GAMG through GL transformation.

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

“…Display of lipase on P. pastoris has been extensively reported for biodiesel production (refer to Yan et al for a detailed review) or synthesis of various kind of esters . Other enzymes displayed on P. pastoris include cellulases, phytase, glycosidases, etc. (Figure D).…”

Concurrent production of glycyrrhetic acid 3-O-mono-β-d-glucuronide and lignocellulolytic enzymes by solid-state fermentation of a plant endophytic Chaetomium globosum

“…Compared with chemical approaches, the biosynthesis of GAMG significantly has more potential and advantages because of its excellent substrate selectivity, high yields, mild reaction conditions, and eco-friendly status. The efficient biosynthesis of GAMG involves the use of β -glucuronidase (GUS) (Zou et al 2013 ; Xu et al 2018a , b ; Wang et al 2013 ; Park et al 2005 ). At present, GUSs are mainly screened from microorganisms (Zou et al 2013 ; Xu et al 2018a , b ; Wang et al 2013 ; Park et al 2005 ).…”

“…The efficient biosynthesis of GAMG involves the use of β -glucuronidase (GUS) (Zou et al 2013 ; Xu et al 2018a , b ; Wang et al 2013 ; Park et al 2005 ). At present, GUSs are mainly screened from microorganisms (Zou et al 2013 ; Xu et al 2018a , b ; Wang et al 2013 ; Park et al 2005 ). However, most of reported GUSs exhibit a low hydrolytic selectivity and cause further transformation of GAMG to GA. On the other hand, GUS is generally prepared by microorganic fermentation.…”

“…In general, most enzymes are produced by microorganic submerged fermentation (SF). GUS can also be produced by SF of Aspergillus terreus Li-20 (Xu et al 2018a , b ), Streptococcus LJ-22 (Park et al 2005 ), and Penicillium purpurogenum Li-3 (Zou et al 2013 ). However, their applications in the manufacturing industry have been met with several processing challenges, including low productivity, especially in terms of ineffective costs of the mass production of enzymes.…”

Improving the activity and thermostability of GH2 β‐glucuronidases via domain reassembly

“…Most identified GUSs have been classified into the GH2 family, making GUSs important members of this family (Bhatt et al, 2020; Biernat et al, 2019; Pellock et al, 2018). GUSs have been widely used as gene reporters, in gene therapy and for natural product modification (Bramwell et al, 2015; Honarbakhsh et al, 2012; Li et al, 2019; Naz et al, 2013; Wang et al, 2019; Xu et al, 2018; Zhao et al, 2017). All the characterized GH2 GUSs are composed of three domains, an SBD, an ISD, and a TIM, but these GUSs show dramatic differences in activity, substrate scope, stability, and expression level.…”