Beta-D-glucosidases and related enzymic activities in pig kidney.
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ABSTRACT:
SUBMITTER: Abrahams HE
PROVIDER: S-EPMC1187604 | biostudies-other | 1969 Mar
REPOSITORIES: biostudies-other
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Project description:BackgroundFungal beta-glucosidases (BGs) from glucoside hydrolase family 3 (GH3) are industrially important enzymes, which convert cellooligosaccharides into glucose; the end product of the cellulolytic process. They are highly active against the β-1,4 glycosidic bond in soluble substrates but typically reported to be inactive against insoluble cellulose.ResultsWe studied the activity of four fungal GH3 BGs on cellulose and found significant activity. At low temperatures (10 ℃), we derived the approximate kinetic parameters k cat = 0.3 ± 0.1 s-1 and K M = 80 ± 30 g/l for a BG from Aspergillus fumigatus (AfBG) acting on Avicel. Interestingly, this maximal turnover is higher than reported values for typical cellobiohydrolases (CBH) at this temperature and comparable to those of endoglucanases (EG). The specificity constant of AfGB on Avicel was only moderately lowered compared to values for EGs and CBHs.ConclusionsOverall these observations suggest a significant promiscuous side activity of the investigated GH3 BGs on insoluble cellulose. This challenges the traditional definition of a BG and supports suggestions that functional classes of cellulolytic enzymes may represent a continuum of overlapping modes of action.
| S-EPMC7350674 | biostudies-literature
Project description:β-Glucosidases are enzymes that hydrolyze β-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides, and thus have significant application potential in industries. However, most β-glucosidases are feedback inhibited by the glucose product, which restricts their application. Remarkably, some β-glucosidases of the glycoside hydrolase (GH) 1 family are tolerant to or even stimulated by glucose. Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 β-glucosidases will be crucial to improve their application through enzyme engineering. In this study, by comparing the primary and tertiary structures of two GH1 β-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were mutated to investigate their exact roles. Both biochemical and structural characterization of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose, and the relative binding affinity/preference of these sites to glucose modulates the glucose dependence. A mechanism was therefore proposed to interpret the glucose dependence of GH1 β-glucosidases. This research provides fresh insight into our current understanding of the properties and mechanisms of GH1 β-glycosidases and related enzymes that modulate their activity via feedback control mechanism.
| S-EPMC4658561 | biostudies-literature
Project description:Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus, and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5-6.0 (CIB12) and 4.5-5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma.
| S-EPMC11376072 | biostudies-literature
Project description:Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of β-glucosidases. Evaluation of previously characterized recombinant rice β-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher β-glucosidase activities on ABA-GE and gibberellin A4 glucose ester (GA4-GE). The β-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular β-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.
| S-EPMC8303963 | biostudies-literature
Project description:The porcine interferon (PoIFN) complex represents an ideal model for studying IFN evolution which has resulted from viral pressure during domestication. Bama and Banna miniature pigs are the two Chinese miniature pig breeds that have been developed as laboratory animal models for studying virus infection, pathogenesis, and vaccine evaluation. However, the PoIFN complex of such miniature pig breeds remains to be studied. In the present study, we cloned PoIFN-β genes from Bama and Banna miniature pigs, detected their PoIFN-β tissue expression profiles, prepared recombinant PoIFN-β (rPoIFN-β) using the E. coli expression system, and measured their antiviral activities against three different pig viruses. At the amino acid sequence level, PoIFN-βs of the two miniature pig breeds were identical, which shared 100% identity with that of Congjiang Xiang pigs, 99.4-100% identity with that of domestic pigs, and 99.5% identity with that of three species of African wild boars. The tissue expression profiles of PoIFN-β mRNA differed not only between the two miniature pig breeds but between miniature pigs and domestic pigs as well. The four promoter domains of PoIFN-β of the two miniature pig breeds were identical with that of humans, domestic pigs, and three species of African wild boars. The recombinant PoIFN-β prepared from the two miniature pig breeds showed dose-dependent pre-infection and post-infection antiviral activities against vesicular stomatitis virus, porcine respiratory and reproductive syndrome virus, and pig pseudorabies virus. This study provided evidence for the high sequence conservation of PoIFN-β genes within the Suidae family with different tissue expression profiles and antiviral activities.
| S-EPMC9030596 | biostudies-literature
Project description:BackgroundPretreating biomass with ionic liquids (IL) increases enzyme accessibility and cellulose is typically recovered through precipitation with an anti-solvent. An industrially feasible pretreatment and hydrolysis process requires robust cellulases that are stable and active in the presence of either small amounts of ILs co-precipitated with recovered cellulose or for saccharifications in the presence of IL. β-glucosidase (BG) hydrolyzes cellobiose into two molecules of glucose (Glc) and is the last step of biomass hydrolysis. These enzymes are prone not only to product inhibition by glucose but also to inactivation by ILs. With increasing interest in IL-based pretreatment methods, there is increasing focus toward a search for Glc-tolerant and IL-tolerant BG.ResultsWe identified a BG belonging to the GH1 family, H0HC94, encoded in Agrobacterium tumefaciens 5A, and cloned and overexpressed the protein in Escherichia coli. H0HC94 exhibited high enzymatic activity with β-glycosidic substrates (248 µmol/min/mg on pNPGlc and 262 µmol/min/mg on cellobiose) and tolerant to Glc (apparent K i = 686 mM). Further evidence of Glc-based stabilization came from the increase in melting temperature of H0HC94, with increasing Glc concentrations. The half-life of H0HC94 also increased between 2- and 20-fold in the presence of increasing concentrations of Glc. In the presence of 0.9 M of different [C2mim]-based ionic liquids, the specific activity of H0HC94 decreased by around 20-30 %. However, the addition of 100 mM glucose to the IL-enzyme mix resulted in a more stable enzyme as evidenced by the slight recovery of H0HC94 melting temperature and up to tenfold increase in half-life. This higher stability came at a cost of 2-10 % decrease in specific activity. The steady-state kinetic analyses for a subset of the ionic liquids tested indicate that the enzyme undergoes uncompetitive inhibition by glucose and ionic liquid, indicating the possibility of binding of the ionic liquid and glucose to the enzyme-substrate complex.ConclusionsH0HC94 is a Glc-stabilized BG that is also tolerant up to 0.9 M concentrations of different IL's and indicates the possibilities of using an IL-Glc-based cellulose solvent that displays enzyme-compatibility.
| S-EPMC4802596 | biostudies-literature
Project description:β-glucosidases catalyze the selective cleavage of glucosidic linkages and are an important class of enzymes having significant prospects in industrial biotechnology. These are classified in family 1 and family 3 of glycosyl hydrolase family. β-glucosidases, particularly from the fungus Trichoderma, are widely recognized and used for the saccharification of cellulosic biomass for biofuel production. With the rising trends in energy crisis and depletion of fossil fuels, alternative strategies for renewable energy sources need to be developed. However, the major limitation accounts for low production of β-glucosidases by the hyper secretory strains of Trichoderma. In accordance with the increasing significance of β-glucosidases in commercial applications, the present review provides a detailed insight of the enzyme family, their classification, structural parameters, properties, and studies at the genomics and proteomics levels. Furthermore, the paper discusses the enhancement strategies employed for their utilization in biofuel generation. Therefore, β-glucosidases are prospective toolbox in bioethanol production, and in the near future, it might be successful in meeting the requirements of alternative renewable sources of energy.
| S-EPMC3747355 | biostudies-literature
Project description:In recent years, minor ginsenosides have received increasing attention due to their outstanding biological activities, yet they are of extremely low content in wild ginseng. Ginsenoside Rb1, which accounts for 20% of the total ginsenosides, is commonly used as a precursor to produce minor ginsenosides via β-glucosidases. To date, many research groups have used different approaches to obtain β-glucosidases that can hydrolyze ginsenoside Rb1. This paper provides a compilation and analysis of relevant literature published mainly in the last decade, focusing on enzymatic hydrolysis pathways, enzymatic characteristics and molecular mechanisms of ginsenoside Rb1 hydrolysis by β-glucosidases. Based on this, it can be concluded that: (1) The β-glucosidases that convert ginsenoside Rb1 are mainly derived from bacteria and fungi and are classified as glycoside hydrolase (GH) families 1 and 3, which hydrolyze ginsenoside Rb1 mainly through the six pathways. (2) Almost all of these β-glucosidases are acidic and neutral enzymes with molecular masses ranging from 44-230 kDa. Furthermore, the different enzymes vary widely in terms of their optimal temperature, degradation products and kinetics. (3) In contrast to the GH1 β-glucosidases, the GH3 β-glucosidases that convert Rb1 show close sequence-function relationships. Mutations affecting the substrate binding site might alter the catalytic efficiency of enzymes and yield different prosapogenins. Further studies should focus on elucidating molecular mechanisms and improving overall performances of β-glucosidases for better application in food and pharmaceutical industries.
| S-EPMC9858181 | biostudies-literature
Project description:The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains.
| S-EPMC1134175 | biostudies-other
Project description:The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.
| S-EPMC4897640 | biostudies-literature