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Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level. Pompe disease is caused by mutations in lysosomal acid α-glucosidase (GAA) and patients are being treated with recombinant human α-glucosidase (rhGAA). Here the authors present the crystal structures of rhGAA and its complexes with inhibitors and a pharmacological chaperone, which is important for drug development.

Metal–organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely β-glucosidase, invertase, β-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH 2 , or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes. Metal–organic frameworks (MOFs) are attractive for encapsulating enzymes for industrial purposes because they can increase selectivity, stability, and/or activity of the enzymes. Here, the authors developed an economical solid-state mechanochemical method to encapsulate enzymes during MOF synthesis.

Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)₈ TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na⁺ or K⁺. The enzyme showed strong ability to hydrolyze cellobiose with a K ₘ and V ₘₐₓ of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K ᵢ of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Recent studies have suggested a correlation between genotype groups of Brettanomyces bruxellensis and their source of isolation. To further explore this relationship, the objective of this study was to assess metabolic differences in carbon and nitrogen assimilation between different B. bruxellensis strains from three beverages, including beer, wine, and soft drink, using Biolog Phenotype Microarrays. While some similarities of physiology were noted, many traits were variable among strains. Interestingly, some phenotypes were found that could be linked to strain origin, especially for the assimilation of particular α- and β-glycosides as well as α- and β-substituted monosaccharides. Based upon gene presence or absence, an α-glucosidase and β-glucosidase were found explaining the observed phenotypes. Further, using a PCR screen on a large number of isolates, we have been able to specifically link a genomic deletion to the beer strains, suggesting that this region may have a fitness cost for B. bruxellensis in certain fermentation systems such as brewing. More specifically, none of the beer strains were found to contain a β-glucosidase, which may have direct impacts on the ability for these strains to compete with other microbes or on flavor production.

β-Glucosidases are useful for hydrolysis of glycosidically-bound volatiles (GBV), thereby facilitating the release of aroma chemicals from the fruit matrices. In this study, 10 putative glycosyl hydrolases belonging to GH1 family from Lactiplantibacillus plantarum NCIM 2903 were cloned and recombinantly expressed. Interestingly, only one (LpBgl5) of the nine soluble proteins, previously characterized as a 6-phospho-β-glucosidase showed β-glucosidase activity which was further characterized. The enzyme had an optimum pH and temperature of 6 and 40°C, respectively, and was categorized as aryl-β-glucosidase due to its ability to hydrolyze different natural as well as synthetic glucosides except cellobiose. The enzyme exhibited functional activity across multiple substrates, with relative activity decreasing sequentially from β-xylosidase to β-glucosidase and finally β-mannosidase. The β-xylosidase and β-glucosidase activities of LpBgl5 were stimulated up to 300% and 700% in the presence of 4 M xylose and 4 M glucose, respectively. The enzyme could also hydrolyze GBV from mango. To our knowledge, this is the first recombinant β-glucosidase/β-xylosidase/β-mannosidase from L. plantarum to have potential for aroma enhancement in fruit products. Key points • A recombinant β-glycosidase from Lactiplantibacillus plantarum was characterized. • The enzyme showed higher β-xylosidase activity than β-glucosidase activity. • The enzyme could also hydrolyze glycosidically bound volatiles from mango.

Use of recombinant glycosidases is a promising approach for the production of minor ginsenosides, e.g., Compound K (CK) and F 1 , which have potential applications in the food industry. However, application of these recombinant enzymes for food-grade preparation of minor ginsenosides are limited by the lack of suitable expression hosts and low productivity. In this study, Corynebacterium glutamicum ATCC13032 , a GRAS strain that has been used extensively for the industrial-grade production of additives for foodstuffs, was employed to express a novel β-glucosidase (MT619) from Microbacterium testaceum ATCC 15829 with high ginsenoside-transforming activity. A cellulose-binding module was additionally fused to the N-terminus of MT619 for immobilization on cellulose, which is an abundant and safe material. Via one-step immobilization, the fusion protein in cell lysates was efficiently immobilized on regenerated amorphous cellulose at a high density (maximum 984 mg/g cellulose), increasing the enzyme concentration by 286-fold. The concentrated and immobilized enzyme showed strong conversion activities against protopanaxadiol- and protopanaxatriol-type ginsenosides for the production of CK and F 1 . Using gram-scale ginseng extracts as substrates, the immobilized enzyme produced 7.59 g/L CK and 9.42 g/L F 1 in 24 h. To the best of our knowledge, these are the highest reported product concentrations of CK and F 1 , and this is the first time that a recombinant enzyme has been immobilized on cellulose for the preparation of minor ginsenosides. This safe, convenient, and efficient production method could also be effectively exploited in the preparation of food-processing recombinant enzymes in the pharmaceutical, functional food, and cosmetics industries.

Here we demonstrate that Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol), largely used as a buffering agent, is a linear mixed inhibitor ( K i = 12 ± 2 mM and α = 3 ± 1) of the GH1 β-glucosidase from the insect Spodoptera frugiperda (Sfβgly). Such an inhibition mechanism implies the formation of a non-productive ESI complex involving Sfβgly, substrate, and Tris. In addition, Tris binding reduces by 3 fold the enzyme affinity for the substrate. Hence, at concentrations higher than the K i , Tris can completely abolish Sfβgly activity, whereas even at lower concentrations the presence of Tris causes underestimation of β-glucosidase kinetic parameters ( K m and k cat ). In agreement with the inhibition mechanism, computational docking showed that Tris could bind to a pocket placed at the lateral of the active site opening in the Sfβgly-substrate complex, hence leading to the formation of an ESI complex. In agreement with the crystallographic data available, computational docking also showed that Tris may find binding spots in the interior of the active site of the Sfβgly and several GH1 β-glucosidases. Moreover, the variety of their active site shapes results in a multiplicity of binding profiles, foreseeing different inhibition mechanisms. Thus, Tris inhibition may affect other GH1 β-glucosidases. This remark should be taken into account in their study, highlighting the importance of the appropriate buffer for accurate enzyme characterization.

Forty-four endophytic fungal isolates obtained from marine sponge, Hyrtios erectus, were evaluated and screened for their hydrolase activities. Most of the isolates were found to be prolific producers of hydrolytic enzymes. Only 11 isolates exhibited maximum cellular contents of lipids, rhamnolipids, and protein in the fungal isolates under the isolation numbers MERVA5, MERVA22, MERVA25, MERVA29, MERVA32, MERVA34, MERV36, MERVA39, MERVA42, MERVA43, and MERVA44. These isolate extracts exhibit the highest reducing activities against carbohydrate-metabolizing enzymes including α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase. Consequently, based on morphological and cultural criteria, as well as sequence information and phylogenetic analysis, these isolates could be identified and designated as Penicillium brevicombactum MERVA5, Arthrinium arundinis MERVA22, Diaporthe rudis MERVA25, Aspergillus versicolor MERVA29, Auxarthron alboluteum MERVA32, Dothiorella sarmentorum MERVA34, Lophiostoma sp. MERVA36, Fusarium oxysporum MERVA39, Penicillium chrysogenum MERVA42, Penicillium polonicum MERVA43, and Trichoderma harzianum MERVA44. The endophytic fungal species, D. rudis MERVA25, P. polonicum MERVA43, Lophiostoma sp. MERVA36, A. alboluteum MERVA32, T. harzianum MERVA44, F. oxysporum MERVA39, A. versicolor MERVA29, and P. chrysogenum MERVA42 extracts, showed significant hepatitis C virus (HCV) inhibition. Moreover, D. sarmentorum MERVA34, P. polonicum MERVA43, and T. harzianum MERVA44 extracts have the highest antitumor activity against human hepatocellular carcinoma cells (HepG2).

A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in glycogen storage disease type II [a severe metabolic myopathy, Pompe disease (PD)] as the currently available therapy, replacement of the missing enzyme acid alpha‐glucosidase, fails to reverse skeletal muscle pathology. PD, a paradigm for LSDs, is characterized by both lysosomal abnormality and dysfunctional autophagy. Here, we show that TFEB is a viable therapeutic target in PD: overexpression of TFEB in a new muscle cell culture system and in mouse models of the disease reduced glycogen load and lysosomal size, improved autophagosome processing, and alleviated excessive accumulation of autophagic vacuoles. Unexpectedly, the exocytosed vesicles were labelled with lysosomal and autophagosomal membrane markers, suggesting that TFEB induces exocytosis of autophagolysosomes. Furthermore, the effects of TFEB were almost abrogated in the setting of genetically suppressed autophagy, supporting the role of autophagy in TFEB‐mediated cellular clearance. Graphical Abstract New Pompe disease models allow testing of novel therapeutic approach for lysosomal storage disorders. The transcription factor EB promotes clearing of muscles from excessive oxygen and autophagic debris, by inducing exocytosis of autophagolysosomes.

β-Glucosidase (BG) is widely applied in the biofuel’s industry, as part of a cellulase cocktail to catalyze the hydrolysis of the β-1,4 linkages that join two glucose molecules in a cellulose polymer. The hydrolysis step is generally recognized as the major limiting step in the development of efficient enzyme - based technologies for the conversion of lignocellulosic biomass to sugars and the production of biofuels due to the accumulation of the reaction product, glucose. Relieving this glucose inhibition of BG is therefore a major challenge. In this study, O08324, a putative BG gene encoded in the hyperthermophilic archaeon Thermococcus sp., was cloned and overexpressed in Escherichia coli . O08324 showed maximum activity between pH 5–6.8 and at 78 °C and was thermostable with a half-life of 860 min at 78 °C in the presence of 1.5 M glucose. O08324 was not inhibited by glucose up to the highest assayable concentration of 4 M and also shows no decrease in activity in the presence of up to 4 M of sodium chloride or potassium chloride. O08324 supplementation of Trichoderma viride cellulase enhanced glucose production by more than 50 % compared to a commercially available BG, when Avicel (10 %, w / v ) was used as a substrate at 37 °C. Multiple sequence alignments across previously reported glucose - tolerant BGs shows that many conserved residues previously implicated in glucose tolerance are not conserved in this BG suggesting a need for a relook at understanding the molecular basis of glucose tolerance.