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1.
Enzyme Microb Technol ; 164: 110188, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36584665

RESUMO

In order to improve the degradation activity of ß-glucosidase (CpBgl) from Coniophora puteana, the structural modification was conducted. The enzyme activity of mutants CpBgl-Q20C and CpBgl-A240S was increased by 65.75% and 58.58%, respectively. These mutants exhibited maximum activity under the same conditions as wild-type CpBgl (65 â„ƒ and pH 5.0), slightly improved stabilities compared that of the wild-type, and remarkably enhanced activities in the presence of Mn2+ or Fe2+. The Vmax of CpBgl-Q20C and CpBgl-A240S was increased to 138.18 and 125.14 µmol/mg/min, respectively, from 81.34 µmol/mg/min of the wild-type, and the catalysis efficiency (kcat/Km) of CpBgl-Q20C (335.79 min-1/mM) and CpBgl-A240S (281.51 min-1/mM) was significantly improved compared with that of the wild-type (149.12 min-1/mM). When the mutant CpBgl-Q20C were used in the practical degradation of different biomasses, the glucose yields of filter paper, corncob residue, and fungi mycelia residue were increased by 17.68%, 25.10%, and 20.37%, respectively. The spatial locations of the mutation residues in the architecture of CpBgl and their unique roles in the enzyme-substrate binding and catalytic efficiency were probed in this work. These results laid a foundation for evolution of other glycoside hydrolases and the industrial bio-degradation of cellulosic biomass in nature.


Assuntos
Celulose , beta-Glucosidase , Biomassa , beta-Glucosidase/metabolismo , Mutação , Celulose/metabolismo
2.
Arch Microbiol ; 204(12): 694, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36346465

RESUMO

Ginsenosides are the main active components of ginseng, including many types and different contents. Among them, minor ginsenosides have better biological functions and pharmacological activities than those of the major ginsenosides. However, minor ginsenosides cannot be obtained in large quantities, but by means of enzymatic transformation technology, some major ginsenosides can be de-glycosylated at a specific position to generate minor ginsenosides. In this study, we report two glycosidase genes associated with the conversion of ginsenoside Rd to ginsenosides F2 or CK. SWMU-CK-1 was identified among the total genes extracted from the feces of plum deer by local Blast screening for putative ginsenoside conversion function, which could cause the conversion of ginsenoside Rd → F2 → CK. The other gene was found in the Bifidobacterium breve 689b SGAir 0764 chromosome genome, which might have the same function as the ß-glucosidase gene testified by the gene matching, named SWMU-F2-2, and can achieve the Rd → F2 transformation. This study reports two genes that enable achieving the biotransformation of rare ginsenosides, while it provides a new insight and a promising approach to explore new genes and develop new functions of existing genes.


Assuntos
Cervos , Ginsenosídeos , Panax , Animais , Ginsenosídeos/metabolismo , Panax/genética , Panax/microbiologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Biotransformação , Metagenômica
3.
Int J Mol Sci ; 23(19)2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36232734

RESUMO

Bermudagrass (Cynodon spp.) is one of the most widely distributed warm-season grasses globally. The growth habits and plant type of bermudagrass are strongly associated with the applied purpose of the landscape, livestock, and eco-remediation. Therefore, persistent efforts are made to investigate the genetic basis of plant type and growth habits of bermudagrass. Here, we dissect the genetic diversity of 91 wild bermudagrass resources by genome-wide association studies (GWAS) combined with weighted gene co-expression analysis (WGCNA). This work is based on the RNA-seq data and the genome of African bermudagrass (Cynodon transvaalensis Burtt Davy). Sixteen reliable single-nucleotide polymorphisms (SNPs) in transcribed regions were identified to be associated with the plant height and IAA content in diverse bermudagrass by GWAS. The integration of the results from WGCNA indicates that beta-glucosidase 31 (CdBGLU31) is a candidate gene underlying a G/A SNP signal. Furthermore, both qRT-PCR and correlation coefficient analyses indicate that CdBGLU31 might play a comprehensive role in plant height and IAA biosynthesis and signal. In addition, we observe lower plant height in Arabidopsis bglu11 mutants (homologs of CdBGLU31). It uncovers the breeding selection history of different plant types from diverse bermudagrass and provides new insights into the molecular function of CdBGLU31 both in plant types and in IAA biosynthetic pathways.


Assuntos
Arabidopsis , Cynodon , Arabidopsis/genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Cynodon/genética , Cynodon/metabolismo , Estudo de Associação Genômica Ampla , Melhoramento Vegetal
4.
Carbohydr Res ; 522: 108682, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36194965

RESUMO

Glycoside hydrolase family 12 endocellulase (GH family12) plays a key role in the degradation of ß-glucan and cellulose. Hyperthermostable GH family 12 endocellulase from the archaeon Pyrococcus furiosus (EGPf) catalyzes the hydrolysis of ß(1 â†’ 4) glucosidic linkages in cellulose and ß-glucan containing ß(1 â†’ 3),(1 â†’ 4) mixed-linkages. Therefore, in the combination with the hyperthermophilic ß-glucosidase from P. furiosus (BGLPf), non-crystalline cellulose and ß-glucan can be degraded to glucose completely by EGPf at high temperature. X-ray crystallography and protein engineering were used to reveal how the ß(1 â†’ 4) and ß(1 â†’ 3) linkages in ß-glucan substrates are recognized by the enzyme. Structural and functional analyses clarified that the active site of EGPf consists of six subsites: the reducing end subsites (+1 and + 2) recognize both ß(1 â†’ 4) and ß(1 â†’ 3) linkages of various substrates in a productive binding mode, and recognition is controlled by Trp121 and Gln208 located at subsite +2. It was also revealed that the deep cleft in subsite -4 can accommodate the torsion angles of substrates consisting of ß(1 â†’ 3),(1 â†’ 4) mixed-linkages due to the changing tilt of the Trp62 side chain. From the structural similarity, it is proposed that the substrate specificity of family 12 endocellulases towards ß(1 â†’ 3),(1 â†’ 4) mixed-linkage substrates are controlled by the subsites (+1, +2, and -4). Furthermore, the function of family 12 endocellulase could be improved by protein engineering method using the information of the analysis.


Assuntos
Pyrococcus furiosus , beta-Glucanas , Pyrococcus furiosus/metabolismo , Cristalografia por Raios X , beta-Glucosidase/metabolismo , Especificidade por Substrato , Celulose/química
5.
Chin J Nat Med ; 20(9): 712-720, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36162956

RESUMO

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3ß-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.


Assuntos
Epimedium , Animais , Epimedium/química , Flavonoides/química , Furanos , Glicosídeos/química , Hidrólise , Masculino , Simulação de Acoplamento Molecular , Estrutura Molecular , Ratos , Testosterona , beta-Glucosidase/metabolismo
6.
Biosci Biotechnol Biochem ; 86(12): 1695-1698, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36102655

RESUMO

A combined intake of cooked sweet potato and fried onion in humans was found to suppress the increase of plasma quercetin metabolite concentration. Experiments using rat ß-glucosidase indicated that excess carbohydrate digestion products, especially glucose-containing saccharides, interfere with the deglycosylation of quercetin glucosides during intestinal epithelial uptake. Combined meals of sweet potato and onion may lower the bioavailability of onion quercetin glucosides.


Assuntos
Ipomoea batatas , Cebolas , Humanos , Ratos , Animais , Quercetina/metabolismo , Glucosídeos/metabolismo , beta-Glucosidase/metabolismo
7.
ChemSusChem ; 15(21): e202201354, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-35934832

RESUMO

Due to the poor enzyme thermal stability, the efficient conversion of high crystallinity cellulose into glucose in aqueous phase over 50 °C is challenging. Herein, an enzyme-induced MOFs encapsulation of ß-glucosidase (ß-G) strategy was proposed for the first time. By using various methods, including SEM, XRD, XPS, NMR, FTIR and BET, the successful preparation of a porous channel-type flower-like enzyme complex (ß-G@MOFs) was confirmed. The prepared enzyme complex (ß-G@MOFs) materials showed improved thermal stability (from 50 °C to 100 °C in the aqueous phase) and excellent resistance to ionic liquids (the reaction temperature was as high as 110 °C) compared to the free enzyme (ß-G). Not only the catalytic hydrolysis of cellulose by single enzyme (ß-G) in ionic liquid was realized, but also the high-temperature continuous reaction performance of the enzyme was significantly improved. Benefiting from the significantly improved heat resistance, the ß-G@MOFs exhibited 32.1 times and 34.2 times higher enzymatic hydrolysis rate compared to ß-G for cellobiose and cellulose substrates, respectively. Besides, the catalytic activity of ß-G@MOFs was retained up to 86 % after five cycles at 110 °C. This was remarkable because the fixation of the enzyme by the MOFs ensured that the folded structure of the enzyme would not expand at high temperatures, allowing the native conformation of the encapsulated protein well-maintained. Furthermore, we believe that this structural stability was caused by the confinement of flower-like porous MOFs.


Assuntos
Líquidos Iônicos , Estruturas Metalorgânicas , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Celulose/química , Hidrólise , Temperatura , Temperatura Alta , Líquidos Iônicos/química , Água
8.
Sci Total Environ ; 850: 158118, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-35987234

RESUMO

Paddy soils regularly experience redox oscillations during the wetting and draining stages, yet the effects of short-term presence of oxygen (O2) on in-situ microbial hotspots and enzyme activities in anoxic ecosystems remain unclear. To fill this knowledge gap, we applied soil zymography to localize hotspots and activities of phosphomonoesterase (PME), ß-glucosidase (BG), and leucine aminopeptidase (LAP) in three compartments of rice-planted rhizoboxes (top bulk, rooted, and bottom bulk paddy soil) under oxic (+O2) and anoxic (O2) conditions. Short-term (35 min) aeration decreased PME activity by 13-49 %, BG by 4-52 %, and LAP by 12-61 % as compared with O2 in three soil compartments. The percentage of hotspot area was higher by 3-110 % for PME, by 10-60 % for BG, and by 12-158 % for LAP under +O2 vs. O2 conditions depending on a rice growth stage. Irrespective of the aeration conditions, the rhizosphere extent of rice plants for three enzymes was generally greater under higher moisture conditions and at earlier growth stage. Higher O2 sensitivity for the tested enzymes at bottom bulk soil versus other compartments suggested that short-term aeration during conventional zymography may lead to underestimation of nutrient mobilization in subsoil compared to top bulk soil. The intolerance of anaerobic microorganisms against the toxicity of O2 in the cells and the shift of microbial metabolic pathways may explain such a short-term suppression by O2. Our findings, therefore, show that anoxic conditions and soil moisture should be kept during zymography and probably other in-situ soil imaging methods when studying anoxic systems.


Assuntos
Oryza , Solo , Ecossistema , Leucil Aminopeptidase , Oxigênio , Monoéster Fosfórico Hidrolases , Microbiologia do Solo , beta-Glucosidase/metabolismo
9.
mBio ; 13(4): e0093522, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35913158

RESUMO

Cellulose being the most abundant polysaccharide on earth, beta-glucosidases hydrolyzing cello-oligosaccharides are key enzymes to fuel glycolysis in microorganisms developing on plant material. In Streptomyces scabiei, the causative agent of common scab in root and tuber crops, a genetic compensation phenomenon safeguards the loss of the gene encoding the cello-oligosaccharide hydrolase BglC by awakening the expression of alternative beta-glucosidases. Here, we revealed that the BglC compensating enzyme BcpE2 was the GH3-family beta-glucosidase that displayed the highest reported substrate promiscuity and was able to release the glucose moiety of all tested types of plant-derived heterosides (aryl ß-glucosides, monolignol glucosides, cyanogenic glucosides, anthocyanosides, and coumarin heterosides). BcpE2 structure analysis highlighted a large cavity in the PA14 domain that covered the active site, and the high flexibility of this domain would allow proper adjustment of this cavity for disparate heterosides. The exceptional substrate promiscuity of BcpE2 provides microorganisms a versatile tool for scavenging glucose from plant-derived nutrients that widely vary in size and structure. Importantly, scopolin was the only substrate commonly hydrolyzed by both BglC and BcpE2, thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes would also fine-tune the strength of virulence. IMPORTANCE Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides. In this work, structural, enzymatic, and expression studies revealed the existence of a "swiss-army knife" enzyme, BcpE2, that was able to retrieve the glucose moiety of a multitude of plant-derived substrates that vary in size, structure, and origin. This enzyme would provide the microorganisms with a tool that would allow them to find nutrients from any type of plant-derived material.


Assuntos
Glucose , beta-Glucosidase , Glucose/metabolismo , Glucosídeos/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
10.
Bioorg Chem ; 127: 106016, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35841671

RESUMO

A series of novel benzimidazole-iminosugars linked a (substuituted) phenyl group on benzene ring of benzimidazole 5(a-p) and 6(a-p) have been rationally designed and conveniently synthesized through Suzuki coupling reaction in high yields. All compounds have been evaluated for their inhibitory activities against ß-glucosidase (almond). Six compounds 5d, 6d, 6e, 6i, 6n, and 6p showed more significant inhibitory activities with IC50 values in the range of 0.03-0.08 µM, almost 10-fold improved than that of the parent analogue 4, and much higher than that of the positive control castanospermine. The additional phenyl ring and the electron donating groups on it would be beneficial for the activity. Compounds 6d, 6n, and 4 had been chosen to be tested for their inhibition types against ß-glucosidase. Interestingly, three compounds have different inhibition types although they had very similar structure. Their Ki values were calculated to be 0.02 ± 0.01 µM, 0.02 ± 0.01 µM, and 0.66 ± 0.14 µM, respectively. The equilibrium dissociation constant (KD) for 6d, 6n, and 4 and ß-glucosidase was 0.04 µM, 0.03 µM and 0.45 µM by the ITC-based assay, respectively. Molecular docking work suggests that such benzimidazole-iminosugars derivatives might bind to the active site of ß-glucosidase mainly through hydrogen bonds, the additional phenyl ring towards the solvent-exposed region played an important effect on their inhibitory activity against ß-glucosidase.


Assuntos
Benzimidazóis , beta-Glucosidase , Benzimidazóis/química , Inibidores de Glicosídeo Hidrolases/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , alfa-Glucosidases/metabolismo , beta-Glucosidase/metabolismo
11.
Colloids Surf B Biointerfaces ; 217: 112694, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35841802

RESUMO

In this study, the upper critical solution temperature (UCST)-responsive polymers poly (ethylene oxide) monomethyl ether-block-poly(acrylamide-co-acrylonitrile) (PEG-b-p(AAM-co-AN) were synthesized and successfully utilized to immobilize ß-glucosidase in crude enzyme solution. These UCST-responsive ß-glucosidase biocatalysts (PEG-b-p(AAM-co-AN@LytA-Glu) have specific UCST with tunable transition temperature, which could be tuned the separation temperature to the desired temperature range. The P2 @ LytA-Glu with an UCST of about 42.9 â„ƒ was exploited by one-step covalent immobilization of ß-glucosidase in crude enzyme solution. The prepared P2 @ LytA-Glu exhibited significantly improved temperature, pH, storage, and operation stabilities compared with that of free enzyme. The catalytic rate of P2 @ Glu-LytA was 14.5% higher than that of P2-Glu (immobilized pure ß-glucosidase), which indicated that one-step immobilization of crude enzyme directly from crude enzyme solution was feasible, and it can greatly save the purification step and reduce the experimental cost. The engineered UCST-responsive immobilized enzymes are potentially useful for the practical green biocatalysis.


Assuntos
Polímeros , beta-Glucosidase , Biocatálise , Enzimas Imobilizadas , Polietilenoglicóis , Temperatura , beta-Glucosidase/metabolismo
12.
Microbiol Spectr ; 10(4): e0153522, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35863007

RESUMO

Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by ß-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable ß-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of ß-glucosidase, the whole genome of B. altitudinis JYY-02 was sequenced and annotated; it is 3,727,518 bp long and contains 3,832 genes. The gene encoding ß-glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant ß-glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and Km values for the recombinant ß-glucosidase were 60°C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl-ß-d-glucopyranoside (pNPG) was used as the substrate. The recombinant ß-glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE ß-Glucosidases are enzymes capable of hydrolyzing ß-glucosidic linkages present in saccharides and glycosides and have many agricultural and industrial applications. Although they are found in all domains of living organisms, commercial ß-glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable ß-glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant ß-glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified ß-glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB production.


Assuntos
Bacillus , Gardenia , Bacillus/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Gardenia/genética , Gardenia/metabolismo , Concentração de Íons de Hidrogênio , Especificidade por Substrato , beta-Glucosidase/química , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
13.
Toxins (Basel) ; 14(7)2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35878196

RESUMO

As one of the most important conjugated mycotoxins, zearalenone-14-glucoside (Z14G) has received widespread attention from researchers. Although the metabolism of Z14G in animals has been extensively studied, the intracellular toxicity and metabolic process of Z14G are not fully elucidated. In this study, the cytotoxicity of Z14G to human ovarian granulosa cells (KGN) and the metabolism of Z14G in KGN cells were determined. Furthermore, the experiments of co-administration of ß-glucosidase and pre-administered ß-glucosidase inhibitor (Conduritol B epoxide, CBE) were used to clarify the mechanism of Z14G toxicity release. Finally, the human colon adenocarcinoma cell (Caco-2) metabolism model was used to verify the toxicity release mechanism of Z14G. The results showed that the IC50 of Z14G for KGN cells was 420 µM, and the relative hydrolysis rate of Z14G on ZEN was 35% (25% extracellular and 10% intracellular in KGN cells). The results indicated that Z14G cannot enter cells, and Z14G is only hydrolyzed extracellularly to its prototype zearalenone (ZEN) by ß-glucosidase which can exert toxic effects in cells. In conclusion, this study demonstrated the cytotoxicity of Z14G and clarified the toxicity release mechanism of Z14G. Different from previous findings, our results showed that Z14G cannot enter cells but exerts cytotoxicity through deglycosylation. This study promotes the formulation of a risk assessment and legislation limit for ZEN and its metabolites.


Assuntos
Adenocarcinoma , Neoplasias do Colo , Zearalenona , beta-Glucosidase , Células CACO-2 , Matriz Extracelular/metabolismo , Feminino , Glucosídeos , Humanos , Zearalenona/metabolismo , Zearalenona/toxicidade , beta-Glucosidase/metabolismo
14.
J Agric Food Chem ; 70(28): 8713-8724, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35793414

RESUMO

The enzymatic pathway of xanthan depolymerization has been predicted previously; however, the ß-glucosidase and unsaturated glucuronyl hydrolase in this system have not been cloned and characterized. This lack of knowledge hinders rational modification of xanthan and exploration of new applications. In this work, we report on the properties of Mibgl3, a xanthan-degrading enzyme isolated from Microbacterium sp. XT11. Mibgl3 exhibits typical structural features of the GH3 family but shares low sequence identity with reported GH3 enzymes. The activity of Mibgl3 can be inhibited by Cu2+, Fe2+, Zn2+, and glucose. Unlike most ß-glucosidases, Mibgl3 can tolerate a wide pH range and is activated by high concentrations of NaCl. This improves the commercial value of Mibgl3. In particular, Mibgl3 exhibits higher substrate specificity toward oligoxanthan than other ß-glucosidases. Ion chromatography, ultrahigh-performance liquid chromatography-mass spectrometry (UPLC-MS), and GC-MS results showed that Mibgl3 could effectively hydrolyze oligoxanthan to release glucose and glucuronate. Therefore, Mibgl3 might play an important role in xanthan depolymerization by functioning as hydrolase of both the xanthan backbone and sidechains. This knowledge of the enzymatic properties and hydrolysis mechanism of a ß-glucosidase will be beneficial for future applications.


Assuntos
Microbacterium , beta-Glucosidase , Cromatografia Líquida , Glucose , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Espectrometria de Massas em Tandem , beta-Glucosidase/metabolismo
15.
Molecules ; 27(13)2022 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-35807351

RESUMO

ß-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant ß-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant ß-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower KM value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields.


Assuntos
Zeolitas , beta-Glucosidase , Estabilidade Enzimática , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura , beta-Glucosidase/metabolismo
16.
Appl Microbiol Biotechnol ; 106(13-16): 5063-5079, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35833950

RESUMO

Beta-glucosidase (Bgl) is an enzyme with considerable food, beverage, and biofuel processing potential. However, as many Bgls are inhibited by their reaction end product glucose, their industrial applications are greatly limited. In this study, a novel Bgl gene (Bgl1973) was cloned from Leifsonia sp. ZF2019 and heterologously expressed in E. coli. Sequence analysis and structure modeling revealed that Bgl1973 was 748 aa, giving it a molecular weight of 78 kDa, and it showed high similarity with the glycoside hydrolase 3 (GH3) family Bgls with which its active site residues were conserved. By using pNPGlc (p-nitrophenyl-ß-D-glucopyranoside) as substrate, the optimum temperature and pH of Bgl1973 were shown to be 50 °C and 7.0, respectively. Bgl1973 was insensitive to most metal ions (12.5 mM), 1% urea, and even 0.1% Tween-80. This enzyme maintained 60% of its original activity in the presence of 20% NaCl, demonstrating its excellent salt tolerance. Furthermore, it still had 83% residual activity in 1 M of glucose, displaying its outstanding glucose tolerance. The Km, Vmax, and kcat of Bgl1973 were 0.22 mM, 44.44 µmol/min mg, and 57.78 s-1, respectively. Bgl1973 had a high specific activity for pNPGlc (19.10 ± 0.59 U/mg) and salicin (20.43 ± 0.92 U/mg). Furthermore, molecular docking indicated that the glucose binding location and the narrow and deep active channel geometry might contribute to the glucose tolerance of Bgl1973. Our results lay a foundation for the studying of this glucose-tolerant ß-glucosidase and its applications in many industrial settings. KEY POINTS: • A novel ß-glucosidase from GH3 was obtained from Leifsonia sp. ZF2019. • Bgl1973 demonstrated excellent glucose tolerance. • The glucose tolerance of Bgl1973 was explained using molecular docking analysis.


Assuntos
Actinomycetales , beta-Glucosidase , Actinomycetales/genética , Actinomycetales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Especificidade por Substrato , beta-Glucosidase/metabolismo
17.
Appl Microbiol Biotechnol ; 106(12): 4539-4551, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35723691

RESUMO

Metagenomic MeBglD2 is a glycoside hydrolase family 1 (GH1) ß-glycosidase that has ß-glucosidase, ß-fucosidase, and ß-galactosidase activities, and is highly activated in the presence of monosaccharides and disaccharides. The ß-glucosidase activity of MeBglD2 increases in a cellobiose concentration-dependent manner and is not inhibited by a high concentration of D-glucose or cellobiose. Previously, we solved the crystal structure of MeBglD2 and designed a thermostable mutant; however, the mechanism of substrate recognition of MeBglD2 remains poorly understood. In this paper, we report the X-ray crystal structures of MeBglD2 complexed with various saccharides, such as D-glucose, D-xylose, cellobiose, and maltose. The results showed that subsite - 1 of MeBglD2, which contained two catalytic glutamate residues (a nucleophilic Glu356 and an acid/base Glu170) was common to other GH1 enzymes, but the positive subsites (+ 1 and + 2) had different binding modes depending on the type of sugar. Three residues (Glu183, Asn227, and Asn229), located at the positive subsites of MeBglD2, were involved in substrate specificity toward cellobiose and/or chromogenic substrates in the presence of additive sugars. The docking simulation of MeBglD2-cellobiose indicated that Asn229 and Trp329 play important roles in the recognition of + 1 D-glucose in cellobiose. Our findings provide insights into the unique substrate recognition mechanism of GH1, which can incorporate a variety of saccharides into its positive subsites. KEY POINTS: • Metagenomic glycosidase, MeBglD2, recognizes various saccharides • Structures of metagenomic MeBglD2 complexed with various saccharides are determined • MeBglD2 has a unique substrate recognition mechanism at the positive subsites.


Assuntos
Celobiose , Metagenoma , Celobiose/metabolismo , Cristalografia por Raios X , Glucose/metabolismo , Especificidade por Substrato , alfa-L-Fucosidase/metabolismo , beta-Glucosidase/metabolismo
18.
Microb Cell Fact ; 21(1): 124, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729556

RESUMO

Replacement of petrochemical-based materials with microbially produced biodegradable alternatives calls for industrially attractive fermentation processes. Lignocellulosic materials offer non-edible alternatives for cultivated sugars, but require often use of expensive sugar releasing enzymes, such as ß-glucosidases. These cellulose treatment costs could be reduced if microbial production hosts could use short cellodextrins such as cellobiose directly as their substrates. In this study, we demonstrate production of poly(hydroxybutyrate) (PHB) in yeast Saccharomyces cerevisiae using cellobiose as a sole carbon source. Yeast strains expressing PHB pathway genes from Cupriavidus necator and cellodextrin transporter gene CDT-1 from Neurospora crassa were complemented either with ß-glucosidase gene GH1-1 from N. crassa or with cellobiose phosphorylase gene cbp from Ruminococcus flavefaciens. These cellobiose utilization routes either with Gh1-1 or Cbp enzymes differ in energetics and dynamics. However, both routes enabled higher PHB production per consumed sugar and higher PHB accumulation % of cell dry weight (CDW) than use of glucose as a carbon source. As expected, the strains with Gh1-1 consumed cellobiose faster than the strains with Cbp, both in flask and bioreactor batch cultures. In shake flasks, higher final PHB accumulation % of CDW was reached with Cbp route (10.0 ± 0.3%) than with Gh1-1 route (8.1 ± 0.2%). However, a higher PHB accumulation was achieved in better aerated and pH-controlled bioreactors, in comparison to shake flasks, and the relative performance of strains switched. In bioreactors, notable PHB accumulation levels per CDW of 13.4 ± 0.9% and 18.5 ± 3.9% were achieved with Cbp and Gh1-1 routes, respectively. The average molecular weights of accumulated PHB were similar using both routes; approximately 500 kDa and 450 kDa for strains expressing either cbp or GH1-1 genes, respectively. The formation of PHB with high molecular weights, combined with efficient cellobiose conversion, demonstrates a highly potential solution for improving attractiveness of sustainable polymer production using microbial cells.


Assuntos
Celobiose , Saccharomyces cerevisiae , Carbono/metabolismo , Celobiose/metabolismo , Fermentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Glucosidase/metabolismo
19.
Antonie Van Leeuwenhoek ; 115(8): 955-968, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35661053

RESUMO

Levilactobacillus (L.) brevis TMW 1.2112 is an isolate from wheat beer that produces O2-substituted (1,3)-ß-D-glucan, a capsular exopolysaccharide (EPS) from activated sugar nucleotide precursors by use of a glycosyltransferase. Within the genome sequence of L. brevis TMW 1.2112 enzymes of the glycoside hydrolases families were identified. Glycoside hydrolases (GH) are carbohydrate-active enzymes, able to hydrolyse glycosidic bonds. The enzyme ß-glucosidase BglB (AZI09_02170) was heterologous expressed in Escherichia coli BL21. BglB has a monomeric structure of 83.5 kDa and is a member of the glycoside hydrolase family 3 (GH 3) which strongly favoured substrates with ß-glycosidic bonds. Km was 0.22 mM for pNP ß-D-glucopyranoside demonstrating a high affinity of the recombinant enzyme for the substrate. Enzymes able to degrade the (1,3)-ß-D-glucan of L. brevis TMW 1.2112 have not yet been described. However, BglB showed only a low hydrolytic activity towards the EPS, which was measured by means of the D-glucose releases. Besides, characterised GH 3 ß-glucosidases from various lactic acid bacteria (LAB) were phylogenetically analysed to identify connections in terms of enzymatic activity and ß-glucan formation. This revealed that the family of GH 3 ß-glucosidases of LABs comprises most likely exo-active enzymes which are not directly associated with the ability of these LAB to produce EPS.


Assuntos
Glicosídeo Hidrolases , Lactobacillaceae/enzimologia , beta-Glucanas , Cerveja , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Especificidade por Substrato , beta-Glucanas/química , beta-Glucanas/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
20.
Mol Plant Microbe Interact ; 35(9): 779-790, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35617509

RESUMO

Asian soybean rust, caused by the fungus Phakopsora pachyrhizi, is one of the most important diseases affecting soybean production in tropical areas. During infection, P. pachyrhizi secretes proteins from haustoria that are transferred into plant cells to promote virulence. To date, only one candidate P. pachyrhizi effector protein has been characterized in detail to understand the mechanism by which it suppresses plant defenses to enhance infection. Here, we aimed to extend understanding of the pathogenic mechanisms of P. pachyrhizi based on the discovery of host proteins that interact with the effector candidate Phapa-7431740. We demonstrated that Phapa-7431740 suppresses pathogen-associated molecular pattern-triggered immunity (PTI) and that it interacts with a soybean glucan endo-1,3-ß-glucosidase (GmßGLU), a pathogenesis-related (PR) protein belonging to the PR-2 family. Structural and phylogenetic characterization of the PR-2 protein family predicted in the soybean genome and comparison to PR-2 family members in Arabidopsis thaliana and cotton, demonstrated that GmßGLU is a type IV ß-1,3-glucanase. Transcriptional profiling during an infection time course showed that the GmßGLU mRNA is highly induced during the initial hours after infection, coinciding with peak of expression of Phapa-7431740. The effector was able to interfere with the activity of GmßGLU in vitro, with a dose-dependent inhibition. Our results suggest that Phapa-7431740 may suppress PTI by interfering with glucan endo-1,3-ß-glucosidase activity. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.


Assuntos
Arabidopsis , Phakopsora pachyrhizi , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Interações Hospedeiro-Patógeno , Padrões Moleculares Associados a Patógenos/metabolismo , Phakopsora pachyrhizi/metabolismo , Filogenia , Doenças das Plantas/microbiologia , RNA Mensageiro/metabolismo , Soja/microbiologia , Virulência , beta-Glucosidase/metabolismo
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