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1.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810098

RESUMO

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl ß-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.


Assuntos
Bifidobacterium/metabolismo , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/biossíntese , Bifidobacterium/genética , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Glicosídeo Hidrolases/genética , Glicosilação , Humanos , Hidrólise , Cinética , Espectrometria de Massas , Leite Humano/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Oligossacarídeos/química , Conformação Proteica , Engenharia de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato
2.
Carbohydr Polym ; 259: 117741, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33674001

RESUMO

Polysialic acid (polySia) is a linear polysaccharide comprised of N-acetylneuraminic acid residues and its over-expression in cancer cells has been correlated with poor clinical prognosis. An assay has been developed for quantitative analysis of cellular polySia expression. This was achieved by extracting and purifying released polySia from glycoproteins by mild acid hydrolysis and optimised organic extraction. The polySia was further hydrolysed into Sia monomers, followed by fluorescent labelling and quantitative analysis. The assay was qualified utilising endoneuraminidase-NF to remove polySia from the surface of C6-ST8SiaII cancer cells (EC50 = 2.13 ng/mL). The result was comparable to that obtained in a polySia-specific cellular ELISA assay. Furthermore, the assay proved suitable for evaluation of changes in polySia expression following treatment with a small molecule inhibitor of polysialylation. Given the importance of polySia in multiple disease states, notably cancer, this is a potentially vital tool with applications in the fields of drug discovery and glycobiology.


Assuntos
Cromatografia de Fase Reversa , Ácidos Siálicos/análise , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ensaio de Imunoadsorção Enzimática , Glicosídeo Hidrolases/metabolismo , Ratos , Ácidos Siálicos/metabolismo , Sialiltransferases/antagonistas & inibidores , Sialiltransferases/metabolismo
3.
Carbohydr Polym ; 260: 117799, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33712147

RESUMO

Bacillus amyloliquefaciens strain PPL shows a potential for the control of phytopathogenic fungi. In the present study, upon growing the strain PPL on various forms of chitosan (0.5 % powder, 0.1 % soluble, and 0.15 % colloidal) as the carbon source, the antifungal activity on tomato Fusarium wilt correlated with the activity of chitosanase and ß-1,3-glucanase. The colloidal substrate-based strain PPL fermentation displayed the highest degree of spore germination inhibition (79.5 %) and biocontrol efficiency (76.0 %) in tomato by increased biofilm formation. The colloidal culture upregulated the expression of chitosanase gene (5.9-fold), and the powder attributed to the expression of cyclic lipopeptides-genes (2.5-5.7 fold). Moreover, the three chitosan cultures induced the morphological changes of Fusarium oxysporum. These results suggest that the choice of growth substrate synergistically affects the production of secondary metabolites by PPL strain, and consequently its antifungal activity.


Assuntos
Quitosana/química , Polímeros/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/crescimento & desenvolvimento , Bacillus amyloliquefaciens/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Biofilmes , Fusarium/efeitos dos fármacos , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Lipopeptídeos/metabolismo , Lycopersicon esculentum/microbiologia
4.
Food Chem ; 352: 128685, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33691998

RESUMO

Neoagarooligosaccharides (NAOs) are drawing more and more attention because of their numerous bioactivities, yet limited number of agarases impedes NAOs production from red algae. In this study, predicted agar polysaccharide utilization loci (agar-PUL) were firstly used as inventory for agarase. 6 agarases were identified from agar-PULs and two of them were successfully expressed and analyzed. Then enzyme cocktail (GH16-1:GH16-2:Aga50D = 2:1:1) was proved to have highest synergistic effect. Finally homogenization was applied to G. amansii and proved to be an efficient way to release agar from tissues. When liquid-to-solid ratio was 9 g/150 mL, homogenization time was 20 min, and enzyme cocktail loading was 150 U/150 mL, maximum NAOs production (90.2 mg per 9 g wet G. amansii) could be achieved. Enzyme supported one-step process (ESOP) proposed in study is environment-friendly, time saving, cost saving and none-destructive, therefore has a potential industrial application in red algae utilization.


Assuntos
Ágar/química , Glicosídeo Hidrolases/metabolismo , Rodófitas/química , Ágar/metabolismo
5.
Arch Insect Biochem Physiol ; 106(3): e21766, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33590531

RESUMO

Among termites, lower termites need symbiotic microorganisms in the digestive tract for digestion and cellulose metabolism. In this symbiotic relationship, the decomposition of cellulose is initiated by endoglucanase in termite salivary glands and completed by ß-glycosidase of symbiotic microorganisms in the hindgut. The expression of ß-glycosidase in lower termites has been reported in recent studies. The expression of two endoglucanases and one ß-glycosidase gene related to cellulose degradation was identified in Reticulitermes speratus, a lower termite, through transcriptomic analysis. The proposed enzyme activities of three identified cellulose degradation genes were confirmed by heterologous expression in Escherichia coli. In addition to the endoglucanase expressed in the salivary gland, additional endoglucanase and ß-glycosidase genes suggest that R. speratus performs the overall cellulose digestion using its own enzymes at all stages.


Assuntos
Celulases/genética , Isópteros/genética , Animais , Celulase/metabolismo , Celulases/metabolismo , Celulose/metabolismo , Trato Gastrointestinal/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Genes de Insetos , Glicosídeo Hidrolases/metabolismo , Isópteros/metabolismo
6.
Poult Sci ; 100(2): 765-775, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33518131

RESUMO

This review discusses the complex nature of the primary nonstarch polysaccharide (NSP) in corn with respect to the merit of debranching enzymes. Celluloses, hemicelluloses, and pectins comprise the 3 major categories of NSP that make up nearly 90% of plant cell walls. Across cereals, the hemicellulose arabinoxylan exists as the primary NSP, followed by cellulose, glucans, and others. Differences in arabinoxylan structure among cereals and cereal fractions are facilitated by cereal type, degree and pattern of substitution along the xylan backbone, phenol content, and cross-linkages. In particular, arabinoxylan (also called glucuronoarabinoxylan) in corn is heavily fortified with substituents, being more populated than in wheat and other cereal grains. Feed-grade xylanases - almost solely of the glycoside hydrolase (GH) 10 and GH 11 families - require at least 2 or 3 contiguous xylose units to be free of attachments to effectively attack the xylan chain. This canopy of attachments, along with a high phenol content and the insoluble nature of corn glucuronoarabinoxylan, confers a significant resistance to xylanase attack. Both in vitro and in vivo studies demonstrate that debranching enzymes appreciably increase xylanase access and fiber degradability by removing these attachments and breaking phenolic linkages. The enzymatic degradation of the highly branched arabinoxylan can facilitate disassembly of other fibers by increasing exposure to pertinent carbohydrases. For cereals, the arabinofuranosidases, α-glucuronidases, and esterases are some of the more germane debranching enzymes. Enzyme composites beyond the simple core mixes of xylanases, cellulases, and glucanases can exploit synergistic benefits generated by this class of enzymes. A broad scope of enzymatic activity in customized mixes can more effectively target the resilient NSP construct of cereal grains in commercial poultry diets, particularly those in corn-based feeds.


Assuntos
Ração Animal/análise , Glicosídeo Hidrolases/metabolismo , Aves Domésticas/metabolismo , Soja , Zea mays , Animais , Galinhas/metabolismo , Fibras na Dieta/administração & dosagem , Fibras na Dieta/metabolismo
7.
Poult Sci ; 100(2): 788-796, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33518133

RESUMO

Non-starch polysaccharides (NSP), especially in water-soluble form, are a common anti-nutritional factor in cereal-based poultry diets. Consequently, carbohydrases are applied to diets to combat the negative effects of NSP on bird performance and health, particularly when feeding viscous grains. This study investigated the effect of supplementing multi-carbohydrases (MC) to broiler diets containing either low (LS) or high (HS) soluble NSP (sNSP) to total NSP (tNSP) ratios on energy partitioning, nitrogen (N) balance, and performance. A 2 × 2 factorial arrangement of treatments (MC, no or yes; sNSP/tNSP, LS vs. HS) was applied, resulting in 4 dietary treatments, each replicated 8 times. These treatments were fed to Ross 308 broilers in closed-circuit indirect calorimetry chambers, with 2 birds (a male and a female) per replicate chamber (n = 64). The results showed that MC addition increased AME, net energy (NE), and AME/gross energy, regardless of sNSP/tNSP content (P < 0.01 for all). There was an MC × sNSP/tNSP interaction for feed intake (FI, P < 0.05), denoting that in the absence of MC, the HS-fed birds had lower FI than LS-fed birds, but this difference was eliminated when MC was present. There were MC × sNSP/tNSP interactions observed for AME intake (AMEi) per metabolic BW (BW0.70, P < 0.05), AMEi/N retention (Nr, P < 0.01), NE intake (NEi)/Nr (P < 0.05), retained energy (RE) as fat per total RE (REf/RE, P < 0.01), and N efficiency (Nr/N intake, P < 0.05). These interactions showed that MC application increased AMEi/BW0.70, AMEi/Nr, NEi/Nr, and REf/RE only in the HS-fed birds, and N efficiency only in the LS-fed broilers. This study demonstrated that MC application markedly increased feed energy utilization in all diets, and increased N efficiency in birds fed an LS diet.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Galinhas/metabolismo , Metabolismo Energético , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/administração & dosagem , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais , Feminino , Masculino
8.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557290

RESUMO

ß-Glucan is widely distributed in various plants and microorganisms and is composed of ß-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with ß-1,6- or ß-1,4-linkage. Numerous studies have investigated different ß-glucans and revealed their bioactivities. To understand the structure-function relationship of ß-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain ß-1,6-branched ß-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived ß-1,3-glucan recognition protein and fungal endo-ß-1,6-glucanase (Neg1)-derived ß-1,6-glucan recognition protein, respectively. In this approach, two ß-glucan recognition proteins bound to ß-glucan molecules come into close proximity, resulting in the assembly of the full-length reporter enzyme and induction of transient luciferase activity, indicative of the structure of ß-glucan. To test the applicability of this assay, ß-glucan and two ß-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a ß-1,3-glucan with a long polymer of ß-1,6-glucan in a dose-dependent manner. This simple test also allows the monitoring of real-time changes in the side chain structure and serves as a convenient method to distinguish between ß-1,3-glucan and long-chain ß-1,6-branched ß-1,3-glucan in various soluble and insoluble ß-glucans.


Assuntos
Técnicas Biossensoriais/métodos , Parede Celular/metabolismo , Glicosídeo Hidrolases/metabolismo , beta-Glucanas/química , beta-Glucanas/metabolismo , Animais , Luciferases/metabolismo , Especificidade por Substrato
9.
Biotechnol Adv ; 47: 107704, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33548454

RESUMO

Xylan is the most abundant hemicellulose in nature and as such it is a huge source of renewable carbon. Its bioconversion requires a battery of xylanolytic enzymes. Of them the most important are the endo-ß-1,4-xylanases which depolymerize the polysaccharide into smaller fragments. Most of the xylanases are members of glycoside hydrolase (GH) families 10 and 11, although they are classified in some other GH families. The relatively new xylanases of GH30 are of special interest. Initially, they appeared to be specific glucuronoxylanases, however, other specificities were found later among prokaryotic and in particular eukaryotic enzymes. This review gives an overview of the substrate and product specificities observed for the GH30 xylanases characterized to date. An emphasis is given to the structure-activity relationship in order to explain how minor differences in catalytic centre and its vicinity can alter catalytic properties from the endoxylanase into the reducing end xylose releasing exoxylanase or into the non-reducing end xylobiohydrolase. Biotechnological potential of the GH30 xylanases is also considered.


Assuntos
Endo-1,4-beta-Xilanases , Glicosídeo Hidrolases , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Especificidade por Substrato , Xilanos , Xilose
10.
Food Chem ; 345: 128771, 2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-33601652

RESUMO

The isothiocyanate sulforaphane (SF) is one of the most potent naturally occurring Phase 2 enzymes inducers derived from brassica vegetables like broccoli, cabbage, brussel sprouts, etc. Ingestion of broccoli releases SF via hydrolysis of glucoraphanin (GRP) by plant myrosinase and/or intestinal microbiota. However, both SF and plant myrosinase are thermal-labile, and the epithiospecifier protein (ESP) directs the hydrolysis of GRP toward formation of sulforaphane nitrile instead of SF. In addition, bacterial myrosinase has low hydrolyzing efficiency. In this review, we discuss strategies that could be employed to improve the stability of SF, increase SF formation during thermal and non-thermal processing of broccoli, and enhance the myrosinase-like activity of the gut microbiota. Furthermore, new cooking methods or blanching technologies should be developed to maintain myrosinase activity, and novel thermostable myrosinase and/or microbes with high SF producing abilities should also be developed.


Assuntos
Brassica/química , Isotiocianatos/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Brassica/metabolismo , Culinária , Estabilidade de Medicamentos , Glucosinolatos/química , Glucosinolatos/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrólise , Imidoésteres/química , Imidoésteres/metabolismo , Isotiocianatos/química , Pressão
11.
Nat Commun ; 12(1): 380, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452262

RESUMO

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.


Assuntos
Bactérias/enzimologia , Eucariotos/enzimologia , Glicosídeo Hidrolases/metabolismo , Heme/metabolismo , Regulação Alostérica , Sequência de Aminoácidos/genética , Bactérias/genética , Cristalografia por Raios X , Eucariotos/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/ultraestrutura , Simulação de Dinâmica Molecular , Filogenia , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
12.
Sheng Wu Gong Cheng Xue Bao ; 37(1): 112-129, 2021 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-33501794

RESUMO

Water solubility, stability, and bioavailability, can be substantially improved after glycosylation. Glycosylation of bioactive compounds catalyzed by glycoside hydrolases (GHs) and glycosyltransferases (GTs) has become a research hotspot. Thanks to their rich sources and use of cheap glycosyl donors, GHs are advantageous in terms of scaled catalysis compared to GTs. Among GHs, sucrose phosphorylase has attracted extensive attentions in chemical engineering due to its prominent glycosylation activity as well as its acceptor promiscuity. This paper reviews the structure, catalytic characteristics, and directional redesign of sucrose phosphorylase. Meanwhile, glycosylation of diverse chemicals with sucrose phosphorylase and its coupling applications with other biocatalysts are summarized. Future research directions were also discussed based on the current research progress combined with our working experience.


Assuntos
Glucosiltransferases , Glicosiltransferases , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Glicosiltransferases/genética
13.
Carbohydr Polym ; 255: 117384, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436213

RESUMO

Prebiotics are known for their ability to modulate the composition of the human microbiome and mediate health-promoting benefits. Endo-levanases, which hydrolyze levan into short-chain FOS, could be used for the production of levan-based prebiotics. The novel endo-levanase (LevB2286) from Azotobacter chroococcum DSM 2286, combines an exceptionally high specific activity with advantageous hydrolytic properties. Starting from levan isolated from Timothy grass, LevB2286 produced FOS ranging from DP 2 - 8. In contrast to endo-levanases described in the literature, LevB2286 formed minor amounts of fructose and levanbiose, even with greatly extended incubation. The combined activity of LevB2286 and the levansucrase LevS1417 from Gluconobacter japonicus LMG 1417 led to a one-step synthesis of levan-type FOS from sucrose. 387.4 ± 17.3 g L-1 FOS were produced within 48 h by the production strategy based on crude cell extract of recombinant Escherichia coli expressing levS1417 and levB2286 simultaneously.


Assuntos
Azotobacter/enzimologia , Proteínas de Bactérias/metabolismo , Gluconobacter/enzimologia , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Oligossacarídeos/biossíntese , Prebióticos/análise , Azotobacter/genética , Proteínas de Bactérias/genética , Dissacarídeos/química , Dissacarídeos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Frutanos/química , Frutanos/metabolismo , Frutose/química , Frutose/metabolismo , Expressão Gênica , Gluconobacter/genética , Glicosídeo Hidrolases/genética , Hexosiltransferases/genética , Humanos , Hidrólise , Oligossacarídeos/química , Phleum/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sacarose/química , Sacarose/metabolismo
14.
J Agric Food Chem ; 69(1): 78-87, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33393308

RESUMO

Chitooligosaccharides (COS) generated from either chitin (chitin oligosaccharides) or chitosan (chitosan oligosaccharides) have a wide range of applications in agriculture, medicine, and other fields. Here, we report the characterization of a chitosanase from Bacillus amyloliquefaciens (BamCsn) and the importance of a tryptophan (Trp), W204, for BamCsn activity. BamCsn hydrolyzed the chitosan polymer by an endo mode. It also hydrolyzed chitin oligosaccharides and interestingly exhibited transglycosylation activity on chitotetraose and chitopentaose. Mutation of W204, a nonconserved amino acid in chitosanases, to W204A abolished the hydrolytic activity of BamCsn, with a change in the structure that resulted in a decreased affinity for the substrate and impaired the catalytic ability. Phylogenetic analysis revealed that BamCsn could belong to a new class of chitosanases that showed unique properties like transglycosylation, cleavage of chitin oligosaccharides, and the presence of W204 residues, which is important for activity. Chitosanases belonging to the BamCsn class showed a high potential to generate COS from chitinous substrates.


Assuntos
Bacillus amyloliquefaciens/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/biossíntese , Bacillus amyloliquefaciens/química , Bacillus amyloliquefaciens/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Quitina/metabolismo , Quitosana/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Especificidade por Substrato
15.
Nat Commun ; 12(1): 367, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446650

RESUMO

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Cinética , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Xanthomonas/química , Xanthomonas/genética , Xilose/química , Xilose/metabolismo
16.
Carbohydr Polym ; 251: 117056, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33142608

RESUMO

We describe a method for permitting efficient modification by transglucosidase (TGA), from glycoside hydrolase family 31 (GH31), sequentially after the pre-treatment by maltogenic α-amylases (MA) from GH13. TGA treatment without MA pre-treatment had negligible effects on native starch, while TGA treatment with MA pre-treatment resulted in porous granules and increased permeability to enzymes. MA→TGA treatments lead to decreased molecular size of amylopectin molecules, increased α-1,6 branching, and increased amounts of amylopectin chains with the degree of polymerization (DP)<10 and decreased amounts of DP 10-28 after debranching. Wide-angle X-ray scattering (WAXS) data showed a general decrease in crystallinity except for a long term (20 h) TGA post-treatment which increased the relative crystallinity back to normal. MA→TGA treatment significantly lowered the starch retrogradation of starch and retarded the increase of storage- and loss moduli during storage. This work demonstrates the potential of sequential addition of starch active enzymes to obtain granular starch with improved functionality.


Assuntos
Glucosidases/química , Glicosídeo Hidrolases/química , Amido/química , Zea mays/química , Amilopectina/química , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrólise , Porosidade , Difração de Raios X/métodos
17.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140523, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853774

RESUMO

Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Thermotoga maritima/química , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Thermotoga maritima/enzimologia
18.
Int J Biol Macromol ; 169: 302-310, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33333093

RESUMO

Neurospora crassa possesses six putative xylanases and four putative xylosidases. qRT-PCR results showed that the expression of all these xylanolytic enzymes was induced by xylan. Except for two intracellular ß-xylosidases, others were shown to be secreted enzymes based on the localization analysis of EGFP-fusion proteins. Among them, GH10-1, GH10-2, GH11-1, and GH11-2 were successfully expressed and characterized as typical endo-ß-1,4-xylanases that hydrolyze the xylooligosaccharides with a polymeric degree not less than three or four. Strains deleted for either gh10-1, gh10-2, gh3-7, or gh3-8 displayed decreased growth in xylan and biomass media. Disruption of gh3-7 or gh43-1 resulted in enhanced-xylanolytic enzyme activity when cultivated in biomass medium. Collectively, these results suggest that xylooligosaccharides released by the actions of xylanases and xylosidases not only serve as the carbon sources to maintain the growth of N. crassa, but they also act as inducers to trigger the expression of hydrolytic enzymes in vivo.


Assuntos
Neurospora crassa/metabolismo , Xilanos/química , Xilanos/metabolismo , Biomassa , Metabolismo dos Carboidratos/fisiologia , Endo-1,4-beta-Xilanases/metabolismo , Glucuronatos/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Especificidade por Substrato , Xilosidases/química , Xilosidases/metabolismo
19.
Int J Biol Macromol ; 168: 223-232, 2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33309660

RESUMO

A novel thermostable xylanase gene from Chaetomium sp. CQ31 was cloned and codon-optimized (CsXynBop). The deduced protein sequence of the gene shared the highest similarity of 75% with the glycoside hydrolase (GH) family 10 xylanase from Achaetomium sp. Xz-8. CsXynBop was over-expressed in Pichia pastoris GS115 by high-cell density fermentation, with the highest xylanase yield of 10,017 U/mL. The recombinant xylanase (CsXynBop) was purified to homogeneity and biochemically characterized. CsXynBop was optimally active at pH 6.5 and 85 °C, respectively, and stable over a broad pH range of 5.0-9.5 and up to 60 °C. The enzyme exhibited strict substrate specificity towards oat-spelt xylan (2, 489 U/mg), beechwood xylan (1522 U/mg), birchwood xylan (1067 U/mg), and showed relatively high activity towards arabinoxylan (1208 U/mg), but exhibited no activity on other tested polysaccharides. CsXynBop hydrolyzed different xylans to yield mainly xylooligosaccharides (XOSs) with degree of polymerization (DP) 2-5. The application of CsXynBop (200 U/g malt) in malt mashing substantially decreased the filtration time and viscosity of malt by 42.3% and 8.6%, respectively. These excellent characteristics of CsXynBop may make it a good candidate in beer industry.


Assuntos
Chaetomium/enzimologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Sequência de Aminoácidos , Cerveja/microbiologia , Chaetomium/genética , Chaetomium/metabolismo , Clonagem Molecular/métodos , Estabilidade Enzimática , Glucuronatos , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Oligossacarídeos , Especificidade por Substrato , Xilanos/química , Xilanos/metabolismo
20.
Food Chem ; 336: 127688, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32768904

RESUMO

The effects of carboxymethylation, hydroxypropylation and dual enzyme hydrolysis combined with heating on some physicochemical and functional properties, and antioxidant activity of coconut cake dietary fibre (CCDF) were studied. Results showed that both the hydroxypropylation and carboxymethylation could effectively improve (p < 0.05) the water retention capacity (WRC), oil retention capacity (ORC), viscosity, α-amylase inhibition activity (α-AAIR), glucose dialysis retardation index (GDRI), cation-exchange capacity, emulsifying capacity index (ECI) and bile adsorption capacity (BAC) of CCDF. Moreover, the cellulase and hemicellulase hydrolysis combination with heating significantly enhanced (p < 0.05) the soluble dietary fibre content, WRC, emulsion stability, GDRI, α-AAIR and BAC of CCDF; but caused decrease in ORC and browning of color. In addition, improvement of total phenol content, Fe2+ chelating ability, ABTS+· and O2-· scavenging activity were obtained in carboxymethylaticted CCDF. These effects were mainly attributed to the composition and structural modifications as evident from SEM, FT-IR and XRD analysis.


Assuntos
Antioxidantes/química , Celulase/metabolismo , Cocos/química , Fibras na Dieta/análise , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Adsorção , Fenômenos Químicos , Glucose/química , Hidrólise , Metilação , Solubilidade , Viscosidade
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