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1.
J Agric Food Chem ; 67(37): 10373-10379, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453692

RESUMO

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many ß-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sefarose/química , Sefarose/metabolismo , Especificidade por Substrato
2.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
3.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
4.
Food Chem ; 295: 311-319, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174763

RESUMO

A novel gene aga3027 from the genome of Flammeovirga sp. OC4, isolated from the deep sea, was screened and expressed in Escherichia coli BL21. This gene encoded the genetic information of a potential agarase that consists of 851 amino acids and belongs to 16 ß-agarase family of glycoside hydrolase. Purified recombinant Aga3027 demonstrated the maximum activity of agarase at 40 °C and pH 9.0, displaying excellent thermostability and pH-stability. The agarase retained more than 80% of its maximum activity after incubation at 30-40 °C for 48 h, or after incubation at pH 6.0-9.0 for 60 min, which indicated that this agarase was suitable for industrial applications. Silica gel chromatography was used to purify the hydrolysates of agar treated by agarase from the recombinant Aga3027. The hydrolysates were identified as neoagarotetraose and neoagarohexaose by thin layer chromatography and further confirmed by ion chromatography.


Assuntos
Ágar/química , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Ágar/metabolismo , Bacteroidetes/enzimologia , Bacteroidetes/genética , Cromatografia em Camada Delgada , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosídeos/metabolismo , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
5.
Food Chem ; 295: 51-57, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174789

RESUMO

To discriminate the trace-rutinosidase variety of Tartary buckwheat 'Manten-Kirari', we developed DNA markers based on RNA polymorphism. Specifically, we mapped 17.76 GB RNA sequences, obtained using HiSeq2000, to create 11,358 large contigs constructed de novo from 'Manten-Kirari' RNA derived from GS-FLX+ titanium. From these, we developed eight DNA markers corresponding to single- to four-nucleotide polymorphisms between 'Manten-Kirari' and 'Hokkai T8', which is representative of normal rutinosidase content varieties in Japan. Using these markers, 'Manten-Kirari' was discriminated from 'Hokkai T8' by eight markers, from major Tartary buckwheat varieties by three markers, and from common buckwheats by two markers. We also performed direct PCR from flour and dried noodle made with 'Manten-Kirari' and 'Hokkai T8'. Based on the results, the DNA markers developed are promising for discriminating 'Manten-Kirari'. This is the first study to develop a DNA marker to discriminate varieties in the Polygonaceae family including buckwheat species.


Assuntos
Fagopyrum/genética , Análise de Alimentos/métodos , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fagopyrum/metabolismo , Glicosídeo Hidrolases/genética , Japão , Proteínas de Plantas/genética , Polimorfismo Genético , RNA de Plantas , Rutina/genética , Rutina/metabolismo
6.
Enzyme Microb Technol ; 127: 22-31, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088613

RESUMO

The recombinant rAgaZC-1 was a family GH50 ß-agarase from Vibrio sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher T5010 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Mutagênese , Vibrio/enzimologia , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica , Estabilidade Proteica , Sefarose/metabolismo
7.
Comput Biol Chem ; 80: 270-277, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054539

RESUMO

Panomycocin is a naturally produced potent antimycotic/antifungal protein secreted by the yeast Wickerhamomyces anomalus NCYC 434 with an exo-ß-1,3-glucanase activity. In this study the three dimensional structure of panomycocin was predicted and the computational site-directed mutagenesis was performed to enhance its thermal stability in liquid formulations over the body temperature for topical therapeutic applications. Homology modeling was performed with MODELLER and I-TASSER. Among the generated models, the model with the lowest energy and DOPE score was selected for further loop modeling. The loop model was optimized and the reliability of the model was confirmed with ERRAT, Verify 3D and Ramachandran plot values. Enhancement of the thermal stability of the model was done using contemporary servers and programs such as SPDBViewer, CNA, I-Mutant2.0, Eris, AUTO-MUTE and MUpro. In the region outside the binding site of the model Leu52 Arg, Phe223Arg and Gly254Arg were found to be the best thermostabilizing mutations with 6.26 K, 6.26 K and 8.27 K increases, respectively. In the binding site Glu186Arg was found to be the best thermostabilizer mutation with a 9.58 K temperature increase. The results obtained in this study led us to design a mutant panomycocin that can be used as a novel antimycotic/antifungal drug in a liquid formulation for topical applications over the normal body temperature.


Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Micotoxinas/química , Micotoxinas/genética , Pichia/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação , Estabilidade Proteica , Estrutura Terciária de Proteína , Temperatura Ambiente
8.
Food Chem ; 294: 293-301, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126466

RESUMO

To construct a high-performance engineered endo-inulinase for fructo-oligosaccharides (FOS) production from inulin, an inulin binding module (IBM) was fused into either N- or C-terminal of an endo-inulinase. After heterologous expression, purification and characterization, the C-terminal fusion one (Eninu-IBM) with better activity, thermostability and inulin binding ability was employed for high-temperature in situ inulin hydrolysis in a 10-L fermentor. During this process, Eninu-IBM was first efficiently produced by the yeast cells at 28 °C for 96 h, and subsequently 1600 g unsterilized inulin per liter fermentation liquor was directly supplemented into the bioreactor for FOS production at 60 °C for 2 h. Finally, high purity of FOS (91.4%) were obtained with FOS titer, yield and productivity of 717.3 g/L, 0.912 gFOS/gInulin and 358.6 g/L/h, respectively. The in vitro prebiotic assay indicated that the final FOS products with main polymerization degrees of 3-5 were preferably fermented by beneficial bifidobacteria and lactobacilli.


Assuntos
Glicosídeo Hidrolases/metabolismo , Inulina/metabolismo , Oligossacarídeos/metabolismo , Reatores Biológicos , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Prebióticos , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Temperatura Ambiente
9.
J Microbiol Biotechnol ; 29(4): 625-632, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30954032

RESUMO

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding ß-agarase and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro- L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.


Assuntos
Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sefarose/metabolismo , Meios de Cultura , Dissacaridases/genética , Dissacarídeos/metabolismo , Enzimas/genética , Escherichia coli/genética , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Glicosídeo Hidrolases/genética , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo
10.
J Microbiol Biotechnol ; 29(4): 562-570, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30955258

RESUMO

ß-Glucosylglycerol (ß-GG) and their derivatives have potential applications in food, cosmetics and the healthcare industry, including antitumor medications. In this study, ß-GG and its unnatural glycosides were synthesized through the transglycosylation of two enzymes, Sulfolobus shibatae ß-glycosidase (SSG) and Deinococcus geothermalis amylosucrase (DGAS). SSG catalyzed a transglycosylation reaction with glycerol as an acceptor and cellobiose as a donor to produce 56% of ß-GGs [ß-D-glucopyranosyl-(1→1/3)-D-glycerol and ß-D-glucopyranosyl- (1→2)-D-glycerol]. In the second transglycosylation reaction, ß-D-glucopyranosyl-(1 → 1/3)-Dglycerol was used as acceptor molecules of the DGAS reaction. As a result, 61% of α-Dglucopyranosyl-( 1→4)-ß-D-glucopyranosyl-(1→1/3)-D-glycerol and 28% of α-D-maltopyranosyl- (1→4)-ß-D-glucopyranosyl-(1→1/3)-D-glycerol were synthesized as unnatural glucosylglycerols. In conclusion, the combined enzymatic synthesis of the unnatural glycosides of ß-GG was established. The synthesis of these unnatural glycosides may provide an opportunity to discover new applications in the biotechnological industry.


Assuntos
Glucosídeos/biossíntese , Glucosiltransferases/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/biossíntese , Biotecnologia , Celobiose/metabolismo , Deinococcus/enzimologia , Deinococcus/genética , Escherichia coli/genética , Glucosidases/metabolismo , Glucosídeos/análise , Glucosídeos/química , Glucosiltransferases/genética , Glicerol/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeos/análise , Glicosídeos/química
11.
Crit Rev Biotechnol ; 39(4): 508-523, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30939944

RESUMO

Glucosinolate-myrosinase is a substrate-enzyme defense mechanism present in Brassica crops. This binary system provides the plant with an efficient system against herbivores and pathogens. For humans, it is well known for its anti-carcinogenic, anti-inflammatory, immunomodulatory, anti-bacterial, cardio-protective, and central nervous system protective activities. Glucosinolate and myrosinase are spatially present in different cells that upon tissue disruption come together and result in the formation of a variety of hydrolysis products with diverse physicochemical and biological properties. The myrosinase-catalyzed reaction starts with cleavage of the thioglucosidic linkage resulting in release of a D-glucose and an unstable thiohydroximate-O-sulfate. The outcome of this thiohydroximate-O-sulfate has been shown to depend on the structure of the glucosinolate side chain, the presence of supplementary proteins known as specifier proteins and/or on the physiochemical condition. Myrosinase was first reported in mustard seed during 1939 as a protein responsible for release of essential oil. Until this date, myrosinases have been characterized from more than 20 species of Brassica, cabbage aphid, and many bacteria residing in the human intestine. All the plant myrosinases are reported to be activated by ascorbic acid while aphid and bacterial myrosinases are found to be either neutral or inhibited. Myrosinase catalyzes hydrolysis of the S-glycosyl bond, O-ß glycosyl bond, and O-glycosyl bond. This review summarizes information on myrosinase, an essential component of this binary system, including its structural and molecular properties, mechanism of action, and its regulation and will be beneficial for the research going on the understanding and betterment of the glucosinolate-myrosinase system from an ecological and nutraceutical perspective.


Assuntos
Glicosídeo Hidrolases/química , Mostardeira/enzimologia , Óleos Voláteis/química , Sequência de Aminoácidos/genética , Catálise , Glicosídeo Hidrolases/genética , Humanos , Hidrólise , Mostardeira/química , Proteínas de Plantas/química , Proteínas de Plantas/genética
12.
Nat Commun ; 10(1): 1491, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940816

RESUMO

Poly-ADP-ribosylation, a post-translational modification involved in various cellular processes, is well characterized in eukaryotes but thought to be devoid in bacteria. Here, we solve crystal structures of ADP-ribose-bound poly(ADP-ribose)glycohydrolase from the radioresistant bacterium Deinococcus radiodurans (DrPARG), revealing a solvent-accessible 2'-hydroxy group of ADP-ribose, which suggests that DrPARG may possess endo-glycohydrolase activity toward poly-ADP-ribose (PAR). We confirm the existence of PAR in D. radiodurans and show that disruption of DrPARG expression causes accumulation of endogenous PAR and compromises recovery from UV radiation damage. Moreover, endogenous PAR levels in D. radiodurans are elevated after UV irradiation, indicating that PARylation may be involved in resistance to genotoxic stresses. These findings provide structural insights into a bacterial-type PARG and suggest the existence of a prokaryotic PARylation machinery that may be involved in stress responses.


Assuntos
Proteínas de Bactérias/química , Deinococcus/enzimologia , Glicosídeo Hidrolases/química , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Deinococcus/genética , Deinococcus/metabolismo , Deinococcus/efeitos da radiação , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Poli ADP Ribosilação/efeitos da radiação , Poli Adenosina Difosfato Ribose/química , Poli Adenosina Difosfato Ribose/metabolismo , Especificidade por Substrato , Raios Ultravioleta
13.
Chemistry ; 25(26): 6533-6541, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30820987

RESUMO

Selective chemical modification of proteins plays a pivotal role for the rational design of enzymes with novel and specific functionalities. In this study, a strategic combination of genetic and chemical engineering paves the way for systematic construction of biocatalysts by tuning the product spectrum of a levansucrase from Bacillus megaterium (Bm-LS), which typically produces small levan-like oligosaccharides. The implementation of site-directed mutagenesis followed by a tyrosine-specific modification enabled control of the product synthesis: depending on the position, the modification provoked either enrichment of short oligosaccharides (up to 800 % in some cases) or triggered the formation of high molecular weight polymer. The chemical modification can recover polymerization ability in variants with defective oligosaccharide binding motifs. Molecular dynamic (MD) simulations provided insights into the effect of modifying non-native tyrosine residues on product specificity.


Assuntos
Bacillus megaterium/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Hexosiltransferases/química , Hexosiltransferases/genética , Oligossacarídeos/metabolismo , Tirosina/química , Bacillus megaterium/química , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Reação de Cicloadição , Frutanos/química , Frutanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismo
14.
Int J Mol Sci ; 20(5)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841519

RESUMO

Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.


Assuntos
Aspergillus niger/enzimologia , Biocatálise , Dissacarídeos/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Quercetina/metabolismo , Aspergillus niger/genética , Dissacarídeos/química , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Microbiologia Industrial/métodos , Pichia/genética , Pichia/metabolismo , Quercetina/química , Rutina/química , Rutina/metabolismo
15.
Oncol Rep ; 41(5): 2657-2666, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864743

RESUMO

The present study investigated the effect of poly(ADP­ribose) glycohydrolase (PARG) on the immune response in tumour metastases of colon carcinoma. CT26 cells were transfected with lentivirus PARG­short hairpin RNA (shRNA). A liver metastasis model of colon carcinoma was successfully established by splenic subcapsular inoculation of the various groups of CT26 cells into BALB/c mice. Next, changes in the liver metastases of colon carcinoma nodules and alterations in the survival times were observed in tumour­bearing mice. The numbers of B220+DEC205+ dendritic cells (B220+DEC205+DC) and CD11c+CD11b+ dendritic cells (CD11c+CD11b+DC) in the spleen and liver were measured by the double­label immunofluorescence assay. The distribution pattern of CD4+T cells and CD8+T cells in the spleen and liver was investigated by immunofluorescence staining. The expression levels of PARG, PARP and nuclear factor­κB (NF­κB) proteins in spleen transplant tumours and liver metastases of colon carcinoma were detected by western blotting. An ELISA was used to detect the levels of IL­10 and TGF­ß in the serum of tumour­bearing mice and from the supernatant of tumour cells. The numbers and grading of metastatic liver nodules in the PARG­silenced group were clearly lower than those in the control group. The survival time of the PARG­silenced group mice was longer than that in the control group. In the PARG­silenced group, the levels of B220+DEC205+DC in the spleen and liver were lower and the numbers of CD11c+CD11b+DC in the spleen and liver were more than those in the control group. The ratio of CD4+/CD8+ in the spleen and liver in the PARG­silenced group was increased compared with that in the control group (P<0.05). The levels of PARG, PARP and NF­κB in spleen transplant tumours and liver metastases of colon carcinoma were lower in the PARG­silenced group than in the control group. In addition, the levels of IL­10 and TGF­ß in the serum of tumour­bearing mice and supernatants of tumour cells were both reduced in the PARG­silenced group compared with those in the control group. The present research suggests that the liver metastases of colon carcinoma could be restrained by silencing PARG. Likely, the silencing of PARG could suppress the expression of PARP and NF­κB and subsequently suppress the secretion of IL­10 and TGF­α, finally affecting the proliferation and differentiation of DC and T cells.


Assuntos
Carcinoma/patologia , Neoplasias do Colo/patologia , Glicosídeo Hidrolases/metabolismo , Neoplasias Hepáticas/patologia , Animais , Carcinoma/secundário , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Dendríticas/patologia , Feminino , Técnicas de Inativação de Genes , Glicosídeo Hidrolases/genética , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Appl Microbiol Biotechnol ; 103(9): 3783-3793, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30903213

RESUMO

Arabinofuranosidase plays an essential role in the process of hydrolysis of arabinoxylan (AX). Thermostable, versatile, and efficient arabinofuranosidase is thus of great interest for the biorefinery industry. A GH51 arabinofuranosidase, Abf51, from Hungateiclostridium clariflavum DSM 19732 was heterogeneously expressed in Escherichia coli. Abf51 was found to have an optimal pH and temperature of 6.5 and 60 °C, respectively, with very high thermostability. At the optimal working temperature (60 °C), Abf51 retained over 90% activity after a 2-day incubation and over 60% activity after a 6-day incubation. Abf51 could effectively remove the arabinofuranosyls from three kinds of AX oligosaccharides [23-α-L-arabinofuranosyl-xylotriose (A2XX), 32-α-L-arabinofuranosyl-xylobiose (A3X), and 2333-di-α-L-arabinofuranosyl-xylotriose (A2 + 3XX)], which characterized as either single substitution or double substitution by arabinofuranosyls on terminal xylopyranosyl units. The maximal catalytic efficiency (Kcat/Km) was observed using p-nitrophenyl-α-L-arabinofuranoside (pNPAF) as a substrate (205.0 s-1 mM-1), followed by using A3X (22.8 s-1 mM-1), A2XX (6.9 s-1 mM-1), and A2 + 3XX (0.5 s-1 mM-1) as substrates. Moreover, the presence of Abf51 significantly stimulated the saccharification level of AX (18.5 g L-1) up to six times along with a ß-xylanase as well as a ß-xylosidase. Interestingly, in our survey of top thermostable arabinofuranosidases, most members were found from GH51, probably due to their owning of (ß/α)8-barrel architectures. Our results suggested the great importance of GH51s as candidates for thermostable, versatile, and efficient arabinofuranosidases toward industry application.


Assuntos
Arabinose/metabolismo , Proteínas de Bactérias/química , Clostridiales/enzimologia , Glicosídeo Hidrolases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridiales/química , Clostridiales/genética , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Especificidade por Substrato , Xilanos/metabolismo
17.
J Biotechnol ; 296: 42-52, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30885654

RESUMO

The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-ß-galactanase and an α-l-arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (kcat values of ˜177 and ˜133 s-1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases - endo-1,4-ß-xylanase (XynA), endo-1,4-ß-mannanase (ManB/Man5A) and ß-glucosidase (GghA) - to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases.


Assuntos
Celulase/química , Glicosídeo Hidrolases/química , Lignina/química , Polissacarídeos/química , Biocombustíveis , Biomassa , Celulase/genética , Estabilidade Enzimática/genética , Escherichia coli/genética , Fermentação , Glicosídeo Hidrolases/genética , Hidrólise/efeitos dos fármacos , Polissacarídeos/genética , Temperatura Ambiente , Thermotoga neapolitana/enzimologia , Thermotoga neapolitana/genética
18.
J Anim Sci ; 97(5): 2139-2153, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-30888017

RESUMO

This study aimed to evaluate the effects of a source of dietary soluble (SF) and insoluble fiber (IF) without or with exogenous carbohydrases (xylanase, ß-glucanase, and pectinase) on diarrhea incidence, selected immune responses, and growth performance in enterotoxigenic Escherichia coli (ETEC)-challenged pigs. Sixty weaned pigs (6.9 ± 0.1 kg BW, ~23 d of age) were blocked by initial BW and placed in individual pens. Pens were randomly assigned to one of six treatments (n = 10 per treatment), including a nonchallenged control (NC), a positive challenge control (PC), the PC + a soluble fiber diet (10% sugar beet pulp) without (SF-) or with carbohydrases (SF+), and PC + an IF diet (15% corn distillers dried grains with solubles) without (IF-) or with carbohydrases (IF+). The control diet was primarily based on corn and soybean meal with 13.5% whey powder. The two sources of fiber were added at the expense of cornstarch in the control diet. Pigs were orally inoculated with 6 mL hemolytic F18 ETEC (~3.5 × 109 cfu/mL) or sham infected with 6 mL phosphate-buffered saline on day 7 (0 d postinoculation, dpi) postweaning. All ETEC challenged pigs were confirmed to be genetically susceptible to F18 ETEC. Pigs had free access to feed and water throughout the 14-d trial. Pig BW and feed intake were recorded on dpi -7, 0, and 7 or 8. Fecal swabs were collected on dpi -7, 0, 1, 2, 3, 5, and 7 or 8 to evaluate hemolytic E. coli shedding. Fecal score was visually ranked daily postchallenge to evaluate diarrhea incidence. Blood samples were collected on dpi -1, 3, and 7 or 8 at necropsy and intestinal tissues were collected at necropsy. Pigs on PC had lower dpi 1 to 7 ADG and ADFI than those on NC (P < 0.05). Compared with PC pigs, SF+ pigs had greater ADG during both pre- and postchallenge period (P < 0.05). The IF- increased postchallenge diarrhea incidence compared with PC (P < 0.05). Pigs on SF- had lower ileal E. coli attachment than PC (P < 0.05). The SF+ reduced haptoglobin and IF+ reduced C-reactive protein on dpi 3 compared with PC (P < 0.05). Compared with PC pigs, SF+ pigs tended to have lower ileal tumor necrosis factor alpha and greater ileal occludin (OCLN) mRNA (P < 0.10) and had greater (P < 0.05) colonic OCLN mRNA levels. Collectively, IF- increased incidence of diarrhea and fecal E. coli shedding compared with PC. The SF+ pigs had improved growth compared with PC pigs, likely due in part to a reduction in inflammatory intermediates.


Assuntos
Ração Animal/análise , Diarreia/veterinária , Fibras na Dieta/metabolismo , Escherichia coli Enterotoxigênica/fisiologia , Glicosídeo Hidrolases/metabolismo , Animais , Diarreia/metabolismo , Dieta/veterinária , Infecções por Escherichia coli/metabolismo , Fezes/microbiologia , Fermentação , Glicosídeo Hidrolases/genética , Íleo/metabolismo , Intestinos/microbiologia , Soja , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia , Desmame , Zea mays
19.
Food Chem ; 286: 696-702, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30827665

RESUMO

A gene encoding chitosanase from Streptomyces albolongus was cloned, sequenced and expressed in Escherichia coli. The novel recombinant enzyme (Csn21c) was purified by Ni-NTA Superflow Column and showed a molecular mass of 29.6 kDa by SDS-PAGE. The enzyme Csn21c showed the optimal activity in 50 mmol/L Tris-HCl buffer, pH 8.0, and 50 °C and it was strongly activated (2-fold) by Mn2+. It belonged to glycoside hydrolase 46 family according to NCBI database (http://www.ncbi.nlm.nih.gov/) and displayed an exo-type cleavage pattern, hydrolyzing chitosan mainly into d-glucosamine (GlcN) and chitobiose ((GlcN)2) as confirmed by TLC and MS analysis. This study demonstrated that Csn21c can be an effective tool to produce abundant glucosamine and chitooligosaccharides (COS) from chitosan.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Quitosana/metabolismo , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/genética , Glucosamina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/genética
20.
J Microbiol Biotechnol ; 29(1): 37-43, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30798571

RESUMO

The gene encoding an α-L-arabinofuranosidase (BvAF) GH51 from Bacillus velezensis FZB42 was cloned and expressed in Escherichia coli. The corresponding open reading frame consists of 1,491 nucleotides which encode 496 amino acids with the molecular mass of 56.9 kDa. BvAF showed the highest activity against sugar beet (branched) arabinan in 50 mM sodium acetate buffer (pH 6.0) at 45°C. However, it could hardly hydrolyze debranched arabinan and arabinoxylans. The time-course hydrolyses of branched arabinan and arabinooligosaccharides (AOS) revealed that BvAF is a unique exo-hydrolase producing exclusively L-arabinose. BvAF could cleave α-(1,2)- and/or α-(1,3)-L-arabinofuranosidic linkages of the branched substrates to produce the debranched forms of arabinan and AOS. Although the excessive amount of BvAF could liberate L-arabinose from linear AOS, it was extremely lower than that on branched AOS. In conclusion, BvAF is the arabinan-specific exo-acting α-L-arabinofuranosidase possessing high debranching activity towards α-(1,2)- and/or α-(1,3)-linked branches of arabinan, which can facilitate the successive degradation of arabinan by endo-α-(1,5)-L-arabinanase.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Arabinose/metabolismo , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Beta vulgaris/química , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura Ambiente
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