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1.
Artigo em Inglês | MEDLINE | ID: mdl-34775631

RESUMO

Maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the hydrolysis and transglycosylation of maltooligosaccharides and soluble starch into maltose, the sole hydrolysate. This process yields pure maltose with potentially wide applications. Here, we identified and evaluated the role of phenylalanine 314 (F314), a key amino acid located near the active center, in the catalytic activities of the CoMA. Site-directed mutagenesis analysis showed that the activity of a F314L mutant on potato starch substrate decreased to 26% of that of wild-type protein. Compared with the wild-type, F314L exhibited similar substrate specificity, hydrolysis pattern, pH, and temperature requirements. Circular dichroism spectrum data showed that the F314L mutation did not affect the structure of the folded protein. In addition, kinetic analysis demonstrated that F314L exhibited an increased Km value with lower substrate affinity. Homology modeling showed that the benzene ring structure of F314L was involved in π-π conjugation, which might potentially affect the affinity of CoMA toward starch. Taken together, these data demonstrated that F314 is essential for the hydrolytic activity of the CoMA from Corallococcus sp. strain EGB.

2.
J Plant Physiol ; 265: 153493, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34403886

RESUMO

As members of the pathogenesis-related protein (PR)-2 family, ß-1,3-glucanases play pivotal roles in plant defense. Previous study showed that the rice genome contains 16 genes encoding putative ß-1,3-glucanases, and the ß-1,3-glucanases in subfamily A were deduced to be involved in plant defense. However, there was limited direct evidence. In this study, the expression of rice ß-1,3-glucanases Gns2-Gns6 belonging to subfamily A in rice plant infection with Magnaporthe oryzae was investigated, and the enhanced expression of Gns6 during infection confirmed its crucial role in the defense of rice seedlings. Enzymological characterization revealed that Gns6 preferentially hydrolyzed laminarin, pachymaran, and yeast glucan. The ß-1,3; 1,6-glucanase Gns6 exhibited a specific activity of 1.2 U/mg with laminarin as the substrate. In addition, Gns6 could hydrolyze laminarin via an endo-type mechanism, yielding a series of oligosaccharides with various degrees of polymerization that are known immune elicitors in plants. Moreover, Gns6 exhibited a significant inhibitory effect against the formation of the germ tubes and appressoria, with potential applications in plant protection. Taken together, this study shows that Gns6 is an essential effector in the defensive response of rice against pathogenic fungi.


Assuntos
Antifúngicos/farmacocinética , Magnaporthe/efeitos dos fármacos , Oryza/química , Oryza/genética , Doenças das Plantas/prevenção & controle , Extratos Vegetais/genética , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacocinética , Regulação da Expressão Gênica de Plantas , Genes de Plantas
3.
J Agric Food Chem ; 69(33): 9571-9584, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34378924

RESUMO

Fungal cell wall synthesizing enzymes or remodeling enzymes represent key factors for the interaction of plant pathogen and antifungal agents, which are regarded as potential biocontrol agents. In this study, a novel endo-ß-1,3-glucanase from Magnaporthe oryzae was expressed and characterized. The expression of MoGluB was significantly upregulated after 2 days of liquid culture and 48 h after infection, indicating that it may be involved in cell wall reconstitution. Purified MoGluB exhibited high activity on insoluble ß-glucans, with a specific activity of 8.18 U/mg toward yeast glucan at pH 9.0 and 50 °C. MoGluB hydrolyzed pachymaran and yeast glucan into oligosaccharides dominated by laminaripentaose, suggesting that it is an endo-ß-1,3-glucanase. Incubation of 8 µg of MoGluB with 106 spores/mL resulted in the inhibition of conidial germination and appressorium formation of M. oryzae, illustrating effective biocontrol activity. Hydrolysates of pachymaran induced the expression of defense genes restricting M. oryzae infection in rice plants, indicating an immunostimulatory effect of MoGluB hydrolysates.


Assuntos
Ascomicetos , Magnaporthe , Oryza , Parede Celular , Proteínas Fúngicas/genética , Doenças das Plantas
4.
Int J Biol Macromol ; 184: 551-557, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34171255

RESUMO

Modified potato starch with slower digestion may aid the development of new starch derivatives with improved nutritional values, and strategies to increase nutritional fractions such as resistant starch (RS) are desired. In this study, a correspondence between starch structure and enzymatic resistance was provided based on the efficient branching enzyme AqGBE, and modified starches with different amylose content (Control, 100%; PS1, 90%; PS2, 72%; PS3, 32%; PS4, 18%) were prepared. Through SEM observation, NMR and X-ray diffraction analyses, we identified that an increased proportion of α-1,6-linked branches in potato starch changes its state of granule into large pieces with crystallinity. Molecular weight and chain-length distribution analysis showed a decrease of molecular weight (from 1.1 × 106 to 1.1 × 105 g/mol) without an obvious change of chain-length distribution in PS1, while PS2-4 exhibited an increased proportion of DP 6-12 with a stable molecular weight distribution, indicating a distinct model of structural modification by AqGBE. The enhancement of peak viscosity was related to increased hydrophobic interactions and pieces state of PS1, while the contents of SDS and RS in PS1 increased by 37.7 and 49.4%, respectively. Our result provides an alternative way to increase the RS content of potato starch by branching modification.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Burkholderiales/enzimologia , Solanum tuberosum/química , Amido/química , Amilose/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Estrutura Molecular , Peso Molecular , Viscosidade , Difração de Raios X
5.
Protein Expr Purif ; 179: 105798, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33232801

RESUMO

A pectinase-producing bacterial isolate, identified as Paenibacillus xylanexedens SZ 29, was screened by using the soil dilution plate with citrus pectin and congo red. A pectin methylesterase gene (Pxpme) was cloned and expressed in Escherichia coli. The gene coded for a protein with 334 amino acids and a calculated molecular mass of 36.76 kDa. PxPME showed the highest identity of 32.4% with the characterized carbohydrate esterase family 8 pectin methylesterase from Daucus carota. The recombined PxPME showed a specific activity with 39.38 U/mg against citrus pectin with >65% methylesterification. The optimal pH and temperature for PxPME activity were 5.0 and 45 °C. Its Km and Vmax value were determined to be 1.43 mg/mL and 71.5 µmol/mg·min, respectively. Moreover, PxPME could increase the firmness of pineapple cubes by 114% when combined with CaCl2. The acidic and mesophilic properties make PxPME a potential candidate for application in the fruit processing.


Assuntos
Proteínas de Bactérias , Hidrolases de Éster Carboxílico , Paenibacillus , Pectinas/metabolismo , Proteínas Recombinantes , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Escherichia coli/genética , Manipulação de Alimentos , Frutas/química , Concentração de Íons de Hidrogênio , Paenibacillus/enzimologia , Paenibacillus/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
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