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Artigo em Inglês | MEDLINE | ID: mdl-34775631


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.

Genes (Basel) ; 12(9)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34573403


Corallococcus sp. strain EGB is a Gram-negative myxobacteria isolated from saline soil, and has considerable potential for the biocontrol of phytopathogenic fungi. However, the detailed mechanisms related to development and predatory behavior are unclear. To obtain a comprehensive overview of genetic features, the genome of strain EGB was sequenced, annotated, and compared with 10 other Corallococcus species. The strain EGB genome was assembled as a single circular chromosome of 9.4 Mb with 7916 coding genes. Phylogenomics analysis showed that strain EGB was most closely related to Corallococcus interemptor AB047A, and it was inferred to be a novel species within the Corallococcus genus. Comparative genomic analysis revealed that the pan-genome of Corallococcus genus was large and open. Only a small proportion of genes were specific to strain EGB, and most of them were annotated as hypothetical proteins. Subsequent analyses showed that strain EGB produced abundant extracellular enzymes such as chitinases and ß-(1,3)-glucanases, and proteases to degrade the cell-wall components of phytopathogenic fungi. In addition, 35 biosynthetic gene clusters potentially coding for antimicrobial compounds were identified in the strain EGB, and the majority of them were present in the dispensable pan-genome with unexplored metabolites. Other genes related to secretion and regulation were also explored for strain EGB. This study opens new perspectives in the greater understanding of the predatory behavior of strain EGB, and facilitates a potential application in the biocontrol of fungal plant diseases in the future.

Int J Biol Macromol ; 184: 551-557, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34171255


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.

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
J Agric Food Chem ; 69(11): 3351-3361, 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33688732


Enzymes that degrade fungal cell walls and the resulting oligosaccharides are promising weapons to combat plant fungal disease. In this study, we identified a novel endo-chitosanase, AqCoA, from Aquabacterium sp. A7-Y. The enzyme showed a specific activity of 18 U/mg toward 95% deacetylated chitosan at pH 5.0 and 40 °C. AqCoA also showed activity toward sodium carboxymethylcellulose, indicating substrate promiscuity. AqCoA hydrolyzed chitosan into chitooligosaccharides (CoA-COSs) with degrees of polymerization (DPs) of 3-5 but showed no activity toward CoA-COSs with DPs <6, indicating an endo-type activity. At 2.5 µg/mL, AqCoA inhibited appressorium formation of Magnaporthe oryzae; the produced CoA-COSs also inhibited the growth of M. oryzae and Fusarium oxysporum. Furthermore, CoA-COSs acted as immune elicitors in rice by inducing the reactive oxygen species burst and the expression of defense genes. These results demonstrated that AqCoA and its resulting CoA-COSs might be effective tools for protecting plants against pathogenic fungi.

Quitina , Quitosana , Glicosídeo Hidrolases , Doenças das Plantas/microbiologia , Ascomicetos , Quitina/análogos & derivados , Fusarium , Oligossacarídeos , Doenças das Plantas/prevenção & controle
ISME J ; 13(9): 2223-2235, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31065029


Myxobacterial predation on bacteria has been investigated for several decades. However, their predation on fungi has received less attention. Here, we show that a novel outer membrane ß-1,6-glucanase GluM from Corallococcus sp. strain EGB is essential for initial sensing and efficient decomposition of fungi during predation. GluM belongs to an unstudied family of outer membrane ß-barrel proteins with potent specific activity up to 24,000 U/mg, whose homologs extensively exist in myxobacteria. GluM was able to digest fungal cell walls efficiently and restrict Magnaporthe oryzae infection of rice plants. Genetic complementation with gluM restored the fungal predation ability of Myxococcus xanthus CL1001, which was abolished by the disruption of gluM homolog oar. The inability to prey on fungi with cell walls that lack ß-1,6-glucans indicates that ß-1,6-glucans are targeted by GluM. Our results demonstrate that GluM confers myxobacteria with the ability to feed on fungi, and provide new insights for understanding predator-prey interactions. Considering the attack mode of GluM, we suggest that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents.

Membrana Externa Bacteriana/enzimologia , Proteínas de Bactérias/metabolismo , Fungos/química , Glicosídeo Hidrolases/metabolismo , Myxococcus xanthus/enzimologia , Proteínas de Bactérias/genética , Glucanos/metabolismo , Glicosídeo Hidrolases/genética , Myxococcus xanthus/fisiologia
Int J Biol Macromol ; 132: 1235-1243, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980875


As the main component of the fungal cell wall, chitin has been regarded as an optimal molecular target for the biocontrol of plant-pathogenic fungi. In this study, the chitin hydrolase CcCti1, which belongs to the glycoside hydrolase family 18 (GH 18) and exhibits potential antifungal activity, was identified from Corallococcus sp. EGB. CcCti1 lacks a fibronectin type-III (FN3) domain that is present in similar enzymes from most genera of myxobacteria, indicating that CcCti1 may have acquired chitinase activity due to the FN3 domain deletion during myxobacterial evolution. CcCti1 was expressed in Escherichia coli BL21 (DE3) with a specific activity of up to 10.5 U/µmol with colloidal chitin as the substrate. Product analysis showed that CcCti1 could hydrolyze chitin into N-acetylated chitohexaose (GlcNAc)6 as the major product, in addition to chitooligosaccharides. The analysis of biochemical properties indicated that the CBD and FN3 domains in CcCti1 determine the substrate affinity and pH stability. Otherwise, CcCti1 exhibited efficient biocontrol activity against the plant pathogen Magnaporthe oryzae in a dose-dependent manner, inhibiting the conidia germination and appressoria formation at a concentration of 0.08 mg/mL. Overall, the chitohexaose-producing chitinase CcCti1 with hydrolytic features may find potential application in chitin conversion and biocontrol of fungal plant diseases.

Antifúngicos/metabolismo , Antifúngicos/farmacologia , Quitinases/genética , Quitinases/farmacologia , Myxococcales/efeitos dos fármacos , Sequência de Aminoácidos , Antifúngicos/química , Quitinases/química , Clonagem Molecular , Evolução Molecular , Hidrólise , Filogenia , Domínios Proteicos