RESUMO
BACKGROUND: Psychrophiles have evolved to adapt to freezing environments, and cold-adapted enzymes from these organisms can maintain high catalytic activity at low temperature. The use of cold-adapted enzymes has great potential for the revolution of food and molecular biology industries. RESULTS: In this study, four different strains producing protease were isolated from traditional fermented shrimp paste, one of which, named Planococcus maritimus XJ11 by 16S rRNA nucleotide sequence analysis, exhibited the largest protein hydrolysis clear zone surrounding the colonies. Meanwhile, the strain P. maritimus XJ11 was selected for further investigation because of its great adaptation to low temperature, low salinity and alkaline environment. The enzyme activity assay of P. maritimus XJ11 indicated that the optimum conditions for catalytic activity were pH 10.0 and 40 °C. Moreover, the enzyme also showed an increasing activity with temperatures from 10 to 40 °C and retained more than 67% activity of the maximum over a broad range of salinity (50-150 g L-1 ). Genome sequencing analysis revealed that strain XJ11 possessed one circular chromosome of 3 282 604 bp and one circular plasmid of 67 339 bp, with a total number of 3293 open reading frames (ORFs). Besides, 21 genes encoding protease, including three serine proteases, were identified through the NR database. CONCLUSION: Cold-adapted bacterium P. maritimus XJ11 was capable of producing alkaline proteases with high catalytic efficiency at low or moderate temperatures. Furthermore, the favorable psychrophilic and enzymatic characters of strain P. maritimus XJ11 seem to have a promising potential for industrial application. © 2020 Society of Chemical Industry.
Assuntos
Proteínas de Bactérias/genética , Alimentos Fermentados/microbiologia , Produtos Pesqueiros/microbiologia , Genoma Bacteriano , Palaemonidae/microbiologia , Peptídeo Hidrolases/genética , Planococáceas/enzimologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Temperatura Baixa , DNA Bacteriano/genética , Estabilidade Enzimática , Produtos Pesqueiros/análise , Hidrólise , Fases de Leitura Aberta , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Planococáceas/química , Planococáceas/genética , Planococáceas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
To improve the flavor profile and sensory quality of baijiu, the utilization of bioaugmented fermentation inoculated with functional microbiota normally serves as an effective method for directional regulation during the baijiu fermentation process. In this study, a systematic analysis of the succession patterns and volatile flavor compound profiles of microbial communities was carried out by high-throughput sequencing and solid-phase microextraction gas chromatography-mass spectrometry, respectively. The results demonstrated that the Saccharomyces cerevisiae YS222-related bioaugmentation clearly altered the microbial composition, particularly the assembly of bacteria, and promoted the quantity of the most volatile flavoring compounds, including alcohols, esters, and pyrazines. In addition, the correlation analysis showed that Saccharomyces and Lactobacillus in the augmented group were the main biomarkers associated with the dynamics of microbial community and greatly contributed to the brewing of sauce-flavor baijiu, which congruent with the outcomes of the enrichment analysis of integrated metabolic pathway. Thus, this work is beneficial for promoting the quality of baijiu and will serve as a useful reference for clarifying the possible mechanism of augmented fermentation on flavor development.
Assuntos
Ésteres , Alimentos , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Sequenciamento de Nucleotídeos em Larga Escala , Saccharomyces cerevisiaeRESUMO
Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp31, Ser72, Asn121, Asp471, and Glu485 were crucial for maintaining PG5-myosin interaction, with Ser72 and Asn121 making significant energy contributions.
RESUMO
In this study, we successfully obtained a novel source protein glutaminase PG5 with specific activity of 10.4 U/mg, good tolerance and broad substrate profile through big data retrieval. Structural analysis and site-directed mutagenesis revealed that the catalytic pocket of Mature-PG5 contained a large number of aromatic amino acids and hydrophobic amino acids, and that Ser72 greatly affects the properties of the catalytic pocket and the affinity of PG5 for the substrate. In addition, molecular dynamics analysis revealed that the opening and closing between amino acid residues Gly65 and Thr66 with Cys164 at the catalytic cleft could affect substrate binding and product release. In addition, PG5 effectively improved the solubility of fish myofibrillar proteins under low-salt conditions while enhancing their foaming and emulsification properties. This study offers valuable insights into the catalytic mechanism of PG5, which will contribute to its future directed evolution and application in the food industry.
Assuntos
Glutaminase , Glutaminase/metabolismo , Glutaminase/química , Glutaminase/genética , Animais , Simulação de Dinâmica Molecular , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Domínio Catalítico , Mutagênese Sítio-Dirigida , Especificidade por Substrato , Peixes/genética , Cinética , BiocatáliseRESUMO
The fermentation process of traditional shrimp paste is closely associated with the production of flavor substances, but the formation mechanism of key aroma components is still unclear. In this study, a comprehensively flavor profile analysis of traditional fermented shrimp paste was carried out by E-nose and SPME-GC-MS. A total of 17 key volatile aroma components with OAV > 1 contributed greatly to the overall flavor formation of shrimp paste. In addition, high-throughput sequencing (HTS) analysis revealed that Tetragenococcus was the dominant genera in the whole fermentation process. Moreover, metabolomics analysis showed that the oxidation and degradation of lipids, protein, organic acids and amino acids produced a large number of flavor substances and intermediates, which laid the foundation for the Maillard reaction in term of generating the distinct aroma of the traditional shrimp paste. This work will provide theoretical support for the realization of flavor regulation and quality control in traditional fermented foods.
Assuntos
Alimentos Fermentados , Multiômica , Fermentação , Odorantes , ÁcidosRESUMO
Microbial contamination is one of the most common food safety issues that lead to food spoilage and foodborne illness, which readily affects the health of the masses as well as gives rise to huge economic losses. In this study, Channa argus was used as a source of antimicrobial agent that was then analyzed by untargeted metabolomics for its antibacterial mechanism against Staphylococcus aureus. The results indicated that the skin mucus extract of C. argus had great inhibitory action on the growth of S. aureus, and the morphology of S. aureus cells treated with the skin mucus extract exhibited severe morphological damage under scanning electron microscopy. In addition, metabolomics analysis revealed that skin mucus extract stress inhibited the primary metabolic pathways of S. aureus by inducing the tricarboxylic acid cycle and amino acid biosynthesis, which further affected the normal physiological functions of biofilms. In conclusion, the antimicrobial effect of the skin mucus extract is achieved by disrupting cell membrane functions to induce an intracellular metabolic imbalance. Hence, these results conduce to amass novel insights into the antimicrobial mechanism of the skin mucus extract of C. argus against S. aureus.