Journal of General and Applied Microbiology最新文献

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two β-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-β-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-β-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

To enhance the value of surimi, efforts have been made to develop a fermentation method with lactic acid bacteria (LAB) to proteolyze fish protein. However, fermenting unheated surimi poses a spoilage risk due to its high bacterial content. Surimi heat treatment can prevent spoilage, but gel formation induced by heating introduces another technical issue: it hinders uniform fermentation. Thus, this study aims to observe the proteolysis and enhance the functionality of seafood product through lactic acid fermentation of kamaboko, a heated surimi. Upon analyzing the kamaboko fermented with Lactobacillus helveticus JCM1004, we observed that LAB produced protease, resulting in the degradation of myosin heavy chain and actin during fermentation. Lactic acid fermentation significantly augmented the peptide content of kamaboko, subsequently elevating the angiotensin Ⅰ-converting enzyme (ACE) inhibitory activity in 200-fold diluted extract of fermented kamaboko to approximately 70% and higher. Notably, our investigation revealed that proteolysis was confined to the surface of kamaboko, as evidenced by SDS-PAGE analysis. This observation implies that the surface area of kamaboko influences the ACE inhibitory activity. Through a comparative analysis of various bacterial strains, we demonstrated that the increase in ACE inhibitory activity is contingent on the protease generated by LAB. These results suggest that LAB-mediated proteolysis of fish proteins liberates bioactive peptides, thereby manifesting in the ACE inhibitory activity. In summary, this study underscores that the fermentation of kamaboko employing proteolytic LAB holds promise in the development of novel functional seafood products.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 β-1,3-glucanase.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The K_m value and k_cat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s^-1 respectively. The K_m value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

The culture filtrates of the predominant bacterial strains isolated from soil samples have been shown to increase the microbial colony counts on agar plates used for the isolation of uncultured bacteria. One of the factors in the culture filtrates responsible for this increase was identified to be superoxide dismutase (SOD). The generation of reactive oxygen species (O₂^-, H₂O₂, and ・OH) was detected from conventional laboratory agar media. The use of agar media supplemented with radical scavengers (SOD, catalase, ascorbic acid, or rutin) effectively increased the colony counts and kinds of microbial strains that grew from soil samples. Taxonomical studies on these isolates revealed new taxa for phylum Actinomycetota; one family, three genera, and nine species were newly described. One of the strains, Patulibacter minatonensis KV-614^T belonging to the new family Patulibacteraceae, was isolated on agar medium supplemented with SOD. P. minatonensis KV-614^T represents a novel lineage within the phylum Actinomycetota. A polymerase chain reaction (PCR) study using specific primers for the detection of strains related to the genus Patulibacter, order Solirubrobacterales, showed a high distribution frequency, with detection in over 70% of the soil samples tested. These data suggest that the use of radical scavengers may facilitate the isolation of some hitherto-uncultivated microorganisms widely distributed in soil.

Naphthalene is a persistent environmental pollutant for its potential teratogenic, carcinogenic and mutagenic effects. In this study, 10 strains of bacteria capable of degrading naphthalene were isolated from crude-oil contaminated soil. Among them, Pseudomonas plecoglossicida 2P exhibited prominent growth with 1000 mg/L naphthalene as the sole carbon source and degraded 94.15% of naphthalene in 36 h. Whole genome sequencing analysis showed that P. plecoglossicida 2P had a total of 22 genes related to naphthalene degradation, of which 8 genes were related to the salicylic acid pathway only, 5 genes were related to the phthalic acid pathway only, 8 genes were common in both the salicylic acid and phthalic acid pathways, and 1 gene was related to the gentisic acid pathway. P. plecoglossicida 2P was applied in a two-phase partition bioreactor (TPPB) to degrade naphthalene in wastewater. The optimal operating conditions of the reactor were obtained through response surface optimization: initial naphthalene concentration (C₀) =1600 mg/L, bacterial liquid concentration (OD₆₀₀) = 1.3, and polymer-to-wastewater mass ratio (PWR) = 2%. Under these conditions, the naphthalene degradation rate was 98.36% at 24 h. The degradation kinetics were fitted using the Haldane equation with a high coefficient of determination (R²=0.94). The present study laid foundations for naphthalene degradation mechanism of genus Pseudomonas and its potential application in TPPB.

We have successfully isolated two novel compounds, 24R005A (1, C₁₃H₁₄O₄) and 24R005B (2, C₁₃H₁₃ClO₄), from Streptomyces sp. 24R005, using fish (anchovy) powder as a medium. In this study, we evaluated the use of fish (anchovy) powder as a fermentation material for producing bioactive compounds. Spectroscopic analyses revealed that the two compounds share a common skeletal structure. However, each compound contains unique branched side chains. Furthermore, compounds 1 and 2 exhibit moderate radical-scavenging activity for 1,1-diphenyl-2-picrylhydrazyl (DPPH), with ED₅₀ values of 200 and 130 μM, respectively.

Most cyanobacterial genomes possess more than two copies of genes encoding cyAbrBs (cyanobacterial AbrB-like proteins) having an AbrB-like DNA-binding domain at their C-terminal region. Accumulating data suggest that a wide variety of metabolic and physiologic processes are regulated by cyAbrBs. In this study, we investigated the function of the essential gene cyabrB1 (sll0359) in Synechocystis sp. PCC 6803 by using CRISPR interference technology. The conditional knockdown of cyabrB1 caused increases of cyAbrB2 transcript and protein levels. However, the effect of cyabrB1 knockdown on global gene expression profile was quite limited compared to the previously reported profound effect of knockout of cyabrB2. Among 24 up-regulated genes, 16 genes were members of the divergently transcribed icfG and sll1783 operons related to carbon metabolism. The results of this and previous studies indicate the different contributions of two cyAbrBs to transcriptional regulation of genes related to carbon, hydrogen and nitrogen metabolism. Possession of a pair of cyAbrBs has been highly conserved during the course of evolution of the cyanobacterial phylum, suggesting physiological significance of transcriptional regulation attained by their interaction.

Bacteria represent an attractive source for the isolation and identification of potentially useful microorganisms for lignin depolymerization, a process required for the use of agricultural waste. In this work, ten autochthonous bacteria isolated from straw, cow manure, and composts were characterized for potential use in the biodelignification of the waste. A comparison of the ability to degrade lignin and the efficiency of ligninolytic enzymes was performed in bacteria grown in media with lignin as a sole carbon source (LLM, 3.5g/L lignin-alkali) and in complex media supplemented with All-Ban fiber (FLM, 1.5g/L). Bacterial isolates showed different abilities to degrade lignin, they decreased the lignin concentration from 7.6 to 18.6% in LLM and from 11.1 to 44.8% in FLM. They also presented the activity of manganese peroxidase, lignin peroxidases, and laccases with different specific activities. However, strain 26 identified as Paenibacillus polymyxa by sequencing the 16S rRNA showed the highest activity of lignin peroxidase and the ability to degrade efficiently lignocellulose. In addition, P. polymyxa showed the highest potential (desirability ≥ 0.795) related to the best combination of properties to depolymerize lignin from biomass. The results suggest that P. polymyxa has a coordinated lignin degradation system constituted of lignin peroxidase, manganese peroxidase, and laccase enzymes.

High salt wastewater is produced in industries, including seafood and pickling processing. The salinity in such wastewaters has been shown to negatively impact biological treatment efficacy. Little is known about the changes in the microbial community structure in the mature biological 2 treatment systems, the impacts of salinity on community composition, and the shifts over time during operation. This study aimed to identify the changes in the microbial community due to both salt and days of operation through 16s rRNA sequencing and KEGG functional predictions. Intermittent sand bioreactors (ISBs) with a focus on ammonia treatment were utilized. Results showed that the overall community structure and diversity were distinct as wastewater salinity varied from 0%-1.3%. At 1.3% salinity Zoogloea, a common genus in wastewater treatment plants, was not present and Aequorovita, Thauera and Dokdonella became the dominant genera. Nitrosomonas, an important ammonia oxidizing bacteria, increased in abundance with days of operation but was not significantly impacted by an increase in salinity. This finding was further supported by an increase in predicted nitrification potential with time of operation within all intermittent sand bioreactors tested. These results provide a deeper understanding of the impacts of salinity on microbial community development in biological treatment systems and elucidate the shifts in community structure occurring during early operations and into system maturity.