Dietary Supplementation of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 Combinations Enhance Growth and Cellular and Humoral Immunity in Olive Flounder (Paralichthys olivaceus).

Experiments were conducted to identify different ratios of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 mixtures at a concentration of 1 × 108 CFU/g diet; the effects on growth and cellular and humoral immune responses and the characteristics of disease protection in olive flounder (Paralichthys olivaceus). Flounder were divided into six groups and fed control diet D-1 (without Bacillus sp. SJ-10 and L. plantarum KCCM 11322), positive control diets D-2 (Bacillus sp. SJ-10 at 1 × 108 CFU/g feed) and D-3 (L. plantarum KCCM 11322 at 1 × 108 CFU/g feed); or treatment diets D-4 (3:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.75 + 0.25 × 108 CFU/g feed), D-5 (1:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.50 + 0.50 × 108 CFU/g feed), or D-6 (1:3 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.25 + 0.75 × 108 CFU/g feed) for 8 weeks. Group D-4 demonstrated better growth and feed utilization (P < 0.05) compared with the controls and positive controls. Similar modulation was also observed in respiratory burst for all treatments and in the expression levels of TNF-α, IL-1ß, IL-6, and IL-10 in different organs in D-4. D-4 and D-5 increased respiratory burst, superoxide dismutase, lysozyme, and myeloperoxidase activities compared with the controls, and only D-4 increased microvilli length. When challenged with 1 × 108 CFU/mL Streptococcus iniae, the fish in the D-4 and D-5 groups survived up to 14 days, whereas the fish in the other groups reached 100% mortality at 11.50 days. Collectively, a ratio-specific Bacillus sp. SJ-10 and L. plantarum KCCM 11322 mixture (3:1) was associated with elevated growth, innate immunity, and streptococcosis resistance (3:1 and 1:1) compared with the control and single probiotic diets.

Effects of dietary non-viable Bacillus sp. SJ-10, Lactobacillus plantarum, and their combination on growth, humoral and cellular immunity, and streptococcosis resistance in olive flounder (Paralichthys olivaceus).

Heat-killed (HK) Bacillus sp. SJ-10 (B), HK Lactobacillus plantarum (P), and their combination were dietary supplemented to olive flounder (Paralichthys olivaceus) to quantify the effects on growth, innate immunity, and disease resistance. Four test diets were supplied: a control feed free of HK probiotics, 1 × 108 CFUs g-1 single treatments of each of HK B (HKB) and HK P (HKP), and an equal proportion of (0.5 HKB + 0.5 HKP) × 108 CFUs g-1 (HKB0.5 HKP0.5). At 8 weeks of completion feeding trail, HKB0.5 HKP0.5 significantly (P < .05) improved growth, feed utilization, and nonspecific immune parameters (respiratory burst and superoxide dismutase) compared to the control group. Similarly, serum lysozyme and myeloperoxidase activities were higher in both HKB and HKB0.5HKP0.5 groups. The levels of pro-inflammatory cytokine IL-6 in the liver and IL-1ß in the liver, kidney, and spleen were also improved in the treatments, but microvilli length was only increased in HKB0.5HKP0.5. After Streptococcus iniae 1 × 108 CFUs mL-1 challenged; HKB and HKB0.5HKP0.5 had a higher survival than control and HKP. Overall, dietary administration of synergy HK probiotics elevated growth, cellular and humoral immunity, and streptococcosis resistance in olive flounder.

Effect of Polar Amino Acid Residue Substitution by Site-Directed Mutagenesis in the N-terminal Domain of Pseudomonas sp. Phytase on Enzyme Activity.

The N-terminal domain of the Pseudomonas sp. FB15 phytase increases low-temperature activity and catalytic efficiency. In this study, the 3D structure of the N-terminal domain was predicted and substitutions for the amino acid residues of the region assumed to be the active site were made. The activity of mutants, in which alanine (A) was substituted for the original residue, was investigated at various temperatures and pH values. Significant differences in enzymatic activity were observed only in mutant E263A, suggesting that the amino acid residue at position 263 of the N-terminal domain is important in enzyme activity.

Antioxidant and gastrointestinal cytoprotective effect of edible polypeptide poly-γ-glutamic acid.

The antioxidant effects and cytoprotection of the poly-γ-glutamic acid (γ-PGA) were investigated. γ-PGA with a molecular weight of ~400 kDa showed hydroxyl radical (OH•) averting capacity (HORAC), 85.2% at 1 mg/mL, which was caused by the chelation of transition metal ions and scavenging hydrogen peroxide. Moreover, this γ-PGA showed 94.1% superoxide anion radical (O2•-) scavenging and 96.0% lipid peroxidation inhibition activity at the same concentrations. The IC50 values were 130 ± 4.2, 107 ± 3.5 and 128 ± 3.8 µg/mL against OH•, O2•- and lipid peroxidation, respectively. There was no significant variation in the HORAC of γ-PGA after 9 h, the end point of the simulated digestion model. Furthermore, γ-PGA showed a completely protective effect in Caco-2 cells and probiotic bacteria against oxidative damage at 1 mg/mL. These data suggest that γ-PGA has a potential use as a cytoprotectant in food and feed supplements, cosmetics and biomedical fields.

Effects of Bacillus subtilis WB60 and Lactococcus lactis on Growth, Immune Responses, Histology and Gene Expression in Nile tilapia, Oreochromis niloticus.

An eight-week feeding trial was conducted to evaluate the effects of a basal control diet (CON), Bacillus subtilis at 107 (BS7) and at 108 CFU/g diet (BS8), Lactococcus lactis at 107 CFU/g (LL7) and at 108 CFU/g diet (LL8), and oxytetracycline (OTC) at 4 g/kg diet on Nile tilapia. Fish with initial body weight of 2.83 ± 0.05 g (mean ± SD) were fed two times a day. Weight gain, specific growth rate, feed efficiency, protein efficiency ratio and lysozyme activity of fish fed BS8, LL8 and LL7 diets were significantly higher than those of fish fed CON diet (p < 0.05). Superoxide dismutase and myeloperoxidase activity of fish fed BS8, LL8, BS7, LL7 and OTC diets were significantly higher than those of fish fed CON diet. Intestinal villi length and muscular layer thickness of fish fed BS8, LL8 and LL7 diets were significantly higher than those of fish fed CON and OTC diets. Also, heat shock protein 70 (HSP70), interleukin (IL-1ß), interferon-gamma (IFN-γ) and tumour necrosis factor (TNF-α) gene expression of fish fed BS8 and LL8 diets were significantly higher than those of fish fed CON diet. After 13 days of challenge test, cumulative survival rate of fish fed BS8 and LL8 diets were significantly higher than those of fish fed CON, BS7 and OTC diets. Based on these results, B. subtilis and L. lactis at 108 (CFU/g) could replace antibiotics, and have beneficial effects on growth, immunity, histology, gene expression, and disease resistance in Nile tilapia.

Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65°C for 30 min, 63°C for 30 min, and 63°C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics

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Effects of probiotic supplementation of a plant-based protein diet on intestinal microbial diversity, digestive enzyme activity, intestinal structure, and immunity in olive flounder (Paralichthys olivaceus).

The aim of this study was to investigate the effects of intestinal microbial manipulation by dietary probiotic supplementation on digestive enzyme activity, immune-related gene transcription, intestinal structure alteration, and viability against pathogenic challenge in olive flounder. Similar-sized flounders (14.92 ±â€¯0.21 g) were divided into three groups and supplemented with a control (without probiotic) or 1 × 108 CFU/g diet of each of Bacillus sp. SJ-10 (ProB) and Lactobacillus plantarum (ProL) for eight weeks. At the end of the feeding trial, the estimated intestinal microbial richness (Chao1) and diversity (Shannon) demonstrated a significant (P < 0.05) abundance in the ProB group (484.80 ±â€¯88.75, 5.08 ±â€¯0.17) compared to the ProL (285.32 ±â€¯17.78, 4.54 ±â€¯0.09) and control groups (263.23 ±â€¯20.20, 4.30 ±â€¯0.20). A similar alteration phenomenon was also found at the phylum level, with a higher abundance of Proteobacteria, Actinobacteria, and Acidobacteria. Trypsin and lipase activities were elevated in both the ProB and ProL groups compared to the control, but amylase was only higher in the ProB group. The expression levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 were significantly higher in the ProB group than in the other two groups. There was a significant increase in transcription of IL-10 in both the ProB and ProL groups compared to the control. The length of villi and microvilli of probiotic-fed olive flounder was increased but was not significantly different from the control group. In an in vivo challenge experiment with Streptococcus iniae (1 × 108 CFU/mL), the survival rates of the ProB and ProL groups were 29.17% and 12.50%, respectively, when control mortality reached 100%. Therefore, intestinal microbiota manipulation by probiotic supplementation increased the richness of the bacterial population, digestive enzyme activity, intestinal immune gene transcription, and infectious disease protection in olive flounder.

Expression in Lactococcus lactis of a ß-1,3-1,4-glucanase gene from Bacillus sp. SJ-10 isolated from fermented fish.

Bacterial ß-1,3-1,4-glucanase (BG) is an endoglucanase that hydrolyzes linear ß-glucans containing ß-1,3 and ß-1,4 linkages, such as barley ß-glucans. In this study, a BG gene was transformed into the food-grade plasmid pNZ8149 and successfully expressed in Lactococcus lactis NZ3900 using the nisin-controlled gene expression system. To facilitate extracellular secretion, the signal peptide Usp45 was added during vector construction. A histidine tag was also added for affinity purification. BG was extracellularly secreted and was also present in the cells in soluble form. N-terminal amino acid residue analysis of secreted BG revealed that the Usp45 peptide was removed. The optimum temperature and pH for both intracellular and extracellular BG were 40 °C and 6, respectively. The enzyme kinetic parameters, Vmax, Km, kcat, and kcat/Km, of extracellular BG were 1317.51 µmol min-1, 1.97 mg ml-1, 588.54 s-1, and 298.26 ml s-1∙mg-1, respectively. There was no significant difference in the enzyme kinetic parameters of intracellular and extracellular BG. The growth pattern of transformed L. lactis NZ3900 in ß-glucan-containing liquid medium confirmed ß-glucan degradation by BG. The transformed strain degraded ß-glucans, produced gluco-oligosaccharide, and produced lactic acid. The strain and expression system constructed in this study could be applied to industrial fields requiring BG produced in food-grade lactococcal secretory expression system.

Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes.

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65 °C for 30 min, 63 °C for 30 min, and 63 °C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics.

Fusion of the N-terminal domain of Pseudomonas sp. phytase with Bacillus sp. phytase and its effects on optimal temperature and catalytic efficiency.

The beta-propeller phytase (BPP) is an enzyme that hydrolyzes phyate to release inorganic phosphorus. The BPP produced by Pseudomonas sp. FB15 (PSphy) possesses an additional N-terminal domain that is not present in BPP produced by other Bacillus species. In this study, BPP produced by Bacillus sp. SJ-10 (SJ-10phy) was fused with the N-terminal domain of PSphy and the enzymatic properties of the resulting fusion protein (FUSJ-10phy) were investigated. FUSJ-10phy exhibited an optimal temperature that was 10 °C lower than that of the wild-type enzyme. A comparison of kinetic parameters showed that the turnover rate of FUSJ-10phy was 2.39-fold higher than that of SJ-10phy, representing a 1.79-fold increase in catalytic efficiency. In addition, BPP produced by Bacillus sp. SJ-48 has relatively low sequence similarity with SJ-10phy, was fused with N-terminal domain (FUSJ-48phy). FUSJ-48phy also increased catalytic efficiency and changed the optimal temperature. These results indicate that, when fused to other BPPs, the N-terminal domain of PSphy increases catalytic efficiency and enzyme activity at lower temperatures.

Efficient production of poly γ-d-glutamic acid from the bloom-forming green macroalgae, Ulva sp., by Bacillus sp. SJ-10.

Numerous studies on poly γ-d-glutamicacid (γ-PGA) production have investigated terrestrial renewable sources for reducing production costs, but there are no studies using waste marine resources so far. We aimed to develop a cost-effective production method of γ-d-PGA by Bacillus sp. SJ-10 using green macroalgae (Ulva sp.) as a major substrate without hydrolysis pretreatment. The SJ-10 was shown to not only cause immediate tissue degradation of the Ulva membrane but also grew well as a sole substrate. The γ-d-PGA yield was 6.29 ± 0.34 g/L under optimized conditions via the response surface method, and the produced γ-d-PGA had a thermal decomposition temperature of 310°C and molecular weight of 250-1780 kDa. The calculated cost efficiency for the final yield was 32% when compared with complex media. Therefore, the present study provided a strategy for promoting an ecofriendly and cost-effective means to produce γ-d-PGA via a marine renewable resource.

Heat-killed Bacillus sp. SJ-10 probiotic acts as a growth and humoral innate immunity response enhancer in olive flounder (Paralichthys olivaceus).

Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 108 CFU g-1) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced weight gain and protein efficiency ratio, 1.17 and 1.11 folds respectively. Humoral innate immune parameters, lysozyme and superoxide dismutase in treatment group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1ß, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with Streptococcus iniae (1 × 108 CFU mL-1), the HKBSJ-10-fed group exhibited significantly higher protection (P < 0.05) against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Therefore, HKBSJ-10 increases growth, modulates innate immune parameters, and protects olive flounders against streptococcosis.

Immunomodulatory Effects of Monascus spp.-Fermented Sacccharina japonica Extracts on the Cytokine Gene Expression of THP-1 Cells.

The immunomodulatory effects of Monascus-fermented Saccharina japonica extract on anti- and pro-inflammatory cytokines gene expression of THP-1 cells were evaluated. Extracts of fermented samples showed higher phenolic, flavonoid, protein, and reducing sugar contents than unfermented one. Fermented samples were rich in many bioactive compounds determined by GC-MS analyses and showed cell viability greater than 85% in MTS assay. Regarding the anti-inflammatory and pro-inflammatory activities of the different samples, Q-PCR analyses revealed that IL-10 gene expression in THP-1 cells was significantly higher (p < 0.05) in cells treated with the SjMp or SjMk sample than those treated with the unfermented sample. Cells treated with the SjMp extract or lipopolysaccharide (LPS) showed significantly (p < 0.05) higher relative gene expression of IL-4 cytokine than cells treated with SjMk or SjU extracts. The relative gene expression of IFN-α was higher in cells treated with SjMp followed by LPS, SjMk, and SjU. TGF-ß expression was higher in LPS-stimulated cells followed by SjMk and other samples. Cells treated with SjMp exhibited significantly higher pro-inflammatory (IL-6, IL-8, TNF-α, and NF-κB) cytokine gene expression than cells treated with SjU. These results revealed that extracts from S. japonica fermented with Monascus spp. regulate cytokine gene expression. Graphical abstract ᅟ.

Characterization of a Bacillus sp. isolated from fermented food and its synbiotic effect with barley ß-glucan as a biocontrol agent in the aquaculture industry.

The purpose of this study was to evaluate Bacillus sp. SJ-10, isolated from traditional Korean seafood, as a probiotic. Strain SJ-10 was demonstrated to be safe, on the basis of in vitro tests confirming the absence of cytotoxicity, hemolysis, and genes with toxigenic potential, and was susceptible to antibiotics. It met the probiotic prerequisites of a spore count that remained almost constant, acid and bile tolerance under simulated gastrointestinal conditions, and significant adhesion to Caco-2 cells. Moreover, SJ-10 demonstrated beneficial properties as a probiotic: broad-spectrum antimicrobial activity, hydroxyl radical, antioxidant activity, production of functional enzymes such as ß-galactosidase and phytase, and selective growth via ß-glucan fermentation. The fish-feeding trial demonstrated that olive flounder fed diets containing SJ-10 alone or in combination with ß-glucan exhibited significantly higher growth performance and pathogenic disease resistance compared with those fed diets containing ß-glucan alone, indicating that SJ-10 diets exerted a beneficial effect as an antibiotic replacer in terms of growth performance and disease resistance in olive flounder.

Synergistic effects of dietary Bacillus sp. SJ-10 plus ß-glucooligosaccharides as a synbiotic on growth performance, innate immunity and streptococcosis resistance in olive flounder (Paralichthys olivaceus).

Bacillus sp. SJ-10 (BSJ-10) was identified from traditional Korean fermented fish, the previously recognized prebiotic ß-glucooligosaccharides (BGO), and their combination as a synbiotic were prepared to evaluate their individual and synergistic effects in olive flounder (Paralichthys olivaceus). Four diets (one control and three treatments) were formulated containing neither BSJ-10 nor BGO (control), 1 × 108 CFU g -1 BSJ-10 (BSJ-10), 0.1% BGO (BGO), and 1 × 108 CFU g-1 BSJ-10 + 0.1% BGO (BSJ-10 + BGO). Triplicates of 15 fish (weight 10 ± 0.25 g) were randomly allocated to the four diet groups and fed one of the diets for 8 weeks. At the end of the experiment, fish weight gain (WG), specific growth rate (SGR), feed conversion ratio, and protein efficiency ratio in BSJ-10, BGO and BSJ-10 + BGO diets were positively modulated (P < 0.05) compared with control. Specially, WG and SGR were significantly (P < 0.05) higher in BSJ-10 + BGO than that of BSJ-10 and BGO (individual component). The innate immune parameters such as respiratory burst, superoxide dismutase, and lysozyme activity (LSZ) of fish fed BSJ-10 and BSJ-10 + BGO (both groups) were significantly (P < 0.05) higher than the control. Moreover, myeloperoxidase activity (MPO) and LSZ of fish fed BSJ-10 + BGO were significantly higher compared with individual component. Compared with control, intestinal BSJ-10 content, expression of interleukin (IL)-1ß in liver and kidney, and tumor necrosis factor (TNF)-α in liver were higher in both groups, but microvillus length was increased (P < 0.05) only in BSJ-10 + BGO. During in vivo challenge experiment with Streptococcus iniae (1 × 108 CFU ml-1), survival rate of fish was significantly higher in all treatment groups versus control. Moreover, in BSJ-10 + BGO, protection against S. iniae infection and transcription of TNF-α and IL-6 in gill were significantly (P < 0.05) higher than the individual component. Collectively, an improved WG, SGR, MPO, LSZ, transcription of IL-6 and TNF-α, and cumulative survival rate against streptococcosis clearly demonstrates a synergistic outcome of diet BSJ-10 + BGO as synbiotic in olive flounder.

Effects of Lactococcus lactis subsp. lactis I2 with ß-Glucooligosaccharides on Growth, Innate Immunity and Streptococcosis Resistance in Olive Flounder (Paralichthys olivaceus).

To identify and quantify the effects of a combination of dietary 1 × 108 CFU/g Lactococcus lactis subsp. lactis I2 (LI2) and 0.1% ß-glucooligosaccharides (BGO) on the growth and immunity of olive flounder (Paralichthys olivaceus), a feeding experiment was conducted. Flounder (14 ± 0.5 g) were divided into two groups and fed control and synbiotic feeds for 8 weeks. Investigations were carried out on growth and feed utilization, innate immunity, serum biochemical parameters, intestinal lactic acid bacterial (LAB) viability, microvillus length, and changes in the expression levels of genes encoding pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, and IL-6). Results demonstrated the synbiotic diet had significantly better (p < 0.05) responses in terms of weight gain and specific growth rate, three innate immune parameters (respiratory burst, serum lysozyme, and superoxide dismutase), intestinal LAB viability, and the relative TNF-α expression level (p < 0.05). Moreover, after challenge with Streptococcus iniae (1 × 108 CFU/ml), the synbiotically fed group exhibited significantly higher (p < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Therefore, according to the results of the present study, synbiotic feed (LI2 + BGO) increased growth, modulated innate immune parameters and protected olive flounder against streptococcosis.

N-terminal domain of the beta-propeller phytase of Pseudomonas sp. FB15 plays a role for retention of low-temperature activity and catalytic efficiency.

Pseudomonas sp. FB15 producing phytase with a high activity at low temperature was isolated from foreshore soil. The phytase gene was cloned and sequenced. Molecular modeling predictions using its amino acid sequence indicated that it belongs to the group of beta-propeller phytases (BPPs). However, it differs from the conventional structure of BPPs in the number of propeller-shaped domains. Most BPPs have a propeller with six blades that consist of four or five beta-sheets. However, the BPP from the isolated strain contained a C-terminal domain containing a conserved BPP sequence and also an N-terminal domain consisting of an additional 275 amino acids. The characterization of recombinant BPP from Pseudomonas sp. FB15 (PSphy) revealed the highest activity to be at pH 6 and 40 °C. Additionally, it showed that more than 80% of optimal activity was retained at a relatively low temperature of 25 °C. In addition, CaCl2 was required for activity, and the optimal concentration of CaCl2 was 4 mM. The activity at low temperature was reduced in a recombinant protein from which the N-terminal domain was removed, as was catalytic efficiency. These results suggest that the N-terminal domain is required for the low temperature activity and high catalytic efficiency of PSphy.

Complete Genome Sequence of Bacillus sp. SJ-10 (KCCM 90078) Producing 400-kDa Poly-γ-glutamic Acid.

Bacillus sp. SJ-10 (KCCM 90078, JCM 15709) is a halotolerant bacterium isolated from a traditional Korean food, i.e., salt-fermented fish (jeotgal). The bacterium can survive and engage in metabolism at high salt concentrations. Here, we reported complete genome sequence of Bacillus sp. SJ-10, which has a single circular chromosome of 4,041,649 base pairs with a guanine-cytosine content of 46.39%. Bacillus sp. SJ-10 encodes a subunit of poly-γ-glutamic acid (γ-PGA) with a molecular weight of approximately 400 kDa, which contains four γ-PGA synthases (pgsB, pgsC, pgsAA and pgsE) and one γ-PGA-releasing gene (pgsS). This bacterium also able to produce salt-stable enzymes such as protease, ß-glucosidase, and ß-1,3-1,4-glucanase. This affords significant insights into strategies employed by halotolerant bacteria to survive at high salt concentrations. The sequence contains information on secondary metabolites biosynthetic gene cluster, and most importantly enzymes produced by the bacterium may be valuable with respect to food, beverage, detergent, animal feed, and certain commercial contexts.

Immobilization of ß-1,3-1,4-glucanase from Bacillus sp. on porous silica for production of ß-glucooligosaccharides.

(1,3)(1,4)-ß-d-glucan has been determined to have various beneficial effects due to its unique structure. ß-glucooligosaccharides (ß-GOS), which are hydrolysates of barley (1,3)(1,4)-ß-d-glucan, provide a useful prebiotic material for selective growth of probiotic bacteria. In this study, recombinant ß-1,3-1,4-glucanase (Bg1314) from Bacillus sp. SJ-10 (KCCM 90078) was immobilized on porous silica using glutaraldehyde as a crosslinking reagent to achieve efficient production of ß-GOS. We investigated the effects of factors such as the amounts of enzyme and glutaraldehyde, reaction temperature, and pH on catalytic activity. Enzyme activity decreased sharply at high concentrations of glutaraldehyde, likely due to the reaction of glutaraldehyde with lysine residues at the catalytic site of Bg1314, because lysine-substituted Bg1314 retained its activity under the same conditions. Immobilized Bg1314 protein (ImBg1314) was stable over a wide range of pH and could be stored long term at 4 °C. The optimal conditions of ImBg1314 were similar to those of Bg1314. However, the optimal temperature of ImBg1314 differed from that of Bg1314. The products were ß-GOS composed of 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-cellotriosyl-d-glucose. After ImBg1314 was reused for 10 cycles, it retained 42% of its initial catalytic activity. This study showed that the Imbg1314 applied economical production of ß-GOS.