Production of bacteriolytic enzymes by mycoparasitic Trichoderma strains

Eighteen mycoparasitic Trichoderma strains were tested for their ability to degrade heat-inactivated Bacillus cereus var. mycoides, B. megaterium, B. subtilis, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa and Serratia marcescens cells. The non-inductive and inductive ferment broths of five strains with good degrading abilities towards B. subtilis were investigated for specific degrading enzyme activities. In addition to trypsin- and chymotrypsin-like protease activities, -1,4-N-acetyl-glucosaminidase (NAGase) was also secreted. Strain Trichoderma harzianum T19 had the most outstanding degrading abilities. The extracellular degrading enzymes of this strain were separated on a Sephadex G-150 column, and their preliminary characterization was performed. The results demonstrated that muramidase-like activities are present in the ferment broth of this T. harzianum strain.     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

Effectiveness of honey bees in delivering the biocontrol agent Bacillus subtilis to blueberry flowers to suppress mummy berry disease

Honey bees are important pollinators of commercial blueberries in the southeastern United States, and blueberry producers often use supplemental bees to achieve adequate fruit set. However, honey bees also vector the plant pathogenic fungus Monilinia vaccinii-corymbosi which infects open blueberry flowers through the gynoecial pathway causing mummy berry disease. Here, we report the results of a 3-year field study to test the hypothesis that using bee hives equipped with dispensers containing the biocontrol product Serenade, a commercial formulation of the bacterium Bacillus subtilis which has shown activity against flower infection by M. vaccinii-corymbosi in laboratory experiments, can reduce mummy berry disease incidence when honey bees are used as pollinators in blueberries. Individual honey bees carried 5.1–6.4 × 10⁵ colony-forming units (CFU) of B. subtilis when exiting hive-mounted dispensers with Serenade. On caged rabbiteye blueberry bushes in the field, population densities of B. subtilis vectored by honey bees reached a carrying capacity of <10³ CFU per flower stigma within 2 days of exposure, and there was a highly significant non-linear relationship between B. subtilis populations per stigma and bee activity, expressed as number of legitimate flower visits per time interval per cage (R = 0.6928, P < 0.0001, n = 32). Honey bee density (1600 or 6400 individuals per 5.8-m³ cage) and Serenade treatment (presence or absence of the product in hive-mounted dispensers) significantly (P < 0.05) affected the incidence of fruit mummification on caged bushes, whereby increasing bee density increased disease incidence and application of Serenade reduced disease levels. Taken together, results of this study suggest that use of a hive-dispersed biocontrol product such as Serenade as a supplement during pollination can reduce the risk of mummy berry disease. This may be a prudent practice that optimizes the benefits to pollination of high bee densities while reducing the associated disease-vectoring risk.     

Phenotypic diversity and technological properties of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> species isolated from <Emphasis Type="Italic">okpehe</Emphasis>, a traditional fermented condiment

The Bacillus subtilis wild strains isolated from okpehe, a traditional fermented condiment used as seasoning in Nigeria, the reference and typed strains were investigated for their phenotypic diversity and their technological parameters with a view to obtain adequate data that would enable selection of appropriated starter cultures for vegetable protein fermentation in West Africa. All the 7 strains studied demonstrated diverse phenotypic characteristics and they were identified as Bacillus subtilis, based on the API 50 CHB combined with API 20E profile. Specific sugars that indicated a good hydrolytic potential of the wild strains were fermented. The highest proteinase activity of 90 AU/ml determined quantitatively was observed in the strain Bacillus subtilis BFE 5372, the proteinase was identified by the APIZYM gallery as chymotrypsin. Highest amylase activity of 13 AU/ml was noticed in strain Bacillus subtilis DSM 347 while only 4 strains produced polyglutamic acid with the strain Bacillus subtilis BFE 5359 producing the highest polyglutamate activity of 2.5 mm. Although strain Bacillus subtilis BFE 5301 did not release detectable polyglutamate, the strain demonstrated antagonism against different bacteria and the antimicrobial substance produced by strain Bacillus subtilis BFE 5301 was confirmed as a bacteriocin since its activities were lost after treatment with chymotrypsin and pepsin. The data generated showed the technological parameters that can aid selection of wild strains such as Bacillus subtilis BFE 5301, BFE 5359 and BFE 5372 for optimization of condiment production.     

Enhanced production of a thermostable mannanase by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> on various membranes

Cells of the thermophilic Bacillus subtilis WY34 were immobilized on various formaldehyde-activated polymer membranes and the immobilized cells were used for the production of thermostable mannanase in flasks. The results showed that polyethersulfone membranes (PES) and nylon-6 membranes were the most suitable supports for cell immobilization to produce the mannanase. Moreover, PES and nylon-6 membranes immobilized cells provided 1.78- and 1.74-fold higher mannanase activity compared to the control after 4 days of cultivation, respectively. The immobilized cells on PES and nylon-6 membranes had good stability and retained 131.5 and 114.3% of ability of enzyme production even after six cycles of repeated batch fermentation, respectively. Active cell growth was observed by scanning electron microscopy (SEM) after 16 days (four cycles) repeated batch cultivation. Therefore, the membrane-immobilized cells of B. subtilis WY34 can be proposed as an effective biocatalyst for repeated usage for production of the thermostable mannanase.     

Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Gene Cloning and Characterization of a Thermostable Phytase from Bacillus subtilis US417 and Assessment of its Potential as a Feed Additive in Comparison with a Commercial Enzyme

An extracellular phytase from Bacillus subtilis US417 (PHY US417) was purified and characterized. The purified enzyme of 41 kDa was calcium-dependent and optimally active at pH 7.5 and 55°C. The thermal stability of PHY US417 was drastically improved by calcium. Indeed, it recovered 77% of its original activity after denaturation for 10 min at 75°C in the presence of 5 mM CaCl₂, while it retained only 22% of activity when incubated for 10 min at 60°C without calcium. In addition, PHY US417 was found to be highly specific for phytate and exhibited pH stability similar to Phyzyme, a commercial phytase with optimal activity at pH 5.5 and 60°C. The phytase gene was cloned by PCR from Bacillus subtilis US417. Sequence analysis of the encoded polypeptide revealed one residue difference from PhyC of Bacillus subtilis VTTE-68013 (substitution of arginine in position 257 by proline in PHY US417) which was reported to exhibit lower thermostability especially in the absence of calcium. With its neutral pH optimum as well as its great pH and thermal stability, the PHY US417 enzyme presumed to be predominantly active in the intestine has a high potential for use as feed additive.     

Discrimination of riboflavin producing Bacillus subtilis strains based on their fed-batch process performances on a millilitre scale

Forty-eight single-use stirred tank bioreactors on a 10-mL scale operated in a magnetically inductive driven bioreaction block and automated with a liquid handler were applied for discrimination of different riboflavin producing Bacillus subtilis strains based on their performances in the parallel fed-batch processes. It was shown that a discrimination of the B. subtilis riboflavin producer strains can efficiently be achieved within one parallel fermentation run based on the integral riboflavin yield after 48 h. The possibility to perform replicates within the parallel fermentation run allows for a robust statistical analysis and is a prerequisite for the discrimination of producer strains under fed-batch process conditions. Within the estimation error, all of the riboflavin producing B. subtilis strains under study showed the same fed-batch process performances on the litre scale compared to the millilitre scale.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape

A strain of Bacillus subtilis (NJ-18) with broad antimicrobial activity was screened in the laboratory and in the field. NJ-18 inhibited the in vitro radial extension of hyphae of the phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. The bacterium apparently produced antifungal metabolites that diffused through the agar and caused abnormal swelling of hyphae. The in vitro data and observations indicated that one of the mechanisms of inhibition by NJ-18 is antibiosis. In field experiments for control of sheath blight of rice, fermentation of NJ-18 at 5.0 × 10⁷ cfu ml⁻¹ significantly reduced disease incidence and severity; NJ-18 alone or combined with 50% kresoxim-methyl treatment at 225 g ai ha⁻¹ provided better control than 50% kresoxim-methyl at 225 g ai ha⁻¹ or Jinggangmycin at 120 g ai ha⁻¹, and control by NJ-18 alone was as high as 100.0%. In field experiments for control of Sclerotinia stem rot of rape, fermentation of NJ-18 at 1.0 × 10⁷ cfu ml⁻¹ again significantly reduced disease incidence and severity; control by NJ-18 was as high as 77.1% and was comparable with control by 46% dimethachlon and better than control by 50% carbendazim at 750 g ai ha⁻¹. We conclude that strain NJ-18 of B. subtilis is a promising biological control agent and should be further studied and tested for control of sheath blight of rice, Sclerotinia stem rot of rape, and other diseases.     

Biological control of Botrytis cinerea on stem wounds with moderately halophilic bacteria

Infection of tomato stem wounds by Botrytis cinerea is an important problem which can cause severe economic losses in greenhouse tomato crops. Three moderately halophilic bacteria were tested for their ability to protect pruning wounds from attacks by B. cinerea under growth chamber conditions. The severity of the disease estimated by the length of the rotted stem was used to calculate the area under the disease progress curves (AUDPC). Bacterial antagonists (B1, B2 and B3) were very effective in controlling Botrytis-infection on the tomato stems during the first 6 days and later by the end of the experiment. Plants treated with Bacillus subtilis (B1) had the lowest AUDPC (0). It was followed by B. subtilis (B3) and Halomonas sp. (B2) with AUDPC of 9.8 and 17.02, respectively. While the B1 strain best inhibited grey mold development when applied as young culture (24 h), the B3 strain performed better as an older culture (48 h). In contrast to the results obtained with Bacillus species, the efficacy of the bacterial treatment B2 seems to be independent of the growth phase. The co-cultures with fungal spores and either B. subtilis (B1) or Halomonas sp. (B2) applied as a 24 h bacterial culture completely inhibited the germination of B. cinerea after 24 h at 21°C.     

Mn<Superscript>2+</Superscript> enhances theanine-forming activity of recombinant glutamine synthetase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn²⁺ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence of 10 mM Mn²⁺ at optimal pH 7.5. The activity is markedly higher activated by Mn²⁺ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn²⁺ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1 g/L by paper chromatography and HPLC, respectively.     

Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.     

Isolation,identification and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> ZJB-063, a versatile nitrile-converting bacterium

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.     

Croatian barberry (<Emphasis Type="Italic">Berberis croatica</Emphasis> Horvat): a new source of berberine—analysis and antimicrobial activity

By using HPLC/UV–VIS, Croatian barberry (Berberis croatica Horvat) was found to be a new source of the bioactive alkaloid berberine. Comparison of berberine content in roots, leaves, and twigs between wild specimens of B. croatica and B. vulgaris collected in Croatia showed that the roots of both species contained the highest berberine content (B. croatica 1.120–1.217%; B. vulgaris 0.805–1.424%), followed by twigs (B. croatica 0.049–0.216%; B. vulgaris 0.077–0.112%). While the berberine content in the leaves of both species was very low (between 0.002% and 0.044%), they were found to be rich in phenols and flavonols. The Student’s t-test showed a significant difference at P < 0.05 for phenol and flavonol content in the plant organs, both between species and within species. Leaf samples were most variable, while root samples were the least. Extracts from the roots of both barberry species expressed antimicrobial activity against Bacillus subtilis NCTC 8236, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10535, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231. Antimicrobial activity of leaf extracts was species-dependent. Root extracts of both species also showed lower MIC values than other extracts (MIC ≤ 87.5 mg/ml).     

Leucine improves the component of isovalerylspiramycins for the production of bitespiramycin

Bitespiramycin, a group of 4″-O-acylated spiramycins with 4″-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4″-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 ± 0.33% total isovalerylspiramycins was recorded compared to 40.9 ± 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4″ side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.     

Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53

Rice bran and yeast extract were found to be the best combination of carbon and nitrogen sources for the production of carboxymethycellulase (CMCase) by Bacillus subtilis subsp. subtlis A-53. Optimal concentrations of rice bran and yeast extract for the production of CMCase were 5.0% (w/v) and 0.10% (w/v), respectively. Optimal temperature and initial pH of medium for cell growth of B. subtilus subsp. subtilis A-53 were 35 °C and 7.3, whereas those for the production of CMCase by B. subtilus subsp. subtilis A-53 were 30 °C and 6.8. Optimal agitation speed and aeration rate in a 7 L bioreactor were 300 rpm and 1.0 vvm, respectively. The optimal agitation speed and aeration rate for the production of CMCase by B. subtilus subsp. subtilis A-53 were lower than those for cell growth. The highest productions of CMCase by B. subtilus subsp. subtilis A-53 in 7 and 100 L bioreactors were 150.3 and 196.8 U mL⁻¹, respectively.