Induction of production and secretion β(1→4) glucanase with Saccharomyces cerevesiae by replacing the MET10 gene with egl1 gene from Trichoderma reesei

Aims:  To construct novel brewer's yeast strains with the ability to degrade β-glucan and increase sulfite levels in beer brewing by genetic manipulation.
Methods and Results:  The recombinant plasmid pA15ME containing P_met10-egl1-T_met10 expression cassette was constructed. Bam HI-linearized target plasmid pA15ME was transformed into the industrial brewer's yeast strain Z0103 to replace the MET10 locus through one-step gene replacement. The recombinants Z8, Z7 and Z3 with the ability to secrete active endo-β-1,4-glucanase I into the culture medium were isolated by Congo red dyeing. The enzymatic activities of EG I of Z8, Z7 and Z3 were 3·3, 1·5, 1·3 U l⁻¹, and the hydrolysing degrees of β-glucans in wort were increased 11·9%, 8·6% and 6·9%, respectively, than that of original strain Z0103. The MET10 gene deletions were confirmed by real-time PCR, and the sulfite levels of the culture mediums inoculated with Z8, Z7 and Z3 were increased 26%, 16% and 17%, respectively, compared to that of Z0103.
Conclusions:  The novel endoglucanase-producing brewer's yeast strains with inserted endoglucanase gene and deficient MET10 gene led to reduced content of barley β-glucans, enhanced filterability and increased sulfur dioxide in fermenting wort. Thus, the cost for addition of microbial β-glucanase enzyme and sulfite preparations in normal beer brewing processes could be reduced.
Significance and Impact of the Study:  These results suggested that genetic engineering approach is a powerful tool to construct the novel recombinant brewer's yeast strains with different properties to reduce the cost of beer brewing and improve the flavour of a beer, and the strains obtained have potential application value in beer brewing.     

A 5-h screening and 24-h confirmation procedure for detecting Escherichia coli O157 : H7 in beef using direct epifluorescent microscopy and immunomagnetic separation

An antibody-direct epifluorescent filter technique (Ab-DEFT) detected 100% of the raw ground beef samples inoculated with Escherichia coli O157 : H7 cells (0·15 cells g⁻¹) and incubated in a prewarmed, modified buffered peptone water (mBPW) non-selective enrichment broth for 5 h at 42°C in an orbital shaking water bath (200 rev min⁻¹). Over 50% of the microscopic fields viewed were positive (1–10 fluorescent cells field⁻¹) in the Ab-DEFT. All positive screening results were confirmed within 24 h by subjecting 1 ml of the mBPW to the Dynabeads^® anti- E. coli O157 immunomagnetic separation procedure, followed by plating on MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indolyl-β- D -glucuronide. At this cell concentration, 41·7% of the inoculated samples were detected by the conventional method involving a 24-h selective enrichment. Exposure to viable cells before filtration was minimized by using a 0·58% formaldehyde concentration for 5 min at 50°C (killed >4·00 logs of E. coli O157 : H7 cells) without affecting cell fluorescence.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, β-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20–44·5°C and at pH values 5·2–7·4 with optimal growth at 37–41·5°C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5·0, the optimum temperature was 40°C for the endoglucanase and 50°C for the xylanase. Both enzyme activities were inhibited at 70°C. Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

The preparation of ampicillin dextrin agar for the enumeration of Aeromonas in water

Introduction of ampicillin dextrin agar (ADA) has revealed problems in details of the preparation. The final pH of the medium varied substantially between different laboratories. Measuring temperature has a pronounced effect on the pH (0·7 units lower at 50°C than at 6°C). Addition of agar during medium preparation resulted in a fall in pH of 0·5 units. If poured plates were stored in the refrigerator, the pH was reduced by 0·1–0·4 units, in particular during the first day. Recovery of Aeromonas from pure cultures and naturally polluted samples was unaffected by variation in pH between 7·1 and 8·3 but colony differentiation was optimal at a higher pH. The use of ADA at a final pH of 7·8 ± 0·2 (at 25°C) is recommended. Different types of dextrin differed in respect of solubility, fermentability and colony differentiation. Optimal results were obtained with Difco 161 and Merck 3006.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Partial purification, characterization and regulation of cellulolytic enzymes from Trichoderma longibrachiatum

A net purification of 9·46-, 18·6- and 16·7-fold for filter paper (FP) hydrolytic activity, carboxymethyl (CM) cellulase and β-glucosidase, respectively was achieved through ion exchange and gel chromatographies. The purified enzyme preparation showed an optimal pH of 5·0 for CM cellulase and 5·5 for the other two components. The enzyme activities increased up to 60°–65°C for the three enzyme components and they were stable at 30° or 40°C and pH 4·5 to 5·0 after 20–30 min treatment. The four enzyme components, that is, two FP activities (unadsorbed and adsorbed), a CM cellulase and a β-glucosidase, had K_m values of 47·6 mg, 33·3 mg, 4·0 mg and 0·18 mmol/l with V _max of 4, 1·28, 66·5 and 1·28 units per mg protein. The molecular weights as determined with SDS-PAGE were found to be 44000, 38000, 55000 and 63000 for the above four enzyme components in the same sequence. A distinct type of synergistic action was observed between these components by their action on dewaxed cotton. Glycerol at 1% strongly repressed the formation of all the cellulolytic enzymes. The role of proteolytic enzymes in in vitro inactivation of cellulases was not apparent.     

Production of β-glucosidase (cellobiase) by Curvularia sp.

A Curvularia sp. isolated from soil was found to produce extracellular β-glucosidase activity when grown in yeast extract, peptone, carboxymethylcellulose (YPC) medium. An initial medium pH of 6·5 and cultivation temperature of 30°C were found to be most suitable for high enzyme productivity. The pH and temperature optima for the enzyme were 4·0 and 70°C, respectively. Under these conditions, the enzyme exhibited a K_m (0-nitrophenyl-β- d -glucoside) value of 0.20 mmol/l. Several divalent metal ions inhibited enzyme activity at high concentration. EDTA. also inhibited β-glucosidase activity.     

Purification and characterization of galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum

Galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p -aminobenzyl 1-thio-β- d -galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β-galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β-galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The K _m values for o -nitrophenyl-β- d -galactopyranoside and lactose were 14·3 and 5·5 mmol l⁻¹, respectively, and the V _max values for these substrates were 33·4 and 94·5 μmol min⁻¹ mg of protein⁻¹, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml⁻¹ galacto-oligosaccharide was produced from 200 mg ml⁻¹ lactose.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

A two-year study of the distribution of ''thermophilic'' campylobacters in human, environmental and food samples from the Reading area with particular reference to toxin production and heat-stable serotype

The incidence of 'thermophilic' campylobacters in foods and environmental samples has been studied over a two-year period. Of 781 environmental samples, 529 (67%) were found to contain campylobacters, and campylobacters were isolated from 835 (39%) of 2116 food samples. Sewage was almost always contaminated with campylobacters (96·6% of samples) and of the food samples both poultry (55·5%) and offal (47·0%) were commonly contaminated. Determination of the heat-stable serotypes of all strains isolated from these sources and of 921 strains isolated from human faeces showed that there was a wide distribution of serotypes in most types of sample. Serotype Pen 2 was the commonest type found in human faeces (18·9%) and it was also commonest in offal (21·3%), beef (40·0%), sewage (17·7%) and was the third commonest type in poultry. A comparison of culture media and conditions for optimal production of both cytotoxic and cytotonic enterotoxins showed that Brucella Broth incubated under microaerobic conditions for 24 h at 42°C was suitable for both toxins. Detection of cytotoxic activity was most sensitive using HeLa cells. The sensitivities of two ELISA systems and a Chinese Hamster Ovary tissue culture assay for detection of cytotonic enterotoxin were comparable. Not all strains isolated from cases of enteritis in human beings produced toxin; 23·1% produced cytotonic enterotoxin and 17·5% produced cytotoxin. There was no correlation between serotype and toxin production. The wide distribution of campylobacters, indistinguishable from those isolated from cases of enteritis in human beings, leads us to conclude that simplistic statements suggesting that one particular type of food is primarily responsible for cases of human disease should not be made.     

Evaluation of chromogenic media for the isolation of vancomycin-resistant enterococci from stool samples

Aims:  This study sought to evaluate the performance of two chromogenic media designed for the isolation of vancomycin-resistant enterococci (VRE) and compare them with a traditional bile-esculin medium for the isolation of VRE from stool samples.
Methods and Results:  A total of 285 stool samples were inoculated onto Chromogenic VRE Agar (AES VRE agar; AES Chemunex), chromID VRE (bioMérieux) and VRE Agar (Oxoid) both directly and also following broth enrichment. In total 18 strains of vancomycin-resistant Enterococcus faecium were recovered, including 17 harbouring the vanA gene and one with vanB . On direct culture, the sensitivity of the three media was 66·7%, 77·8% and 44·4% and after broth enrichment 66·7%, 83·3% and 77·8% using AES VRE Agar, chromID VRE and Oxoid VRE Agar respectively.
Conclusions:  All three media are useful tools for the isolation of VRE from stool samples. AES VRE Agar and bioMérieux chromID VRE are easier to use than Oxoid VRE Agar due to diffusion of black coloration from the latter.
Significance and Impact of the Study:  This is the first study to evaluate the performance of AES VRE Agar and the first to compare two media containing synthetic chromogens for the isolation of VRE.     

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

The effect of chlorine on β- D- galactosidase activity of sewage bacteria and Escherichia coli was studied. β- D- galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0·05 mg Cl₂ l⁻¹) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0·1 mg Cl₂ l⁻¹, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu⁻¹ increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu⁻¹ decreased when the chlorine dosage was increased from 0·1 to 0·5 mg Cl₂ l⁻¹, but were not significantly different ( P > 0·05) for dosages of 0·2–0·7 mg Cl₂ l⁻¹. After chlorination, β- D- galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.     

Effects of light intensity and addition of carotene rich Dunaliella salina live cells on growth and antioxidant activity of Solea senegalensis Kaup (1858) larval and metamorphic stages

Senegal sole Solea senegalensis larval and metamorphic stages were exposed to a range of light intensities (200, 1000 and 2000 lx) in cultures with or without supplementation of β-carotene-rich live Dunaliella salina cells. Antioxidant biomarkers such as superoxide dismutase (SOD), catalase (KAT), total glutathione peroxidase (t-GPX) and malondialdehyde (MDA) were determined in larval and metamorphic stages. Growth was not affected ( P > 0·05) either by light intensity or D. salina supplementation. Survival after metamorphosis was also unaffected by D. salina supplementation (mean ± s . e . 81·0 ± 2·5% against 80·6 ± 2·9% those fed the control algal diet) or light intensity (mean ± s . e . 74·3 ± 4·9% for 200 lx, 85·1 ± 2·7% for 1000 lx and 82·8 ± 5·2% for 2000 lx, respectively). Light intensity affected ( P < 0·05) KAT and t-GPX throughout development. SOD was only affected in metamorphosing larvae. The highest KAT and t-GPX activities were detected when the lowest light intensity (200 lx) was used. Light had no effect ( P > 0·05) on MDA at any stage. Supplementing the diet with D. salina did not affect SOD, KAT or t-GPX and there was no interaction ( P > 0·05) with light intensity. MDA was the only biomarker whose activity was significantly ( P < 0·05) reduced when D. salina was supplemented to the larval rearing tanks. The effect of D. salina supplementation was only detected in metamorphosing larvae, whose MDA levels were noticeably higher than in earlier stages. These results are evidence of the antiperoxidative effect of β-carotene from live algae in the larval rearing process of marine fishes.     

The immunoglobulin of the brown trout, Salmo trutta and its concentration in the serum of antigen-stimulated and non-stimulated fish

Immunoglobulin production in the primary and secondary immune response of brown trout to keyhole limpet haemocyanin has been investigated including the effect of dose size, route and number of injections, and the use of adjuvant. Antibody activity was found in the first fraction from Sephadex G200 and in the second from Sepharose 6B. Trout immunoglobulin had β₂—Γ₁ electrophoretic mobility, and S_app of 16·7 and an approximate molecular weight of 670 000 daltons. It was sensitive to dithiothreitol and stable at 56°C for 30 min. Immunoglobulin concentrations were measured by single radial immunodiffusion with a specific rabbit antiserum. Sera from non-injected trout had a mean immunoglobulin level of 7·3 ± 0·3 mg ml⁻¹ which accounted for 10% of the total serum protein. Phosphate buffered saline-injected controls contained 6·7 ± 0·2. In fish given a single injection the mean concentration ranged from 7·5 to 12·9 and in those given more than one injection from 12·6 to 16·8. The use of adjuvant resulted in higher immunoglobulin concentrations. Neither dose nor route had any significant effect on the primary response. However, in the secondary response the intramuscular route resulted in significantly increased immunoglobulin production.     

β-Lactam and aminoglycoside production from streptomycetes

Streptomyces cattleya, S. fradiae and S. griseus produced different amounts of growth when cultured sequentially through sporulation, vegetative and antibiotic production media. Only S. griseus grew well on all three types of medium. Streptomyces cattleya grew poorly on both sporulation and vegetative media. Growth was 1·6 and 8·0 mg/1/h respectively. For all three species, biomass yield in the final antibiotic production medium was dependent on amount of inoculum. Antibiotic yields were obtained only from production media. Under slow growth conditions l -cysteine and l -valine supplementation stimulated S. cattleya β-lactam production, giving 1000 μg/ml β-lactam equivalents compared with 45 μg/ml β-lactam equivalents for no supplementation. For aminoglycosides the agar well diffusion bioassay was more sensitive towards the hydrochloride than the neutral salt. Paper chromatography confirmed the main antibiotic classes. R _F values for replicate samples indicated aminoglycoside homogeneity and β-lactam heterogeneity.     

Sensitization of thermally injured spores of Bacillus stearothermophilus to sodium benzoate and potassium sorbate

The effects of sodium benzoate and potassium sorbate added to the recovery medium, at different pH values (6·5, 6·0 and 5·0), on the recovery rates and heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Heated spores of strains 12980 and 7953 were inhibited by sorbate concentrations over 0·05%. Potassium sorbate at concentrations as low as 0·025%, and sodium benzoate at 0·1%, were very effective inhibitory agents for heat-damaged spores. Their effectiveness always increased at pH 5·0, at which no growth occurred, with sodium benzoate for strains 7953, 15951 and 15952, and with potassium sorbate for strains 15951 and 15952. Decimal reduction times, whenever recovery was possible, were not significantly ( P  > 0·05) affected. None of these compounds modified the z -values obtained for the spores of the four strains, which had a mean value of 7·53 ± 0·28.     

Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP

Escherichia coli shifted from broth at external pH (pH₀) 7·0 to pH₀ 7·0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose. Tolerance appeared without the medium pH becoming acidic. Tolerance was most substantial when glucose was added at pH₀ 7·0 but was also appreciable at pH₀ 7·5, 8·0 and 8·5. Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose ; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid. Glucose-induced organisms showed less acid damage to DNA and β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

A New Medium for Bacillus thuringiensis Berliner

S ummary : A new solid medium for culturing Bacillus thuringiensis consists of groundnut cake, 10%; tamarind kernel powder, 1·5% and agar, 0·5%. The quantity of agar in the medium could be decreased from 1·5 to 0·5% by adding tamarind kernel powder. A spore yield (85% sporulation) of 3·2 g/l was obtained. Bacterial spores produced on the new solid medium were pathogenic to the larvae of the almond moth, Cadra cautella.