Antibacterial Effect of Glucose Oxidase on Growth of Pseudomonas fragi as Related to pH

The effect of glucose oxidase (GOX) catalyzed reaction with glucose on Pseudomonas fragi was analyzed in nutrient broth and fish extract media. Growth of P. fiugi in nutrient broth was clearly suppressed by 1.0, 2.0 and 4.0 mg/mL glucose when combined with 0.5–2.0 U/mL GOX. The same GOX/glucose combinations inhibited P. frasi growth in fish extract media. Viable cell numbers in fish media showed clear growth inhibition with combinations of l.0–2.0 U/mL GOX and 8.0–16.0 mg/mL glucose. Higher GOX and glucose rapidly produced 2.0–2.5 unit decreases in pH, but produced enough gluconic acid to precipitate fish proteins. Use of 0.5 U/mL GOX in fish extract media resulted in slow, sustained activity with potential for inhibition of microbial growth in foods without excessive acidity.     

A statistical approach to the optimization of cold‐adapted amylase production by Exiguobacterium sp. SH3

Cold‐adapted enzymes produced by psychrotrophic organisms are interesting from both molecular and biotechnological viewpoints. The enzymes show superior catalytic activity than their mesophilic and thermophilic counterparts at room temperature. Therefore, the enzymes seem interesting for applications where high catalytic activity at ambient temperature is required. In this study, the production of cold‐adapted amylase by Exiguobacterium sp. SH3 was optimized and modeled. In the first step, single factor experiments using shake flask cultures were conducted for primary optimization. These experiments resulted in the improvement of amylase production up to 180 U/mL. In the next step, the Plackett–Burman design was used to identify significant factors affecting the amylase production. Starch concentration, tryptone concentration, and temperature were selected as significant factors; while time, shaking, yeast extract, pH, MnCl₂, CaCl₂, MgCl₂, and KH₂PO₄ were not significant in this step. Finally, the response surface methodology based on central composite design (CCD) was used for further optimization and modeling of the significant factors. The optimization efforts resulted in the maximum amylase production of 730 U/mL, which was four times higher than that achieved by the single‐factor optimization experiments.     

Production of a Lactobacillus plantarum starter with linamarase and amylase activities for cassava fermentation

Lactobacillus plantarum strain A6 isolated from cassava, cultured on cellobiose MRS medium showed a growth rate of 0.41 h^?1, a biomass yield of 0.22 g g^?1, and produced simultaneously an intracellular linamarase (76.4 U g^?1 of biomass) and an extracellular amylase (36 U ml^?1). The synthesis of both enzymes was repressed by glucose. The use of such a strain as a cassava fermentation starter for gari production had the following influences: a change from a hetero-fermentative pattern observed in natural fermentation to a homofermentation, a lower final pH, a faster pH decline rate and a greater production of lactic acid (50 g kg^?1 DM). However, this starter did not appear to play a significant role in cassava detoxification, since it was observed that the level of endogenous linamarase released during the grating of the roots was sufficient to permit the complete and rapid breakdown of linamarin.     

Disruption of the cytochrome c gene in xylose‐utilizing yeast Pichia stipitis leads to higher ethanol production

The xylose‐utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non‐cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild‐type P. stipitis CBS 6054 by cross‐hybridization to CYC1 from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1 showed 74% and 69% identity to ScCYC1 and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1 resulted in a mutant that uses the salicylhydroxamic acid (SHAM)‐sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes c and a·a₃ both disappeared in the cyc1‐Δ mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1‐Δ mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1‐Δ mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0·46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0·38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen‐limiting conditions. Copyright © 1999 John Wiley & Sons, Ltd.     

A Maltooligosaccharides Producing α-Amylase from Bacillus subtilis SDP1 Isolated from Rhizosphere of Acacia cyanophylla Lindley

Maltooligosaccharides producing amylases are required in the food industry, especially in breadmaking. The Bacillus subtilis strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h. Bacillus subtilis strain SDP1 was isolated from the rhizosphere of Acacia cyanophylla Lindley from the Çukurova region of Turkey. The highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37°C. Under optimized culture conditions, 68.49 U/mL activity was obtained. SDP1 α-amylase had molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and was highly active at pH ranging from 5.0 to 9.0. The optimum temperature of the crude enzyme was 60°C, and it retained 83% and 74% of its initial activity after 1 h and 2 h incubation periods, respectively, at 50°C. While, Mn⁺² has a stimulatory effect on the activity, Ca⁺², Mg⁺², Na⁺ did not effect the enzyme activity. Fe⁺³, Ni⁺², Cu⁺² and Co⁺² had an inhibitory effect on SDP1 amylase activity.     

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05–0·40 h⁻¹) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h⁻¹) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h⁻¹) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h⁻¹ to growth at μ_max. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.     

Production of β-galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria

The aim of this work was to establish optimal conditions for the maximum production of β-galactosidase using an industrially suitable medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was cultivated in skim milk, whey and whey permeate basal media, supplemented with whey protein products, yeast extract or De Man–Rogosa–Sharpe (MRS) broth, at pH 5.6 and 43°C. All supplementations of the whey and whey permeate basal media resulted in the enhancement of the specific growth rates, rate of lactic acid production and β-galactosidase activity. However, unsupplemented skim milk gave the greatest rate of lactic acid production (3.50±0.269 mg lactic acid ml⁻¹ media h⁻¹) and the highest β-galactosidase activity (5.491±0.116 U activity ml⁻¹ media); far superior to the best whey-based medium supplemented with MRS (2.71±0.176 mg lactic acid ml⁻¹ media h⁻¹ and 3.091±0.089 U activity ml⁻¹ media, respectively). A technologically feasible approach for the reprocessing of the spent skim milk was tested and a conceptual process scheme is proposed.     

Effect of Medium Composition on Glucoamylase Production During Batch Fermentation of Recombinant Saccharomyces cerevisiae

Plasmid stability of the recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) strain harboring a pGAC9 plasmid with glucoamylase genes has been investigated in shake flasks and in a bioreactor system using various compositions of media containing glucose or starch as the main carbon and energy source. The medium composition affected both the growth characteristics of S. cerevisiae and stability of the plasmid. Superior plasmid stability was obtained in yeast minimal medium and in complex medium with 0.5 to 2% D‐glucose. Plasmid stability of 92% was obtained in complex medium with 2% D‐glucose yielding 48 units of glucoamylase/g of cells compared to 54% plasmid stability achieved with 2% soluble starch, which yielded 23 units of glucoamylase/g of cells. The plasmid stability increased at high growth rates and decreased with increasing starch concentration in the complex media as compared to glucose medium. The kinetic characteristics of biomass and glucoamylase production were investigated, and a growth kinetic model was used to interpret the experimental results.     

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.     

Production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation using spent brewing grains as substrate

BACKGROUND: The optimisation of nutrient levels for the production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation (SSF) with spent brewing grains (SBG), an inexpensive substrate and solid support, was carried out using response surface methodology (RSM) based on Plackett–Burman design (PBD) and Box–Behnken design (BBD). RESULTS: In the first optimisation step a PBD was used to evaluate the influences of related factors. Corn steep liquor, CaCl₂ and MgSO₄ were found to be the most compatible supplements to the substrate of SBG and influenced α‐amylase activity positively. In the second step the concentrations of these three nutrients were optimised using a BBD. The final concentrations (g/g dry substrate basis) in the medium optimised with RSM were 1.8% corn steep liquor, 0.22% CaCl₂ and 0.2% MgSO₄ · 7H₂O using SBG as the solid substrate. The average α‐amylase activity reached 6186 U g^?1 dry substrate under the optimised conditions at 30 °C after 96 h. Under the optimised conditions of SSF an approximately 17.5% increase in enzyme yield was observed. CONCLUSION: SBG was found to be a good substrate for the production of α‐amylase by A. oryzae As 3951 under SSF. Copyright © 2007 Society of Chemical Industry     

Functional Expression of the Raw Starch‐Binding Domain of Bacillus sp. Strain TS‐23 α‐Amylase in Recombinant Escherichia coli

Two DNA fragments encoding the starch‐binding domain (SBD) of Bacillus sp. strain TS‐23 α‐amylase were prepared by polymerase chain reaction and cloned into the Escherichia coli expression vector, pQE‐30, to generate pQE‐N428/C607 and pQE‐N465/ C607. In isopropyl‐β‐D ‐thiogalactopyranoside (IPTG)‐induced E. coli strain M15 harboring these expression plasmids, the recombinant SBDs (N428/C607 and N465/C607) could comprise up to 20% of the total soluble proteins. The His‐tag/SBD fusion proteins were purified to homogeneity with a His‐bind affinity column and had molecular masses of approximately 22.6 and 16.5 kDa, respectively. Starch‐binding assays revealed that about 7.1 and 8.3 μg, respectively, of N428/C607 and N465/C607 were bound by 1 mg of raw corn starch, indicating that the SBD of Bacillus sp. strain TS‐23 α‐amylase retain sufficient function in the absence of a catalytic center.     

SCREENING FOR PECTINOLYTIC CANDIDA YEASTS: OPTIMIZATION AND CHARACTERIZATION OF THE ENZYMES

In screening 72 Candida strains for extracellular enzymes, one acidic and seven alkaline proteases, one amylase and six pectinases were found. Candida kefyr, Candida macedoniensis and four strains from Candida pseudotropicalis were pectinolytically active; Candida kefyr showed considerable activity also under aerobic conditions but the highest activities were attained under strictly anaerobic conditions with N₂ gassing. YNB and peptone were the best nitrogen sources as regards enzyme production. The highest enzyme activities were achieved with succinic acid (aerobic) and inulin (anaerobic) as carbon sources. Enzyme production under aerobic conditions was considerably increased at pH 3.0. The addition of inducers (pectins or pectin ballast substances) led only to very slightly increased enzyme production. From kinetic studies of the enzyme, optimum activity was found to lie at pH 5.0 and 50°C. Subsequent characterization of the enzyme showed it to be an endo-polygalacturonase. Eight to ten bands could be resolved by analytical isoelectric focussing. The K_m value was found to be 1.14 × 10^?5 mol. The maceration test, using potato, carrot and apple tissue as substrate, showed considerable maceration activity, especially in the case of apple.     

The impact of oxygen availability on stress survival and radical formation of Bacillus cereus

Both the growth and stress survival of two model Bacillus cereus strains, ATCC 14579 and ATCC 10987, were tested in three different conditions varying in oxygen availability, i.e., aerobic, microaerobic and anaerobic conditions. Both B. cereus strains displayed highest growth rates and yields under aerobic conditions, whereas the microaerobic and anaerobic cultures showed similar reduced growth performances. The cells grown and exposed microaerobically and anaerobically were more resistant to heat and acid than cells that were cultured and exposed aerobically. On the other hand, the anaerobically grown cells were more sensitive to hydrogen peroxide compared to the (micro)aerobically grown cells. The increased heat- and acid-induced inactivation in aerobic conditions appeared to be associated with intracellular accumulation of excess hydroxyl and/or peroxynitrite radicals, as determined by flow cytometry in combination with the fluorescent reporter dye 3′-(p-hydroxyphenyl) fluorescein. This suggests that radical formation may contribute to inactivation of bacteria in the presence of oxygen, such as in aerobic and microaerobic conditions. No evidence was found for radical formation upon exposure to salt and hydrogen peroxide. The increased resistance to heat and acid in microaerobic and anaerobic conditions shows that oxygen availability should be taken into account when behavior of bacteria, such as B. cereus, in food industry related conditions is investigated, because oxygen availability may affect the efficiency of food preservation conditions.     

Modelling of the Enzymatic Hydrolysis of Potato (Solanum tuberosum) Using Response Surface Methodology

Starch hydrolysates containing glucose can be obtained from potato (Solanum tuberosum). Acid hydrolysis generates a solution that must be neutralized to be used for fermentative purposes while an enzymatic hydrolysis generates a glucose solution which can be used without further treatments. This work deals with the modelling of the enzymatic hydrolysis of dried potatoes with α‐amylase and glucoamylase with temperature, time, activity units/substrate ratio and α‐amylase activity fraction as operational variables and released glucose concentration as dependent variable. The model obtained showed that the optimal conditions were 70ºC, 60 min; 74 activity units/g substrate and 0.13 α‐amylase activity U/total U fraction. Using these conditions, a glucose concentration of 38.9 g/L was predicted which corresponded well with an experimental value of 38.0 ± 0.5 g/L. The model obtained can be used in the development of processes for the hydrolysis‐fermentation of potato for the production of food additives or ethanol.     

ISOLATION AND IDENTIFICATION OF A NOVEL ASPERGILLUS JAPONICUS JN19 PRODUCING β-FRUCTOFURANOSIDASE AND CHARACTERIZATION OF THE ENZYME

A novel strain, Aspergillus sp. JN19, producingβ‐fructofuranosidase (FFase), was isolated from soil. According to the physiological and biochemical characteristics and its 18S rDNA gene sequence analysis, it was identified as Aspergillus japonicus. The optimal conditions for production of fructofuranosidase by A. japonicus JN‐19 were investigated. The initial concentration of sucrose was 15 to 18%. Yeast extract was the best nitrogen source. K₂HPO₄ was effective in increasing enzyme production. The enzyme activity was increased to about 1.3 times by addition of 0.2% carboxymethylcellulose in the medium. The highest FFase activity was 55.42 U/mL at pH 5.5 and 30C, and production yield of fructooligosaccharides was 55.8%. Some characteristics of purified FFase were also studied.     

Isolation of a Recombinant Bacillus sp. TS‐23 α‐Amylase by Adsorption‐Elution on Raw Starch

A Bacillus sp. TS‐23 α‐amylase produced by recombinant Escherichia coli was adsorbed onto raw starch and the adsorbed enzyme was eluted with maltose or maltodextrin in 50 mM Tris/HCl buffer (pH 8.5). The adsorption‐elution procedure resulted in a yield of 53% α‐amylase activity and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS/PAGE) analysis showed that the eluted α‐amylase had a molecular mass of approximately 64 kDa. Raw starch could be used repeatedly in the adsorption‐ elution cycle with good reproducibility. Scanning electron microscopy of the isolated corn starch exhibited a smooth appearance of the granules before adsorption and only a small change in appearance after three adsorption‐elution cycles. These results suggest that the raw starch adsorption‐elution technique has a great potential in the isolation of Bacillus sp. TS‐23 α‐amylase from the culture broth of recombinant E. coli.     

基于低拷贝质粒的高产紫色杆菌素谷氨酸棒杆菌的构建和发酵

该研究以公认安全(Generally Recognized as Safe,GRAS)的谷氨酸棒杆菌(Corynebacterium glutamicum)为宿主,构建高产紫色杆菌素的重组菌株。利用谷氨酸棒杆菌天然大质粒pTET3的复制与分配元件,构建了低拷贝质粒pOK12CG1,该质粒在谷氨酸棒杆菌中的拷贝数约为6拷贝/基因组,且与谷氨酸棒杆菌常用质粒pEC-XK99E和pXMJ19兼容。以低拷贝质粒pOK12CG1为骨架构建了携带紫色杆菌素合成操纵子(vioABCDE)的质粒pCGvio,并分别以谷氨酸棒杆菌标准株ATCC 13032和插入序列(Insertion Sequence,IS)元件删除株为宿主,构建了7株合成紫色杆菌素的重组菌株。通过初步筛选,发现基于低拷贝质粒的重组菌株ATCC13032/p CGvio,其紫色杆菌素产量(508.24 mg/L)高于基于中高拷贝质粒的重组菌株ATCC 13032/pECvio(376.16 mg/L),而基于低拷贝质粒的IS元件删除重组菌株ISDM023/pCGvio紫色杆菌素产量达到了610.13mg/L。进一步采用正交实验设计对重...     

Activation of Lactoperoxidase System in Milk by Glucose Oxidase Immobilized in Electrospun Polylactide Microfibers

ABSTRACT: In this study, glucose oxidase (GOX) was immobilized in polylactide (PLA) fibers that were used to activate the lactoperoxidase (LP) system in milk. The GOX‐containing microfibers were electrospun from emulsions prepared by dispersing aqueous GOX in PLA dissolved in a chloroform and N,N‐dimethylformamide blend, using sorbitan monopalmitate as an emulsifier. The enzymatic activity of GOX‐in‐PLA fibers (1100 ± 400 nm diameter) was more than 19 times higher than that of the GOX‐in‐PLA membrane formed by direct casting, due to the larger surface area of the electrospun fibers. The activation of LP in model solutions using GOX‐in‐PLA fibers provided a more sustained generation of antimicrobial OSCN^? than direct activation using H₂O₂. Preliminary evaluation on milk samples showed that the electrospun GOX‐in‐PLA microfibers are capable of activating the naturally present LP system, indicating that they may be promising for active food packaging applications to extend the shelf life of milk.     

Evaluation of the conditions of carotenoids production in a synthetic medium by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor

This work studied the cultivation conditions for the production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor. A Plackett–Burman design was used for the screening of the most important factors, followed by a complete second order design, to maximise the concentration of total carotenoids. The maximum concentration of 3425.9 μg L^?1 of total carotenoids was obtained in a medium containing 80 g L^?1 glucose, 15 g L^?1 peptone and 5 g L^?1 malt extract, with an aeration rate 1.5 vvm, 180 r.p.m., 25 °C and an initial pH of 4.0. Fermentation kinetics showed that the maximum concentration of total carotenoids was reached after 90 h of fermentation. Carotenoid bio‐production was partially associated with cell growth. The specific carotenoid production (Y_P/X) was 238 μg carotenoids/g cells, whereas Y_P/S (substrate to product yield) was 41.3 μg g^?1. The specific growth rate (μ_x) was 0.045 h^?1. The highest cell and total carotenoid productivity were 0.19 g L^?1 h^?1 and 56.9 μg L^?1 h^?1, respectively.