Formation of glycosidases in batch and continuous culture of Bacteroides fragilis.

Nine strains of bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce glycosidases. B. fragilis subsp. vulgatus B70 was used for optimizing the production of glycosidases. The highest bacterial yield was obtained in proteose peptone-yeast extract medium. The optimum pH for maximal bacterial yield was 7.0, and the optimum temperature for growth was 37 degrees C. The formation of glycosidases was optimal between pH 6.5 and 7.5, and the optimum temperature for synthesis of glycosidases was between 33 and 37 degrees C. Culture under controlled conditions in fermentors gave more reproducible production of glycosidases than static cultures in bottles. The strain was also grown in continuous culture at a dilution rate of 0.1 liter/h at pH 7.0 and 37 degrees C with a yield of 2.0 mg of dry weight per ml in the complex medium. The formation of glycosidases remained constant during the entire continuous process.     

Growth of Bacteroidaceae in Stirred Fermentors

The conditions for increasing bacterial yields in cultures of Bacteroidaceae by the use of stirred fermentors and pH control were investigated by means of three representative species: Sphaerophorus necrophorus, Bacteroides fragilis, and B. melaninogenicus. A medium containing tryptone, yeast extract, and glucose or sucrose was used. Horse serum had to be added to obtain substantial growth of B. melaninogenicus. The optimal pH for growth rate and yield was 7.0 to 7.2. Lysis of the bacteria occurred when the glucose (or sucrose) was exhausted. The rate of lysis was very high in cultures of S. necrophorus, less so in B. fragilis and B. melaninogenicus. Pleomorphism, manifested as large sperical forms of the bacteria, was observed in the late logarithmic phase of S. necrophorus. Great differences in the length of the lag phase and of the mean generation time were found among the three bacterial species. The yield in static cultures of the three species without pH control was approximately 0.4 g of dry cells per liter, but was increased, in stirred fermentors with pH control, to 3.5 g (S. necrophorus), 2.7 g (B. fragilis), and 4.3 g (B. melaninogenicus) per liter. With an inoculum density of 5 to 10 mg (dry weight) per liter, these yields were obtained in approximately 10 (S. necrophorus), 25 (B. fragilis), and 35 hr (B. melaninogenicus), respectively.     

Effects of temperature and phosphorous concentration on microbial sulfate reduction by Desulfovibrio desulfuricans

The effects of temperature and phosphorous concentration on the rate and the extent of microbial sulfate reduction with lactate as carbon and energy source were investigated for Desulfovibrio desulfuricans. The continuous culture experiments (chemostat) were conducted at pH 7.0 from 12 to 48 degrees C. The maximum specific growth rate (mu(max)) was relatively constant in the range 25 degrees C-43 degrees C and dramatically decreased outside this temperature range. The half-saturation coefficient was minimum at 25 degrees C. Cell yield was highest in the optimum temperature range (35 degrees C-43 degrees C) for growth. Maintenance energy requirements for D. desulfuricans were not significant. Two moles of lactate is consumed for every mole of sulfate reduced, and this stoichiometric ratio is not temperature dependent. Steady state rate and stoichiometric coefficients accurately predicted transient behavior during temperature shifts. The extent of extracellular polymeric substance (EPS) is related to the concentration of phosphorous in the medium. EPS production rate increased with decreased phosphorous loading rate. Failure to discriminate between cell and EPS formation by D. desulfuricans leads to significant overestimates of the cell yield. The limiting C:P ratio for D. desulfuricans was in the range of 400:1 to 800:1.     

产胞外邻苯二酚1.2—双加氧酶的菌种筛选和发酵条件的研究

Two strains of Pseudomonus sp. having the extracellular catechol 1, 2-dioxygenase activity were selected from 112 bacterial strains. The conditions for enzyme production of the strains were examined. The optimal temperature and pH for enzyme formation were 30 degrees C and pH 6.8-7.0 respectively. Enzyme formation was enhanced by sodium benzoate, and was markedly inhibited by glucose, maltose and glycerol. Ammoniacal nitrogen sources were essential for cell growth and enzyme production. Sodium succinate was an effective inducer for enzyme formation. When the organism was grown in 0.15% sodium benzoate medium (pH 6.8-7.0) at 30 degrees C for 72 hours, about 10 units of catechol 1,2 dioxygenase per ml was obtained.     

Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K

The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.     

Optimization of extracellular thermotolerant alkaline protease produced by marine <Emphasis Type="Italic">Roseobacter</Emphasis> sp. (MMD040)

Marine endosymbiontic Roseobacter sp. (MMD040), which produced high yields of protease, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in Luria-Bertani broth. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 37 degrees C and 7.0, respectively. The enzyme exhibited maximum activity in pH range of 6-9 with an optimum pH of 8.0 and retained nearly 92.5% activity at pH 9.0. The enzyme was stable at 40 degrees C and showed 89% activity at 50 degrees C. Based on the present findings, the enzyme was characterized as thermotolerant alkaline protease, which can be developed for industrial applications.     

Growth of Bacteroides fragilis in Continuous Culture and in Batch Cultures at Controlled pH

Bacteroides fragilis NCTC 9343 has been grown in continuous cultures with glucose as growth-limiting factor. At pH 7.0 and at a dilution rate of 0.07 per h, glucose limited growth in concentrations up to 0.6%. Maximal cell yield and productivity were obtained with 0.87% glucose in the inflowing medium. A pH of 7.0 was optimal for growth. With 0.6% glucose in the fresh medium and at pH 7.0, cell yield and productivity were highest at a dilution rate of 0.07 per h and 0.11 per h, respectively. At dilution rates higher than 0.07 per h, glucose was no longer growth limiting, and at dilution rates above 0.11 per h, another compound seemed to have replaced glucose also as energy source. When grown in batch cultures at pH 7.0, the best yields of B. fragilis was achieved with 0.6% glucose in the fresh medium. The highest specific growth rate (mum) determined from viable counts was 0.45, corresponding to a mean generation time of 92 min.     

Effect of water activity and pH on growth and toxin production by Clostridium botulinum type G.

The combined effect of water activity (aw) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated. The minimum aw at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose. The minimum pH (at the optimum aw) for growth and toxin production of C. botulinum type G was found to be 5.6. Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37 degrees C. These data did not show evidence of heat-labile spores, since a heat shock of 75 degrees C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6. It was frequently observed that cells grown at reduced aw or pH experienced severe morphological changes.     

Effect of water activity and pH on growth and toxin production by Clostridium botulinum type G

The combined effect of water activity (aw) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated. The minimum aw at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose. The minimum pH (at the optimum aw) for growth and toxin production of C. botulinum type G was found to be 5.6. Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37 degrees C. These data did not show evidence of heat-labile spores, since a heat shock of 75 degrees C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6. It was frequently observed that cells grown at reduced aw or pH experienced severe morphological changes.     

Effect of temperature and pH on immobilized Zymomonas mobilis for continuous production of ethanol

The effects of temperature and inlet pH of the medium on the ethanol productivity and activity of the immobilized Z. mobilis cells during continuous fermentation of glucose have been studied at various temperatures and pH. On changing the temperature from one steady state level to a new one, 6-8 h were required in order to fully experience the effect of a change in temperature; whereas 8-20 h were required on changing the pH. The optimum temperature of 37 degrees C and a broad pH range of 4.4-6.0 were observed for maximum ethanol productivity and ethanol yield.     

Optimization of extracellular psychrophilic alkaline lipase produced by marine Pseudomonas sp. (MSI057)

An endosymbiotic Pseudomonas sp. (MSI057), which could produce high yields of lipase, was isolated from marine sponge Dendrilla nigra, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% tributyrin. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 30 degrees C and 9.0, respectively. The enzyme exhibited maximum activity in pH range of 8-9 with an optimum pH 9.0. The activity of purified enzyme was optimum at 37 degrees C and showed 80% activity at 20 degrees C and the enzyme activity decreased dramatically above 50 degrees C. Based on the present findings, the enzyme was characterized as psychrophilic alkaline lipase, which can be developed for industrial applications.     

Purification and properties of invertase from the flowers of Woodfordia fruticosa

Invertase (beta-fructofuranosidase, EC 3.2.1.26) was purified from the flowers of Woodfordia fruticosa, which is used to prepare certain fermented Ayurvedic drugs. The enzyme was purified to near homogeneity as judged by native PAGE with a yield of 10.7%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.8 and 4.42. The molecular mass of the purified enzyme as determined by elution through Sepharose 4B gel column was found to be approximately 280 kDa. SDS-PAGE of the purified enzyme showed that the enzyme is composed of three subunits with molecular mass of 66, 43 and 40 kDa. The enzyme showed a broad pH optimum between 4.0-7.0. Optimum assay temperature was 37 degrees C and above 45 degrees C, the enzyme activity slowly declined and inactivated around 80 degrees C. The apparent Km value of the enzyme for sucrose was 160 mM.     

Production and partial characterization of elastase of Bacillus subtilis isolated from the cervices of human females

Conditions are described for the production of extracellular elastase by Bacillus subtilis. The yield of enzyme was maximum in shake-cultures grown in Syncase medium at 37 degrees C and was stable in culture supernatants. The enzyme, purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration, showed a molecular weight of 25,000 and activity between pH 6.0 and 9.5, with an optimum of 9.0 in Tris-maleate buffer. Elastinolytic activity was maximum in glycine-NaOH buffer and minimum in phosphate buffer. Enzyme activity was adversely affected by temperature greater than or equal to 40 degrees C.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity

The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days. Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography. Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C. The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively. The production of fumitremorgin C by N. fischeri has not been previously reported. Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C. Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C). Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose. Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose. Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C.     

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity.

The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days. Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography. Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C. The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively. The production of fumitremorgin C by N. fischeri has not been previously reported. Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C. Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C). Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose. Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose. Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C.     

鞘胺醇杆菌肝素酶的产生

肝素类分子是一类结构异常复杂的高度硫酸化的糖胺聚糖,是临床上的一种主要的抗凝剂。除了抗凝血及其相关的抗栓活性以外,肝素还具有多种其他生物学功能,如抗平滑肌细胞及肾小球系膜细胞的增殖^[1]、抗炎症^[2]和阻止肿瘤生长及转移的作用^[3]等。因而肝素可用于防治球囊扩充血管成型术后的血管再狭窄、肾小球系膜细胞增生性肾炎、病理性炎症及肿瘤。但由于完整肝素的抗凝血活性,会引起出血及血小板减少综合症等负作用,限制了肝素在这些方面的临床应用。研究表明,肝素的抗凝血活性依赖于一种独特的肝素五糖序列,约占完整肝素链的1／3,除了五糖序列以外,还需要附近的至少13糖残基,因而不含五糖序列或含五糖序列小于18糖的肝素其抗凝活性大大降低^[4]。而六糖以上的肝素片段就具有抗平滑肌细胞增生、抗炎症及抗肿瘤的活性,并且这些活性与抗凝活性无关。因此,可利用特异性的肝素酶降解肝素长链,产生一系列不同结构及大小的肝素片段,并从中筛选出具有不同生物活性的片段。     

Expression, enzymatic characterization, and high-level production of glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) in Escherichia coli

High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0-9.0. Its optimum temperature was around 85 degrees C, and it exhibited good thermostability when the temperature was lower than 90 degrees C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 microM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool.     

肝素酶产生菌的筛选及发酵条件

从土壤中筛选到一株活性较高的肝素酶产生菌株Corynebacterium sp.。培养及产酶最佳条件表明,最适培养基组成(g/L)：胰蛋白胨20,氯化钠1,磷酸氢二钾25,硫酸镁05,麦芽糖20,pH65。最适生长温度27℃,最佳产酶温度31℃。在500mL三角瓶中装40mL培养基,在30℃,200r/min摇床上培养24 h,每升发酵液可产酶1700u。