Effect of culture media on protease and oxytetracycline production with mycelium and protoplasts of Streptomyces rimosus

For the simultaneous production of protease and oxytetracycline, mycelium and protoplasts of Streptomyces rimosus TM-55 were cultivated in basal medium containing soluble starch, corn steep liquid, ammonium sulphate, calcium carbonate, sodium chloride and soybean oil. Protease and oxytetracycline production increased with decreasing in ratio of culture broth to vessel volume from 1:2 to 1:5. Each ml of broth with 0.286 mg fresh mycelia yielded 168–204 units of protease and 785–972 g of oxytetracycline after replacement of corn steep liquor, sodium chloride and soybean oil with beef extract and sunflower oil, while each ml of broth with 7.5 × 10⁷ protoplasts produced 141–153 units of protease and 504–615 g of oxytetracycline. Protease and oxytetracycline production were low when the pH was 5.1 or 9.0. Soluble starch and ammonium sulphate were the best carbon and nitrogen sources, respectively. Supplementation with calcium carbonate enhanced protease and oxytetracycline production. The productivity of protoplasts decreased sharply when the incubation temperature increased from 28 to 34 °C, while the productivity of mycelium was almost unchanged.     

Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans

A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern in Qingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2 U/ml) was obtained in a medium containing 2.5 g soluble starch and 2.0 g NaNO(3), 100ml seawater, initial pH 6.0, after fermentation at 24.5 degrees C for 30 h. The protease had the highest activity at pH 9.0 and 45 degrees C.     

Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease

While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth) was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production.     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

Production of alkaline protease by a genetically engineered Aspergillus oryzae U1521

The production of alkaline protease of Aspergillus oryzae U1521 was examined in liquid culture. In a culture of defatted soybean only, it gave satisfactory enzyme yields at 584,000 U/g defatted soybean. When various carbohydrates were supplemented, enzyme production was significantly increased. An increase in production by lactose was the most marked. Enrichment with casitone or casein increased productivity, but not cornsteep solid. Media formulation (g/L) of defatted soybean 10, lactose 5, casitone 1, and KH(2)PO(4) 5 enhanced alkaline protease production by A. oryzae U1521 to a maximum of 1,410,000 U/g defatted soybean. Scaling-up experiments indicated the flask-scale results could be reproduced at 40 g of substrate in 5-L fermenter. The enzyme activity was maximum between pH 8-9 and at a temperature of 45 degrees C.     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14

Statistical optimization of culture conditions for production of a widely suited detergent protease from Bacillus sp. RGR-14 was carried out using a two-step approach. A quick identification of the important factors with simple screening experiment was followed by application of complex response surface design for further optimization. The production of extracellular alkaline protease by Bacillus sp. was favored in the presence of complex carbon and nitrogen sources, viz. starch, casamino acid and soybean meal. A reduced quadratic model was found to fit the alkaline protease production. Response surface analysis revealed the significant role of phosphate ions in determining alkaline protease production. A steep, stretched out response surface showed direct relation between the level of protease production and casamino acid and starch concentration in the medium. A 12.85 fold increase in protease production could be obtained within the design space. Protease production was found to be repressed in the presence of high concentrations of casamino acid. The model could be validated in up to 2 l shake flasks (3914 U ml⁻¹). The same statistical design could explain economic protease production in cost-effective medium as well.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.     

Isolation of Alkaline Protease Inhibitor Producing Microorganisms

For the purpose of obtaining microorganisms capable of producing alkaline protease inhibitor, screening test was carried out. One strain of microorganisms (No. S–3253) showed strong ability to produce alkaline protease inhibitor.

The morphological and physiological characteristics of strain S–3253 were studied. This strain was found to belong to Streptomyces albogriseolus.

When the strain S–3253 was cultivated at 45°C with a medium containing 4% polypepton, 2% starch, 0.1% yeast extract, 0.1% NaCl, 0.1% K₂HPO₄ and 0.05% MgSO₄-7H₂O in shake-flasks (pH 7.0), the highest activity was obtained after 40~48 hr cultivation.     

Comparison of culture methods for human-human hybridomas secreting anti-HBsAg human monoclonal antibodies

Human-human hybridomas which secrete a human monoclonal antibody (h-MoAb) against hepatitis B virus surface antigen showed growth associated production kinetics. The rate of h-MoAb production rapidly decreased after cell growth was arrested in a perfusion culture, even if the perfusion rate was increased. A continuous suspended-perfusion culture, in which both culture broth and culture supernatant are continuously harvested and the same volume of fresh medium is continuously fed into the reactor, was developed to maintain continuous growing conditions during cultivation. In this culture system, the production of h-MoAb continued for more than 50 days with an average productivity of 5.0 mg/l of working volume/day. A semicontinuous immobilized-perfusion culture in which parts of the cells are repeatedly removed from the immobilized reactor was another useful technique for the long term cultivation of these h-h hybridomas. As an average h-MoAb production rate, 62 mg/l of immobilized-bed volume/day was achieved for 65 days of cultivation using a ceramic matrix reactor, and 327 mg/l/day was achieved over 47 days of cultivation using a hollow fiber reactor equipped with Cultureflo M^TM Thus, the antibody productivity per reactor volume per day by the semicontinuous immobilized-perfusion culture was much higher than that of the continuous perfusion culture in an agitation reactor.     

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.     

Protease from Sporosarcina sp. RRLJ 1

Protease was isolated from Sporosarcina RRLJ1 which was collected from acid tea (Camellia sinensis) plantations. It showed potential for production of the enzyme for commercial purposes. The study revealed that optimum pH for growth of the organism was 6.5-7 and supplement of casein (1%) in the medium was required for production of protease. Enzyme production and enzyme activity was maximum in 72 hr old broth culture. Maximum activity of the enzyme was found at pH 6.5.     

恒细胞密度发酵策略提高重组毕赤酵母生产碱性果胶酶的表达效率

为了提高重组毕赤酵母生产碱性果胶酶(Alkaline polygalacturonate lyase,PGL)的比速率,开发了一种新的恒细胞密度发酵策略。通过不同的甲醇流加方式,实现发酵过程细胞密度的合理控制。实验结果表明:控制细胞密度为75 g/L的策略为最优,最终单位发酵液体积生产强度和单位菌体生产强度为6.11 U/(mL.h)和81.5 U/(g.h),分别比传统高密度发酵提高了42.1%和191.2%,最终PGL酶活为441.9 U/mL。此外,该策略还具有提高细胞活性和降低蛋白酶降解作用等优势。     

Protease Production by Species of Entomophthora

Ten insect-pathogenic species of Entomophthora showed wide variation in their ability to produce alkaline protease in surface culture. E. coronata, the most active producer, was selected for studies in submerged culture together with E. virulenta. All media tested appeared suitable for mycelial growth of these two organisms, but a liver medium was superior for the production of protease. The effect of the constituents of the liver medium upon yield was investigated. The lag between growth and the production of protease was 24 to 40 hr, and only very small amounts of protease were obtained from sonically treated mycelium. The pH values during growth rose from ranges of 4.5 to 7.5 in the initial medium to 7.2 to 7.9, and did not affect the final yields. The optimal temperature for the production of protease by E. coronata was 24 to 32 C, and good growth was observed at temperatures as low as 16 C. The process with E. coronata was scaled up to fermentors without a decrease in yield; 5 enzyme units/liter were obtained after approximately 33 hr. This corresponds to a maximal productivity of 0.45 enzyme unit per liter per hr during the protease-producing phase. The process was insensitive to changes in aeration rate. The liver in the medium was replaced by various agricultural by-products, meat scrap, rapeseed oil meal, cottonseed nutrients, milk powder, and meat hydrolysate, with approximately the same or higher yields of protease.     

Isolation and Identification of Microorganism,Producing Microbial Alkaline Proteinase Inhibitor (MAPI)

In the screening of actinomycetes’ culture filtrate for inhibitor of subtilisin and various microbial alkaline proteinases, a novel inhibitor was found in a cultured broth of strain WT-27. This inhibitor was named as MAPI, abbreviation of microbial alkaline proteinase inhibitor.Judging from the morphological and physiological properties of the actinomycetes which produced MAPI, this strain was identified as Streptomyces nigrescens.

For the production of MAPI, this strain was aerobically cultured at 25 ~ 27%C in a jar fermentor which contained an optimum medium consisting of polypepton 3 %, meat extract 1%, glucose 1%, NaCl 0.1%, K₂HPO₄ 0.1% and MnSO₄·nH₂O 0.0001%, pH 7.0. The production of MAPI reached its maximum after 21 ~ 24 hr cultivation.

MAPI had an inhibitory activity against various microbial alkaline proteinases, α-chymotrypsin and papain but not against trypsin, kallikrein, thermolysin, or pepsin.     

Production of Thermostable Alkaline Proteases by Thermophilic Streptomyces

Conditions for the production of thermostable proteases (alkaline proteinase and carboxypeptidase) by a thermophilic streptomycete (Streptomyces rectus var. proteolyticus) were investigated in 20-liter volumes. Proteinase production was affected by the concentration of defatted soybean powder, its optimum being 1.2% in medium containing 2.0% soluble starch. Relatively high concentration of phosphate (0.3 to 0.4% K(2)HPO(4)) was needed for the maximum enzyme production. A large inoculum size (5 to 10%) was favorable, but the inoculum age did not significantly influence the production. The yield increase of 20 to 30% was obtained by feeding of medium during fermentation. The optimal temperature for proteinase production was 50 C, at which the maximal rate of production was 66.2 proteinase units per ml per hr, whereas at 40 C it was 9.0. Production at 50 C reached the maximum within 12 to 16 hr. The optimal agitation rate was different for the production of proteinase and carboxypeptidase, 400 rev/min for the former and 500 rev/min for the latter. The optimal aeration for proteinase production was 20 to 30 liters/min at 400 rev/min, whereas carboxypeptidase production was not markedly affected by aeration rate. The possibility that carboxypeptidase production was correlated with the shear of mycelium was discussed.     

Optimization of culture conditions for production of the anti-tubercular alkaloid hirsutellone A by Trichoderma gelatinosum BCC 7579

AIMS: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. METHODS AND RESULTS: The fungus was initially cultured in shake flasks at 25 degrees C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l(-1), obtained from basal condition, to 610.55 mg l(-1) and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min(-1) at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l(-1) of hirsutellone A within 6 days. CONCLUSIONS: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with an initial pH of 7. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future.     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

Optimization and enhanced production of α-amylase and protease by a newly isolated Bacillus licheniformis ZB-05 under solid-state fermentation

Eight different agro-residues were tested for α-amylase and protease production by using Bacillus licheniformis ZB-05. Among them, rice husk (RH) was proved as the best substrate for two enzymes (α-amylase 443 U/g and protease 469,000 U/g). Maximum enzyme production was observed to be 30 % initial moisture, with a growth period of 36 h in 20 and 30 % inoculum volumes for α-amylase and protease, respectively. The best enzyme recovery from solid mass was obtained when extracted with tap water. Among the tested various nitrogen sources, 1 % ammonium sulphate followed by 2 % Bacto liver, 2 % ammonium sulphate and 1 % Bacto casaminoacid served as the best inorganic and organic nitrogen sources for α-amylase and protease production, respectively. As additional carbon sources, 2 % soluble starch enhanced α-amylase production, while 1 % maltose enhanced protease production.