Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.     

Optimization of extracellular xylanase production by Dictyoglomus sp. B1 in continuous culture

The thermophilic, xylanolytic, anaerobic organism, Dictyoglomus sp. B1, was cultivated in batch and continuous cultures in media containing insoluble beech-wood xylan. The extracellular xylanase activity levels obtained for the two cultivation methods were compared. Experiments were performed separately to determine the optimum substrate concentration, dilution rate, pH and temperature for xylanase production. Maximum xylanase activity was found at a substrate concentration of 1.5 g xylan/l, a dilution rate of 0.112 h^–1, pH 8.0 and at 7°C. Different combinations of these optimum values were used in a 2³ factorial experiment to investigate whether an increase in the xylanase production/activity could be achieved. A maximum xylanase activity of 2312 U/l was found when fermentors were operated at 73°C with a substrate concentration of 1.5 g xylan/l, pH 8.0, and a dilution rate of 0.112 h^–1. Thus, the optimum xylanase activity in the factorial experiment was obtained when the conditions that gave the maximum xylanase activities in the individual experiments were combined. Optimum xylanase activity obtained in the 2³ factorial experiment was 6.2 times higher than the activity found in the initial batch culture (373 U/l) and 3.0 times higher than the activity of a batch culture (783 U/l) grown at the same optimum conditions as the factorial experiment. The higher specific xylanase activity (217 U/mg protein) found in the 2³ factorial experiment was 4.1 times higher than the specific activity in the initial batch culture (53 U/mg protein).     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Influence of culture conditions on lipase production byBacillus sp. FH5

Lipases are a class of enzymes, which catalyse the hydrolysis of long chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases have industrial potential in the chemical, pharmaceutical, medical, cosmetic, leather and paper manufacturing industries, biosurfactant synthesis, and agrochemicals. ABacillus strain isolated from soil was tested for the production of extracellular lipase, by batch culturing in shake flask. The growth conditions were optimised for the maximum production of enzyme. Various parameters for the production of lipase, such as temperature, incubation period, pH, carbon source, nitrogen source and lipids were studied. Maximum lipase production was found in 48-h-old culture filtrate at 37 °C, pH 8.0. Among all the carbon sources, salicin gave the maximum activity and among all the nitrogen sources yeast extract gave maximum production/activity. Tween (20 and 80) does not stimulate the growth much but assisted in enzyme production.     

Glucose repression of enterotoxins A, B and C and other extracellular proteins in staphlyococci in batch and continuous culture.

The production of enterotoxins, lipase and total extracellular protein by four strains of Staphylococcus aureus grown in batch culture at a controlled pH of 6.5 in a completely defined medium was markedly reduced by glucose or glycerol constantly maintained at 0.I M. A concomitant increase in the production of deoxyribonuclease, up to 13-fold, showed however that not all extracellular proteins are under the same control mechanism. The presence of glucose and glycerol in the medium also resulted in a rapid increase in the specific growth rate. However, growth of S. aureus s6 in Mgilimited continuous culture showed that glucose repression of enterotoxin B when the growth rate was held constant was more than twice that in batch culture. Therefore glucose repression can occur independently of an increase in growth rate. The specific rate of production of enterotoxin B, lipase, deoxyribonuclease, beta-haemolysin and total extracellular protein by S. aureus s6 increased as the growth rate increased from 0.07 to 0.24 h-1. Non-replicating cells grown in the absence of glucose produced considerable amounts of enterotoxin, and production was not repressed by the presence of glucose in the resuspension medium. In contrast, no enterotoxin B or C was obtained from nonreplicating cells grown in the presence of glucose. Chloramphenicol completely inhibited enterotoxin production by non-replicating cells, indicating that synthesis of new protein was required.     

脂肪酶产生菌Candida rugosa产酶条件研究

脂肪酶(Lipase,EC3.1.1.3)是用来催化酯类化合物的分解、合成和酯交换的特殊酶,具有高度的化学选择性和立体异构性,它广泛应用于食品、轻纺、皮革、香料、化妆品、洗涤剂、有机合成、医药等领域.本世纪80年代,美国科学家发现酶在近无水的有机溶剂中不仅能保存其催化活力,而且还获得许多新的催化特征[1],此后,脂肪酶在非水相酶催化领域的研究和应用逐渐增多.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix thermosphacta

Lipolytic activity of lactobacilli strains and Brochothrix thermosphacta was cellrelated; no significant activity was found in the supernatant fluids. Most lipase was produced during the logarithmic phase of growth and was greatly affected by growth conditions. The optimal temperatures for growth and lipase production were respectively 24°C for B. thermosphacta and 30°C for lactobacilli. For all strains, an initial pH of around 7-0 for the medium and low glucose concentration stimulated lipase production. Tributyrin inhibited both growth and lipase production at a concentration of 0-1% for B. thermosphacta or 1% for lactobacilli. Butyric acid (0-1%) and anaerobic culture inhibited lipase production by B. thermosphacta while these two factors had no effect on enzyme production by lactobacilli.     

Optimal design for the maximization of Penicillium cyclopium lipase production

Penicillium cyclopium triacylglycerol lipase production was maximized in stationary batch culture. We used a surface response methodology based on a Doehlert experimental design to study the effect on the lipase activity released in the culture medium of the three most important factors: substrate concentration, pH and inoculum. Besides reducing the number of experiments required for optimization, this technique allowed us to quantify the lipase activity in any part of the experimental domain.We determined an optimal set of conditions for high lipase production: 1% substrate (corn steep), pH 5.5 and an inoculum of 10(4) spores/ml. Between conditions giving the minimum and the maximum lipase production, we observed a nine-fold increase of both the predicted and measured values.     

Effect of the growth rate on the enzymatic activities of L-lysine-producing cells of Corynebacterium glutamicum

The activity of 6-phosphogluconate dehydrogenase, aspartate kinase and phosphoenolpyruvate carboxylase has been studied at different dilution rates in aerobic continuous culture of Corynebacterium glutamicum. 6-Phosphogluconate dehydrogenase and aspartate kinase reached their maximum values at the lower dilution rates (0.02–0.06 h^–1), when L-lysine was produced. The phosphoenolpyruvate carboxylase activity seemed to be independent of metabolite synthesis. The production of L-lysine was also studied in non-growing cells in batch cultures. In these conditions, statistical analysis revealed significant differences in L-lysine titres when glucose or gluconic acid were used as carbon sources. Higher L-lysine concentration obtained with gluconic acid was found to be associated with a high 6-phosphogluconate dehydrogenase activity.     

Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix thermosphacta

Lipolytic activity of lactobacilli strains and Brochothrix thermosphacta was cell-related; no significant activity was found in the supernatant fluids. Most lipase was produced during the logarithmic phase of growth and was greatly affected by growth conditions. The optimal temperatures for growth and lipase production were respectively 24 degrees C for B. thermosphacta and 30 degrees C for lactobacilli. For all strains, an initial pH of around 7.0 for the medium and low glucose concentration stimulated lipase production. Tributyrin inhibited both growth and lipase production at a concentration of 0.1% for B. thermosphacta or 1% for lactobacilli. Butyric acid (0.1%) and anaerobic culture inhibited lipase production by B. thermosphacta while these two factors had no effect on enzyme production by lactobacilli.     

Production of an extracellular proteinase and α-amylase byBacillus subtilis in chemostat

The formation of two extracellular enzymes (α-amylase and a proteinase) ofB. subtilis was investigated in batch and continuous cultures. Differences were observed in the production of both enzymes in batch culture when studied in flasks and in a fermentor. The values of proteinase activity from continuous cultivation never reached those obtained during batch cultivation. The optimal dilution rate for proteinase synthesis was 0.1 – 0.2/h. The optimal dilution rate for the production of α-amylase was 0.2 –0.3/h.     

Production of extracellular lipase by Candida cylindracea CBS 6330

In this study we investigated the influences of aeration, substrate type and concentration on extracellular lipase production in a batch fermentor. The use of air enriched with pure oxygen is the most suitable for the lipase production. Additionally, we found that the presence of fats in the culture broth did not affect the value of the volumetric mass transfer coefficient of oxygen in our system. Olive oil or oleic acid was used as carbon sources. In both cases, the maximal specific rate of growth, μ_max, was the same but the highest activity was obtained when 10?g/dm³ of olive oil were used as an initial substrate concentration.     

Production and characterization of the agarase ofCytophaga flevensis

Cytophaga flevensis produced an inducible agarase which was extracellular under most conditions tested. The effect of cultural conditions on the production of enzyme was studied in batch and continuous culture. In batch culture, production was optimal whenCytophaga flevensis was incubated at 20C in a mineral medium with agar as the sole carbon source and ammonium nitrate as the nitrogen source at an initial pH of 6.6–7.0. The enzyme appeared to be subject to catabolite repression, since its synthesis was repressed when glucose was added to the medium in batch culture. Furthermore, in continuous culture, enzyme production decreased with increasing growth rate. Extracellular agarase was partially purified and the enzyme preparation obtained was very stable. The enzyme has a molecular weight of 26000 daltons. It is a β-agarase which is highly specific for polysaccharides containing neoagarobiose units. The final products of hydrolysis of agarose by the endo-acting enzyme were neoagarotetraose and neoagarobiose. Optimal conditions for its activity were pH 6.3 and 30C. When agarose was used as a substrate, an apparent temperature optimum of 35C was found, due to gelling of the substrate during the assay procedure.     

A thermostable lipase produced by a newly isolated <Emphasis Type="Italic">Geotrichum</Emphasis>-like strain,R59

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml^–1) was obtained with urea as the nitrogen source. Growth at 30°C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60°C.     

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez     

Mass production of lipase by fed-batch culture of Pseudomonas fluorescens

Summary High concentration production of an extracellular enzyme, lipase, was achieved by a fed-batch culture of Pseudomonas fluorescens. During the cultivation, temperature, pH and dissolved oxygen concentration wwre maintained at 23°C, 6.5 and 2–5 ppm, respectively. Olive oil was used as a carbon source for microbial growth. To produce lipase effectively the specific feed rate of olive oil had to be maintained in a range of 0.04–0.06 (g oil) · (g dry cell)^-1 · h^-1. The CO₂ evolution rate was monitored to estimate the requirement of olive oil. The ratio of feed rate of olive oil to the CO₂ evolution rate was varied in the range of 20–60 g oil/mol CO₂. The higher value of the ratio accelerated microbial growth, but did not favour lipase production. Once the high cell concentration of 60 g/l had been achieved, the ratio was changed from 50 to 30 g oil/mol CO₂ to accelerate the lipase production. By this CO₂-dependent method a very high activity of lipase, 1980 units/ml, was obtained. Both the productivity and yield of lipase were prominently increased compared with a conventional batch culture.     

Production of Streptokinase in Continuous Culture

A method for continuous cultivation of a β-hemolytic streptococcus, strain H 64, is described. The production of cells and streptokinase at various dilution rates, pH, and temperature were studied in a complex medium supplied with excess glucose. At pH 7.0, productivity of cells and streptokinase, as well as the yield constant with respect to glucose, all increased with increasing dilution rate in the range of 0.1 to 0.5 hr^-1. The production of streptokinase was found to be a function of both growth rate and cell concentration. Although higher concentrations of streptokinase were obtained in experiments with batch cultures, the production of streptokinase in continuous cultures was found to be 2.3 times higher. The possible industrial application of a continuous production method is considered.