根霉液态发酵产生纤溶酶的培养条件优化

目的:目前临床使用的溶栓药物疗效肯定,但还存在许多缺陷,而且价格昂贵,因此研制新型溶栓药物的需求迫切。方法:研究了根霉Rhizopus chinensisYY-15液体摇瓶发酵产生纤溶酶的工艺条件。采用单因素试验对液体发酵培养基的碳源、氮源、碳氮比、初始pH进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果:实验范围内菌株液体发酵产纤溶酶的适宜培养基组成为:麸皮水浓度3%(w/v),豆粕浓度5%(w/v),初始培养基pH5.0。适宜培养条件为接种量6%,培养时间72h。优化条件下的摇瓶液体发酵纤溶酶产量平均达98.31 U/ml。     

Modelling and optimization of fermentation factors for enhancement of alkaline protease production by isolated Bacillus circulans using feed-forward neural network and genetic algorithm

Aim: Modelling and optimization of fermentation factors and evaluation for enhanced alkaline protease production by Bacillus circulans. Methods and Results: A hybrid system of feed‐forward neural network (FFNN) and genetic algorithm (GA) was used to optimize the fermentation conditions to enhance the alkaline protease production by B. circulans. Different microbial metabolism regulating fermentation factors (incubation temperature, medium pH, inoculum level, medium volume, carbon and nitrogen sources) were used to construct a ‘6‐13‐1’ topology of the FFNN for identifying the nonlinear relationship between fermentation factors and enzyme yield. FFNN predicted values were further optimized for alkaline protease production using GA. The overall mean absolute predictive error and the mean square errors were observed to be 0·0048, 27·9, 0·001128 and 22·45 U ml^?1 for training and testing, respectively. The goodness of the neural network prediction (coefficient of R²) was found to be 0·9993. Conclusions: Four different optimum fermentation conditions revealed maximum enzyme production out of 500 simulated data. Concentration‐dependent carbon and nitrogen sources, showed major impact on bacterial metabolism mediated alkaline protease production. Improved enzyme yield could be achieved by this microbial strain in wide nutrient concentration range and each selected factor concentration depends on rest of the factors concentration. The usage of FFNN–GA hybrid methodology has resulted in a significant improvement (>2·5‐fold) in the alkaline protease yield. Significance and Impact of the Study: The present study helps to optimize enzyme production and its regulation pattern by combinatorial influence of different fermentation factors. Further, the information obtained in this study signifies its importance during scale‐up studies.     

Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate

An alkalophilic bacterial isolate identified as Bacillus pantotheneticus, isolated from saline-alkali soils of Avadh region of UP, India, was studied for the production of alkaline protease. The mutant of the isolated species showed 44% improved production over the parent strain. Organic nitrogen sources supported better protease production than the inorganic sources. The production of alkaline protease was (242 U/ml) in the medium containing molasses, which was comparable with molasses and wheat bran (285 U/ml) as carbon and nitrogen sources, respectively. Protease production was best at pH 10 and temperature 30 degrees C. The Km (for casein) was 11 mg/ml and Vmax was 380-microg tyrosine/ml/min. The enzyme was stable between pH 7 and 10.7 and temperature between 30 and 60 degrees C with a pH and temperature optimum at 8.4 and 40 degrees C, respectively. The results indicated that molasses was an optimal substrate for alkaline protease production.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Pilot-scale production of intracellular and extracellular enzymes

Optimization of pilot-scale enzyme production is described for the case of an extracellular protease and an intracellular esterase. Media optimization was conducted to reduce medium costs and to determine the effect of various defined ingredients as well as complex nitrogen sources on enzyme production. Fermentation conditions such as inoculum transfer timing, agitation rate, and cultivation temperature were evaluated for their effect on enzyme production. Broths were harvested via microfiltration, diafiltered, and in the case of the extracellular enzyme, lysed via homogenization. An improvement in enzyme titer and reduction in medium costs for the extracellular protease was realized through replacement of Sabouraud dextrose broth medium with more reasonably priced complex nitrogen sources such as N-Z-amine A. An improvement in enzyme titer and reduction in medium costs for the intracellular esterase was realized by decreasing the amount of malt extract and omitting glycerol from the medium. An improvement in the harvest conditions for both enzymes was realized by using large-lumen-diameter hollow-fiber membranes (2.7 mm) which seemed wide enough to pass clumps of fungal and filamentous bacterial fermentation broth without clogging.     

Optimal protease production condition for Prevotella ruminicola 23 and characterization of its extracellular crude protease

In this study, Prevotella ruminicola 23 (ATCC 19189), a ruminal proteolytic bacterium, was used as protease producer to examine the optimal condition for protease production. The best carbon and nitrogen sources for the maximum growth were glucose with peptone. Both sucrose and glucose could stimulate high protease production. Casein and peptone are better nitrogen sources for protease production than other choice in this study. The best enzyme production condition was 18-20 h incubation which was at late log phase in the broth of 5% glucose or sucrose as carbon source with 0.1% ammonium chloride and 0.2% peptone as nitrogen sources. Most of the protease activity was secreted into broth (65%) and on cell surface (18%). The optimal temperature and pH for protease reaction were 40 degrees C and pH 6.8, respectively. After incubation for 6h, the crude extract maintained 50% of original protease activity at 30 and 50 degrees C, and protease activity was stable between pH 6 and 8. The protease inhibitor test showed that serine, aspartic acid and metallo-protease inhibitors could cause inhibition of proteolysis. Protein feedstuff degradation experiments suggested that protease in crude extract had higher degradation ability on fish meal, whey, and feather meal (2.39, 2.60 and 1.76 micromol aminoacid/mg enzyme/h) in comparison to soybean meal and blood meal (1.11 and 1.09 micromol aminoacid/mg enzyme/h). The protease in the crude extract should have application potential in term of improving utilization of fish meal and feather meal for monogastric animals.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

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.     

根霉12号固体发酵产生纤溶酶工艺条件的研究*

研究了根霉12号固体发酵产生纤溶酶的工艺条件。采用单因素试验、均匀设计方法对固体发酵培养基的碳源、氮源、碳氮比、初始pH、加水量、无机盐加量进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果表明,实验范围内根霉12固体发酵产纤溶酶的适宜培养基组成为:麸皮∶豆粕=1∶2,初始pH5.0,加水量0.75ml/g物料, MnSO4H2O和 (NH4)2SO4加量分别为0.25%和 1.42%(对物料)。适宜培养条件为接种量107个孢子/g物料,培养时间72h。优化条件下的纤溶酶产量平均达791.81u/g物料。     

L-asparaginase production by isolated Staphylococcus sp. - 6A: design of experiment considering interaction effect for process parameter optimization

AIMS: Evaluation of fermentation process parameter interactions for the production of l-asparaginase by isolated Staphylococcus sp. - 6A. METHODS AND RESULTS: Fractional factorial design of experimentation (L18 orthogonal array of Taguchi methodology) was adopted to optimize nutritional (carbon and nitrogen sources), physiological (incubation temperature, medium pH, aeration and agitation) and microbial (inoculum level) fermentation factors. The experimental results and software predicted enzyme production values were comparable. CONCLUSION: Incubation temperature, inoculum level and medium pH, among all fermentation factors, were major influential parameters at their individual level, and contributed to more than 60% of total l-asparaginase production. Interaction data of selected fermentation parameters could be classified as least and most significant at individual and interactive levels. Aeration and agitation were most significant at interactive level, but least significant at individual level, and showed maximum severity index and vice versa at enzyme production. SIGNIFICANCE AND IMPACT OF THE STUDY: All selected factors showed impact on l-asparaginase enzyme production by this isolated microbial strain either at the individual or interactive level. Incubation temperature, inoculum concentration, pH of the medium and nutritional source (glucose and ammonium chloride) had impact at individual level, while aeration, agitation and incubation time showed influence at interactive level. Significant improvement (ninefold increase) in enzyme production by this microbial isolate was noted under optimized environment.     

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%。     

Optimization and modeling studies on the production of a new fibrinolytic protease using <Emphasis Type="Italic">Streptomyces radiopugnans</Emphasis>_VITSD8

Background

The current study demonstrated the possibility of statistical design tools combination with computational tools for optimization of fermentation conditions for enhanced fibrinolytic protease production.

Methods

The effects of using different carbon and nitrogen sources for protease production by Streptomyces radiopugnans_VITSD8 were examined by a full factorial design method. The incubation time, temperature, pH of the medium, and RPM were assessed by the predictable one factor at a time (OFAT) method. Optimization was carried out using starch and oat meal as carbon source, nitrogen source as peptic and malt extract using Fractional Factorial Design (FFD). The analysis was further continued for medium volume, temperature, initial medium pH, inoculum concentration, high determination co-efficient as (R’-0.965), and lower determination co-efficient of variation (CV-8.19%), which defines a reliable and accurate experimental value.

Results

Analysis of variance by the fixed slope effect by temperature and starch; temperature and L-aspargine, temperature and oat meal, temperature and peptic extracts, temperature and pH, temperature and duration of incubation were more vital for protease production at an interactive level. Response surface plots revealed that temperature, starch, and peptic extracts affix critical concerning in temperature. Programming estimated a 28% increase in protease production. Incubation temperature and medium volume portrayed extreme impact among all factor. Starch, peptic and temperature play an important regulatory role in protease production. Optimium temperature for protease production was 33°C. The ratio of carbon and nitrogen sources and pH were the major regulatory factors in protease production by Streptomyces radiopugnans_VITSD8. It demonstrated a 4% noteworthy change in condition.

Conclusion

Among all the selected parameters, temperature was the most intuitive factor, demonstrating a notable connection with the type of media and pH, while inoculum fixation had a direct impact on protein production.

     

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.     

Production of thermostable acid protease by Aspergillus niger

Aspergillus niger F2078 produces high levels of extracellular thermostable acid protease within 96 h. Although glucose and peptone were the best carbon and nitrogen sources, respectively, sucrose and a cheap nitrogen source, corn steep liquor, also gave satisfactory enzyme yields. Supplementation of groundnut meal to the basal medium enhanced enzyme production. Temperature and pH optima of the enzyme activity were 60°C and 3.0–4.0, respectively. The enzyme was stable between pH 3.0 and 6.0 and at temperatures up to 60°C.     

Studies on the enhanced production of extracellular lipase by Staphylococcus epidermidis

Staphylococcus epidermidis isolated from spoiled frozen marine fish samples exhibited optimum lipase activity of 8.1 U within 72 h in batch fermentation. Inducible effect of different sugars, nitrogen sources, salts and metal ions were studied on enzyme production. Trybutyrin induced the enzyme production by twofold. Addition of lactose in the production medium further improved lipase production. Sodium chloride increased lipase production whereas the presence of metals in the media had an inhibitory effect. Cells of immobilized S. epidermidis in agar beads (3%) increased lipase production compared with free cells. The optimum temperature and pH for enzyme activity was 20 degrees C and 7.0 respectively. Lipase retained its 85% stability at pH 6.0 and at 40 degrees C. Immobilized cells with high lipolytic activity and stability may provide commercial advantages over conventional methods of lipase production.     

Alkaline protease production by an actinomycete MA1-1 isolated from marine sediments

Alkaliphilic actinomycetes isolated from sediment samples of the Izmir Gulf, Turkey were studied for the production of protease activity. Strain MA1-1 was selected as a good alkaline protease producer as measured by the clear zone diameter by the hydrolysis of skim-milk and casein. The alkaline protease production from the marine alkaliphilic actinomycete MA1-1 was studied by using different carbon and nitrogen sources in medium containing glycerol, peptone, KCl, MgSO₄, K₂HPO₄, and trace elements at 30°C for 72 h. Among the different carbon and nitrogen sources, fructose, starch, maltose, D(+) glucose, yeast extract, malt extract, beef extract and peptone provided higher production of protease. Starch was also found to be effective for growth and enzyme production with highest specific activity at 699 U mg^?1. Purification was achieved by adsorption on Diaion HP 20 which resulted in a recovery rate of 68% with a specific activity of 7618 U mg^?1 protein and 40-fold purification. The optimum pH and temperature of the partially purified protease were determined as pH 9.0 and 50°C, but high activity was also observed at pH 8.0–13.0 and 35–50°C. The inhibition profile exhibited by phenylmethylsulphonyl fluoride (PMSF) showed that this enzyme belongs to the serine-protease group.     

Isolation and screening of Streptomyces sp. Al-Dhabi-49 from the environment of Saudi Arabia with concomitant production of lipase and protease in submerged fermentation

In this study, Streptomyces sp. Al-Dhabi-49 was isolated from the soil sample of Saudi Arabian environment for the simultaneous production of lipase and protease in submerged fermentation. The process parameters were optimized to enhance enzymes production. The production of protease and lipase was found to be maximum after 5 days of incubation (139.2 ± 2.1 U/ml, 253 ± 4.4 U/ml). Proteolytic enzyme increases with the increase in pH up to 9.0 (147.2 ± 3.6 U/ml) and enzyme production depleted significantly at higher pH values. In the case of lipase, production was maximum in the culture medium containing pH 8.0 (166 ± 1.3 U/ml). The maximum production of protease was observed at 40 °C (174 ± 12.1 U/ml) by Streptomyces sp. Lipase activity was found to be optimum at the range of temperatures (30–50 °C) and maximum production was achieved at 35 °C (168 ± 7.8 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (218 ± 12.8 U/ml). Lipase production was maximum when Streptomyces sp. was cultured in the presence of glucose (162 ± 10.8U/ml). Among various concentrations of peptone, 1.0% (w/v) significantly enhanced protease production. The lipase production was very high in the culture medium containing malt extract as nitrogen source (86 ± 10.2 U/ml). Protease production was maximum in the presence of Ca²⁺ as ionic source (212 ± 3.8 U/ml) and lipase production was enhanced by the addition of Mg²⁺ with the fermentation medium (163.7 ± 6.2 U/ml).     

响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

Production of Extracellular Halo-alkaline Protease from a Newly Isolated Haloalkaliphilic Bacillus sp. Isolated from Seawater in Western India

Summary Haloalkaliphilic, gram positive, aerobic, coccoid Bacillus sp. Po2 was isolated from a seawater sample in Gujarat, India. On the basis of 16s rRNA gene homology, Po2 was 95% related to Bacillus pseudofirmus. A substantial level of extracellular alkaline protease was produced by Po2, which corresponded with the growth and reached a maximum level (264 U/ml) during the stationary phase at 24 h. The production thereafter remained nearly static at optimal level till 36 h. Po2 could grow in the range of 0–20% NaCl (w/v) and pH 7–9, optimally at 10% NaCl (w/v) and pH 8. The protease production was salt-dependent and optimum production required 15% NaCl (w/v) and pH 8. Among the organic nitrogen sources, optimum growth and protease production (260 U/ml) were supported by the combination of peptone and yeast extract. However, growth and protease production were highly suppressed by the inorganic nitrogen sources used; with the exception of potassium nitrate, which supported both growth and protease production to limited extent (24 U/ml). Strong inhibition of enzyme production was observed at above 1% glucose (w/v). Wheat flour served as both carbon and nitrogen source supporting growth and protease production.     

Alkaline protease production by an isolated Bacillus circulans under solid-state fermentation using agroindustrial waste: process parameters optimization

Alkaline protease production using isolated Bacillus circulans under solid-state fermentation environment was optimized by using Taguchi orthogonal array (OA) experimental design (DOE) methodology to understand the interaction of a large number of variables spanned by factors and their settings with a small number of experiments in order to economize the process optimization. The software-designed experiments with an OA worksheet of L-27 was selected to optimize fermentation (temperature, particle size, moisture content and pH), nutrition (yeast extract and maltose), and biomaterial-related (inoculum size and incubation time) factors for the best production yields. Analysis of experimental data using Qualitek-4 methodology showed significant variation in enzyme production levels (32,000-73,000 units per gram material) and dependence on the selected factors and their assigned levels. Validation of experimental results on alkaline protease production by this bacterial strain based on DOE methodology revealed 51% enhanced protease production compared to average performance of the fermentation, indicating the importance of this methodology in the evaluation of main and interaction effects of the selected factors individually and in combination for bioprocess optimization.