Effect of bioprocess conditions on growth and alkaline protease production by halotolerant <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> BA17

The effect of bioprocess conditions (pH and temperature) on the growth and alkaline protease production of halotolerant Bacillus licheniformis BA17 bioreactor cultures have been systematically analyzed using response surface methodology in order to assess the importance of these generally disregarded parameters. Two models were proposed differing by the choice of response variable. Under optimized bioprocess conditions, whole alkaline protease activity was about 3 fold higher than the activities obtained in the preliminary studies. Results of this study not only highlight the importance of pH and temperature for further engineering purposes but also serve as basis for understanding the true mechanism lying under the relation between these process parameters and growth and whole alkaline protease production. Published in Russian Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 5, pp. 539–544. The text was submitted by the autors in English.     

Mathematical model based estimation of volumetric oxygen transfer coefficient(s) in the production of proteolytic enzymes in Bacillus amyloliquefaciens

Alkaline protease is a class of important hydrolytic enzymes having wide applications in bioprocess industries. Their optimum pH in the alkaline range and stability at higher temperatures make them ideal in detergent and leather processing industries. These enzymes have excellent depilating capacity. The present study aims at process optimization for the production of alkaline protease from Bacillus amyloliquefaciens ATCC 23844. Information on the optimal operating temperature and pH were elicited from specific growth rates and alkaline protease yields. It was also observed that besides pH and temperature, the oxygen transfer rate is another important limiting variable for the production of protease. Volumetric oxygen transfer coefficient (k _L a) was estimated at various impeller speeds and aeration rates. The optimal impeller speed and aeration rates were determined from k _L a and the relative protease yield data. It was understood that the oxygen transfer rate is one of the crucial parameters for the production of proteolytic enzymes by B. amyloliquefaciens.     

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.     

Statistical Optimization of Alkaline Protease Production from Penicillium citrinum YL-1 Under Solid-State Fermentation

Proteases from halotolerant and halophilic microorganisms were found in traditional Chinese fish sauce. In this study, 30 fungi were isolated from fermented fish sauce in five growth media based on their morphology. However, only one strain, YL-1, which was identified as Penicillium citrinum by internal transcribed spacer (ITS) sequence analysis, can produce alkaline protease. This study is the first to report that a protease-producing fungus strain was isolated and identified in traditional Chinese fish sauce. Furthermore, the culture conditions of alkaline protease production by P. citrinum YL-1 in solid-state fermentation were optimized by response surface methodology. First, three variables including peptone, initial pH, and moisture content were selected by Plackett–Burman design as the significant variables for alkaline protease production. The Box–Behnken design was then adopted to further investigate the interaction effects between the three variables on alkaline protease production and determine the optimal values of the variables. The maximal production (94.30 U/mL) of alkaline protease by P. citrinum YL-1 took place under the optimal conditions of peptone, initial pH, and moisture content (v/w) of 35.5 g/L, 7.73, and 136%, respectively.     

Fuzzy Logic Control of Bioreactor for Enhanced Biosynthesis of Alkaline Protease by an Alkalophilic Strain of Bacillus subtilis

Present studies describe the optimization of some cultural parameters such as medium pH, incubation temperature, and agitation rate for the biosynthesis of alkaline protease by Bacillus subtilis IH-72 in a bioreactor using fuzzy logic control. The process of fermentation was carried out in a 7.5-L bioreactor (New Brunswick Scientific, USA) with a working volume of 5 L. All of the parameters were automatically controlled with the help of attached software. The optimum pH, temperature, and agitation for the production of alkaline protease by B. subtilis IH-72 were found to be 9.0, 35°C, and 175 rpm, respectively. The performance of the fuzzy logic of the bioreactor was found to be encouraging for enhanced production of the enzymes. The maximum production of alkaline protease during the present study was found to be 9.6 U mL⁻¹.     

地衣芽孢杆菌D-1产碱性蛋白酶培养条件的优化

为了对产碱性蛋白酶的地衣芽孢杆菌D-1的培养条件进行优化,利用10 L发酵罐,采用正交设计19(34)试验,对培养温度、pH值、搅拌转速、通气量4条件进行优化,得到地衣芽孢杆菌D-1发酵产碱性蛋白酶的最优培养条件为:培养温度37.0℃,pH值7.5,通气量4L/min,搅拌转速300r/min.利用最优条件组合进行验证...     

Studies on the production of extracellular protease by Alcaligenes faecalis

Alcaligenes faecalis produced extracellular protease when incubated in media containing protein substrates. Enzyme production was found to be influenced by various culture conditions. Enzyme production was growth-associated, expressed linearity with growth and reached a maximum at the end of the growth phase. Carbohydrates and inorganic nitrogen sources could not be utilized by the bacterium for its growth, and organic nitrogen appeared to be a primary determinant in protease production. Enzyme production reached its maximum level of 171.2 U/ml when the culture was incubated at 30 °C at pH 8.0. Ca²⁺ and Mg²⁺ enhanced the enzyme production. The crude enzyme powder was stable at high alkaline pH and stable upto 6 months at the storage temperature of 0–4 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus

Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen-free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed-batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design of bioreactors for production of protease and bulk chemicals by this bacterium.     

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.     

Production of Alkaline Protease by a New Aspergillus flavus Isolate under Solid-Substrate Fermentation Conditions for Use as a Depilation Agent

The production of alkaline protease by an Aspergillus flavus strain isolated in our laboratory by solid-substrate fermentation for use as a depilation agent and the influence of various factors on enzyme production are reported. The optimum conditions for maximum production were a growth temperature of 32°C, 63% substrate moisture, and a growth period of 48 h. Enrichment with corn steep liquor or Casitone increased productivity. Scaling-up experiments indicated that flask-scale results could be reproduced at 1 and 30 kg of substrate. The enzyme preparation exhibited maximum activity at both pH 7.5 and pH 9.5. The use of this enzyme as a depilation agent was confirmed by experiments in a tannery.     

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.     

An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.     

家蚕肠道枯草杆菌产蛋白酶的发酵条件与酶学性质

肠道微生物分泌的蛋白酶可促进家蚕对桑叶养分的消化吸收,枯草芽孢杆菌是家蚕肠道内一种重要的产蛋白酶菌株。为提高枯草芽孢杆菌蛋白酶的高效利用,对该菌株适宜发酵条件及酶学性质进行了研究。结果表明：各因素对枯草芽孢杆菌产酶活性影响的大小顺序依次为：pH值〉培养温度〉培养时间〉装液量;最适的产酶条件为：pH=7,培养温度：30 ℃,培养时间：36 h;对枯草芽孢杆菌产蛋白酶进行初步提纯后并研究得出该酶反应的最适pH 10.0,最适反应温度为：60 ℃;该酶为碱性蛋白酶、不耐高温、不耐酸,但在35 ℃条件下热稳定性较好。     

Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions

AIMS: The isolation and identification of new Bacillus sp. capable of growing under highly alkaline conditions as alkaline protease producers. METHODS AND RESULTS: A Bacillus strain capable of growing under highly alkaline conditions was isolated from compost. The strain is a Gram-positive, spore-forming, motile, aerobic, catalase- and oxidase-positive, alkaliphilic bacterium and designated as GMBAE 42. Good growth of the strain was observed at pH 10. The strain was identified as Bacillus clausii according to the physiological properties, cellular fatty acid composition, G + C content of genomic DNA and 16S rRNA gene sequence analyses. The result of 16S rRNA sequence analyses placed this bacterium in a cluster with B. clausii. The G + C content of the genomic DNA of the isolate GMBAE 42 was found to be 49 mol%. The crude extracellular alkaline protease produced by the isolate showed maximal activity at pH 11.0 and 60 degrees C. CONCLUSIONS: The results suggest that isolated strain GMBAE 42 is a new type of B. clausii capable of growing at pH 10.0 and produce extracellular alkaline protease very active at pH 11.0. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolated strain could be used in commercial alkaline protease production and its enzyme can be considered as a candidate as an additive for commercial detergents.     

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.     

地衣芽孢杆菌TG116胞外蛋白酶产酶条件与酶学性质

【背景】从独角莲中分离得到的地衣芽孢杆菌TG116是一株对植物病原菌具有广谱抗性作用的生防菌株。【目的】优化TG116的产酶条件并探索其酶学性质,进一步了解其抗菌机制。【方法】采用Folin-Phenol显色法与响应曲面法,优化菌株TG116的产酶条件并研究其蛋白酶的酶学性质。【结果】菌株TG116产酶最适条件为:温度40.83°C,p H 8.01,发酵时间53.74 h,增加通气量可以显著提高酶活力。按照优化后的条件培养48 h后,上清液蛋白酶活力从57.46 U/mL达到了254.07 U/mL。酶学性质研究表明:该酶为碱性蛋白酶,最适反应pH为8.5,最适反应温度为50°C,具有良好的温度和pH稳定性,EDTA对酶活具有强烈的抑制作用,金属离子Mg~(2+)、Ca~(2+)、Na~+、Co~(2+)、K~+等对酶活也具有一定的抑制作用。【结论】菌株TG116具有良好的p H与温度稳定性,在实际应用中蛋白酶不易失活,可以分解真菌的细胞壁蛋白成分,破坏细胞壁结构,从而抑制甚至杀死病原菌,达到抗菌作用。     

Biosynthesis of protease from Lactobacillus paracasei: kinetic analysis of fermentation parameters

Fifteen strains of Lactobacillus species, isolated from different samples of curd were screened for their ability to produce more extracellular protease. The proteolytic activities of these strains based on casein hydrolysis showed a variation of 1.26-5.80 U ml(-l), with Lactobacillus IH8 showing the maximum activity and was identified as L. paracasei. Different cultural conditions for enhanced production of protease by L. paracasei were optimized. The optimal conditions for production of the enzyme were an incubation temperature of 35 degrees C and a medium pH of 6.0. The maximum proteolytic activity of L. paracasei (7.28 Uml(-1)) was achieved after 48 h of cultivation. The kinetic parameters such as product yield (Yp/x,), growth yield (Yx/s), specific product yield (qp) and specific growth yield (qs) coefficients also revealed that the values of experimental results were kinetically significant.     

产碱性蛋白酶芽孢杆菌的鉴定

通过测量比较在碱性蛋白平板上产生的蛋白水解圈直径,从土壤中筛选到一株高产蛋白酶菌株Bacillus sp.HFBL0079,根据生理生化特性、16S rDNA序列,鉴定为B.amyloliquefaciens。其最适培养温度为35°C-37°C,最适生长pH 8.0,在特定培养条件下16 h达到稳定期,菌体生长和蛋白酶合成同步进行。以大豆分离蛋白为氮源时发酵液具有最高酶活。发酵液在pH 10时具有最高酶活,表明为碱性蛋白酶。该菌株产生的碱性蛋白酶可水解多种天然蛋白质,对胶原蛋白水解度高于其他蛋白质,对羽毛角蛋白也有一定水解能力,提示该酶具有一定新颖性。