Response surface methodology based optimization of keratinase from Bacillus velezensis strain ZBE1 and nanoparticle synthesis,biological and molecular characterization

The increasing demands of keratinases for biodegradation of recalcitrant keratinaceous waste like chicken feathers has lead to research on newer potential bacterial keratinases to produce high-value products with biological activities. The present study reports a novel keratinolytic bacterium Bacillus velezensis strain ZBE1 isolated from deep forest soil of Western Ghats of Karnataka, which possessed efficient feather keratin degradation capability and induced keratinase production. Production kinetics depicts maximum keratinase production (11.65 U/mL) on 4th day with protein concentration of 0.61 mg/mL. Effect of various physico-chemical factors such as, inoculum size, metal ions, carbon and nitrogen sources, pH and temperature influencing keratinase production were optimized and 3.74 folds enhancement was evidenced through response surface methodology. Silver (AgNP) and zinc oxide (ZnONP) nanoparticles with keratin hydrolysate produced from chicken feathers by the action of keratinase were synthesized and verified with UV–Visible spectroscopy that revealed biological activities like, antibacterial action against Bacillus cereus and Escherichia coli. AgNP and ZnONP also showed potential antioxidant activities through radical scavenging activities by ABTS and DPPH. AgNP and ZnONP revealed cytotoxic effect against MCF-7 breast cancer cell lines with IC₅₀ of 5.47 µg/ml and 62.26 µg/ml respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the thermostability, structure and surface attributes. The study suggests the prospective applications of keratinase to trigger the production of bioactive value-added products and significant application in nanotechnology in biomedicine.     

Optimizing the fermentation conditions and enhanced production of keratinase from Bacillus cereus isolated from halophilic environment

Keratinase degrading Bacillus cereus was isolated from the halophilic environment in Tamilnadu, India and keratinase production was optimized using wheat bran substrate. Of the screened bacterial isolates, four were found to have the ability to produce keratinolytic enzyme. The process parameters were optimized using one-variable-at-a-time approach and response surface methodology. Supplementation of 1% lactose supported more keratinase production (120?U/g). Among the selected nitrogen sources, addition of casein significantly enhanced maximum keratinase production (132.5?U/g). Among the ions, manganese chloride significantly enhanced keratinsase production (102.6?U/g), however addition of zinc sulphate and copper sulphate decreased keratinase production. The maximum keratinase production was obtained in the wheat bran medium containing 1% lactose, 0.5% manganese with 80% moisture (292?U/g). Statistics based contour plots were generated to explore the variations in the response surface and to find the relationship between the keratinase yield and the bioprocess conditions.     

Secretion of keratinolytic enzymes and keratinolysis by Scopulariopsis brevicaulis and Trichophyton mentagrophytes: regression analysis

A survey on keratinophilic fungi from poultry-farm soils at Namakkal and from feather dumping soils at Chennai, India, revealed the existence of 34 species of fungi. Most of the fungi exhibited variable efficiency in producing extracellular keratinase when grown in plates with chicken feathers as the sole carbon and nitrogen source. The fungi Aspergillus flavus, Aspergillus niger, Aspergillus versicolor, Chrysosporium state of Arthroderma tuberculatum, Paecilomyces carneus, Scopulariopsis brevicaulis, Trichoderma viride, and Trichophyton mentagrophytes were efficient candidates to degrade the feathers. However, when cultivating the strains in submerged conditions in a medium containing chicken feathers as the sole nutrients source, Aspergillus glaucus, Chrysosporium keratinophilum, Curvularia lunata, Fusarium solani, and Penicillium citrinum also proved to be potent. Among all species, S. brevicaulis and Trichophyton mentagrophytes produced higher amounts of keratinase in both methods. Conditions for keratinase production were optimized by statistical design and surface plots. The highest keratinase activity was estimated by S. brevicaulis (3.2 KU/mL) and Trichophyton mentagrophytes (2.7 KU/mL) in the culture medium with chicken feathers and shows (79% and 72.2% of degrading ability, respectively).     

Degradation of chicken feathers by Chrysosporium georgiae

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin–salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8~pH at 30 °C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin–salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracelluar keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimization of metallo-keratinase production by Pseudomonas sp. LM19 as a potential enzyme for feather waste conversion

Locally isolated bacterium Pseudomonas sp. LM19, a metallo-keratinase producer was used to hydrolyze the highly rigid keratin recalcitrant in this study. The production of crude keratinase by Pseudomonas sp. LM19 is influenced by both physical and nutritional parameters. The highest keratinase activity of 127?U/ml (2.15-fold) was observed in feather meal medium supplemented with fructose and peptone at a C/N ratio of 40. The optimum pH and temperature for keratinase production were found to be pH 8 and 30?°C, using 1% (w/v) feather as substrate. The degradation rate of the feathers was increased 2.4-fold at optimized physical and nutritional conditions. Feather degradation by Pseudomonas sp. LM19 led to the production of free amino acids such as arginine, glycine, leucine, and serine. The information on the production of keratinase by Pseudomonas sp. LM19 obtained from this study warrants further research for possible commercial application.     

Medium optimization for keratinase production in hair substrate by a new <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KD-N2 using response surface methodology

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO₄ and K₂HPO₄ were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO₄ and K₂HPO₄ concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl₂ 0.05, KH₂PO₄ 0.7, sucrose 3, MgSO₄ 0.91, K₂HPO₄ 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.     

落叶松锈迷孔菌产多糖液体培养基的优化及其体外抗氧化活性

本研究通过响应面设计优化了落叶松锈迷孔菌产胞外多糖的液体培养基,并对其体外抗氧化活性进行了分析。结果显示,以落叶松锈迷孔菌胞外多糖产量为响应值,采用Plackett-Burman设计、最陡爬坡设计和Box-Behnken设计建立数学模型,获得落叶松锈迷孔菌产胞外多糖的最适培养基为：去皮马铃薯415.70g/L、蛋白胨10.80g/L、葡萄糖20.0g/L、吐温80 7.27mL/L、KH₂PO₄ 1.0g/L、MgSO₄?7H₂O 0.5g/L、CaCO₃ 0.57g/L、FeSO₄?7H₂O 0.3g/L、ZnSO₄?7H₂O 0.3g/L、维生素B₁ 0.01g/L、芦丁6.09g/L。在此优化条件下得到的多糖产量为3.07g/L,与理论值3.06g/L相近,相比于优化前提高了2.87倍,说明响应面法对于优化落叶松锈迷孔菌的液体培养基以提高多糖产量行之有效。此外,经过醇沉、透析和去蛋白等获得初步纯化的多糖可有效清除羟自由基、超氧阴离子、1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基和2,2?-连氮-双(3-乙基苯并噻唑-6-磺酸)[2,2?-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid),ABTS]自由基。     

Enhanced antibiotic activity of Xenorhabdus nematophila by medium optimization

Nutrition had highly influence on the antibiotic production by Xenorhabdus nematophila YL001. Glucose and peptone were identified as the best carbon and nitrogen sources that significantly affected antibiotic production using one-factor-at-a-time approach. Response surface methodology was applied to optimize the medium constituents (Glucose, peptone and minerals) for antibiotic production by X. nematophila YL001. Higher antibiotic activity (328.9 U/ml) was obtained after optimizing medium components. The optimal levels of medium components were (g/l): glucose 6.13, peptone 21.29, MgSO(4).7H(2)O 1.50, (NH(4))(2)SO(4) 2.46, KH(2)PO(4) 0.86, K(2)HPO(4) 1.11 and Na(2)SO(4) 1.72. An overall 16% and 35% increase in antibiotic activity were obtained as compared with mean observed response (283.7U/ml) at zero level of all variables and YSG medium.     

Medium optimization for the production of cyclic adenosine 3',5'-monophosphate by Microbacterium sp. no. 205 using response surface methodology

Response surface methodology was employed to optimize medium composition for the production of cyclic adenosine 3',5'-monophosphate (cAMP) with Microbacterium sp. no. 205. A fractional factorial design (2(11-7)) was applied to evaluate the effects of different components in the medium. K(2)HPO(4), MgSO(4) and NaF were found to significantly influence on the cAMP production. The steepest ascent method was used to access the optimal region of the medium composition. The concentrations of the three factors were optimized subsequently using central composite design and response surface methodology. The optimal medium composition to achieve the optimal cAMP production was determined (g/L): K(2)HPO(4), 12.78; MgSO(4), 3.53 and NaF, 0.18. The corresponding cAMP concentration was 8.50 g/L, which was about 1.8-fold increase compared with that using the original medium. Validation experiments were also carried out to prove the adequacy and the accuracy of the model obtained. The cAMP fermentation in 5L fermenter reached 9.87 g/L.     

Application of statistical experimental design for optimization of keratinases production by Bacillus pumilus A1 grown on chicken feather and some biochemical properties

A new keratinolytic enzyme-producing bacterium was isolated from slaughter house polluted water and identified as Bacillus pumilus A1. Medium composition and culture conditions for the keratinases production by B. pumilus A1 were optimized using two statistical methods: Plackett–Burman design applied to find the key ingredients and conditions for the best yield of enzyme production and central composite design used to optimize the concentration of the five significant variables: feathers meal, soy peptone, NaCl, KCl, and KH₂PO₄. The medium optimization resulted in a 3.4-fold increase in keratinase production (87.73 U/ml) compared to that of the initial medium (25.9 U/ml). The zymography analysis shows the presence of at least five keratinolytic enzymes. The keratinolytic activity of the extracellular proteinases was examined by incubation with non-autoclaved chicken feathers. Complete solubilisation of whole feathers was observed after a 6-h incubation at temperatures ranging from 45 °C to 60 °C. The crude enzyme exhibited maximal activity at 60 °C and pH 8.5 or 55 °C and pH 9.0 using casein or keratin as substrates, respectively.     

Screening and optimization of nutritional factors for higher dextransucrase production by Leuconostocmesenteroides NRRL B-640 using statistical approach

To improve dextransucrase production from Leuconostocmesenteroides NRRL B-640 culture medium was screened and optimized using the statistical design techniques of Plackett-Burman and response surface methodology (RSM). Plackett-Burman design with six variables viz. sucrose, yeast extract, K2HPO4, peptone, beef extract and Tween 80 was performed to screen the nutrients that were significantly affecting dextransucrase production. The variables sucrose, K2HPO4, yeast extract and beef extract showed above 90% confidence levels for dextransucrase production and were considered as significant factors for optimization using response surface methodology. 2(4)-central composite design was used for RSM optimization. The experimental results were fitted to a second-order polynomial model which gave a coefficient of determination R2=0.95. The optimized composition of 30g/l sucrose, 18.9g/l yeast extract, 19.4g/l K2HPO4 and 15g/l beef extract gave an experimental value of dextransucrase activity of 10.7U/ml which corresponded well with the predicted value of 10.9U/ml by the model.     

Optimization of the medium composition for production of mycelial biomass and exo-polymer by Grifola frondosa GF9801 using response surface methodology

In this work, a three-level Box-Behnken factorial design was employed combining with response surface methodology (RSM) to optimize the medium composition for the production of the mycelial biomass and exo-polymer in submerged cultures by Grifola frondosa GF9801. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of mycelial biomass and exo-polymer. The model estimated that, a maximal yield of mycelial biomass (17.61 g/l) could be obtained when the concentrations of glucose, KH2PO4, peptone were set at 45.2 g/l, 2.97 g/l, 6.58 g/l, respectively; while a maximal exo-polymer yield (1.326 g/l) could be achieved when setting concentrations of glucose, KH2PO4, peptone at 58.6 g/l, 4.06 g/l and 3.79 g/l, respectively. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yields of mycelial biomass and exo-polymer. Maximum mycelial biomass yield of 22.50 g/l was achieved in a 15-l fermenter using the optimized medium.     

重组谷氨酸棒杆菌发酵L-苯丙氨酸培养基的优化

【目的】提高重组谷氨酸棒杆菌发酵L-苯丙氨酸(L-phenylalanine,L-Phe)的产量。【方法】使用正交试验设计以及响应面优化法分别对种子培养基及发酵培养基进行优化,确定了重组谷氨酸棒杆菌发酵L-Phe的最佳种子培养基及最佳发酵培养基。【结果】重组谷氨酸棒杆菌发酵L-Phe最佳种子培养基(g/L):葡萄糖25.0,玉米浆25.0,硫酸铵15.0,硫酸镁1.0,磷酸二氢钾2.0,尿素2.0,p H 6.8-7.0;最佳发酵培养基(g/L):葡萄糖110.0,玉米浆7.0,硫酸铵25.0,硫酸镁1.0,磷酸二氢钾1.0,柠檬酸钠2.0,谷氨酸1.0,碳酸钙25.0,p H 6.8-7.0;在最佳培养基条件下L-Phe产量最高达到9.14 g/L,较优化前的7.46 g/L提高了22.5%。【结论】通过正交试验和响应面分析对重组谷氨酸棒杆菌发酵L-Phe培养基进行优化,明显提高了L-Phe的产量,并确定了葡萄糖、玉米浆和硫酸铵为发酵培养基中影响L-Phe产量的3个关键因子。研究结果为L-Phe的发酵放大提供了依据。     

Use of response surface methodology to study the effect of media composition on aflatoxin production by Aspergillus flavus

Aflatoxins are one of the most important secondary metabolites. These extrolites are produced by a number of Aspergillus fungi. In this study, we demonstrate the effect of media components and enhanced aflatoxin yield shown by A. flavus using response surface methodology in response to different nutrients. Different components of a chemically defined media that influence the aflatoxin production were monitored using Plackett–Burman experimental design and further optimized by Box–Behnken factorial design of response surface methodology in liquid culture. Interactions were studied with five variables, namely sorbitol, fructose, ammonium sulfate, KH₂PO₄, and MgSO₄.7H₂O. Maximum aflatoxin production was envisaged in medium containing 4.94 g/l sorbitol, 5.56 g/l fructose, 0.62 g/l ammonium sulfate, 1.33 g/l KH₂PO₄, and 0.65 g/l MgSO₄·7H₂O using response surface plots and the point prediction tool of the DESIGN EXPERT 8.1.0 (Stat-Ease, USA) software. However, a production of 5.25 μg/ml aflatoxin production was obtained, which was in agreement with the prediction observed in verification experiment. The other component (MgSO₄.7H₂O) was found to be an insignificant variable.     

Statistical optimization of the medium composition by response surface methodology to enhance schizophyllan production by Schizophyllum commune

The response surface methodology (RSM) involving central composite design (CCD) was employed to optimize the fermentation medium for the cell growth and schizophllan production by Schizophyllum commune CGMCC 5.113 in submerged culture at pH 6.5 and 26 degrees C. The four variables involved in this study were glucose, yeast extract, ammonium nitrate, and magnesium sulfate. The statistical analysis of the results showed that, in the range studied, glucose and yeast extract had a highly significant effect on schizophyllan production. The optimal medium for schizophyllan production calculated from the regression model of RSM was as follows: glucose, 18 g/l; yeast extract, 0.5 g/l; NH4NO3, 0.48 g/l; and MgSO4, 0.05 g/l, with a predicted maximum schizophyllan production of 11.74 g/l. These predicted values were experimentally validated. The excellent correlation between predicted and measured values justifies the validity of the response model. The results of bioreactor fermentation also show that the optimized medium enhanced schizophyllan production (12.80 g/l) by S. commune in a 5-1 fermenter.     

Optimization of chitinase production by a new <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> C9 isolate using response surface methodology

The purpose of this article is to use statistical Plackett–Burman and Box–Wilson response surface methodology to optimize the medium components and, thus, improve chitinase production by Streptomyces griseorubens C9. This strain was previously isolated and identified from a semi-arid soil of Laghouat region (Algeria). First, syrup of date, colloidal chitin, yeast extract and K₂HPO₄, KH₂PO₄ were proved to have significant effects on chitinase activity using the Plackett–Burman design. Then, an optimal medium was obtained by a Box–Wilson factorial design of response surface methodology in liquid culture. Maximum chitinase production was predicted in medium containing 2% colloidal chitin, 0.47% syrup of date, 0.25 g/l yeast extract and 1.81 g/l K₂HPO₄, KH₂PO₄ using response surface plots of the STATISTICA software v.12.0.     

Characterization of a novel <Emphasis Type="Italic">Stenotrophomonas</Emphasis> isolate with high keratinase activity and purification of the enzyme

A feather-degrading bacterium was isolated from poultry decomposition feathers in China. The strain, named L1, showed significant feather-degrading activity because it grew and reproduced quickly on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. According to the phenotypic characteristics and 16S rRNA profile, the isolate belongs to Stenotrophomonas maltophilia. Keratinase activity of the isolate was determined during cultivation on raw feathers at different temperatures and initial pH. Maximum growth and feather-degrading activity of the bacterium were observed at 40°C and initial pH ranging from 7.5 to 8.0. The crude enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 chromatographic and ceramic hydroxyapatite (CHT) chromatographic. Its molecular mass estimated as 35.2 kDa in SDS-PAGE. The enzyme had an optimum activity at the pH was 7.8 and the temperature was 40°C. The keratinase was wholly inhibited by a serine protease inhibitor, PMSF. Its activity was activated or inhibited by different metal ions. The keratinase activity of enzyme from strain L1 functioned on different keratins, such as feather, hair, wool, horn, and so on.     

林可霉素生物合成培养基的优化

以花生粉和棉籽蛋白粉取代了原培养基中的黄豆饼粉,采用响应面法对林可霉素产生菌的发酵培养基进行了优化.首先通过单因素试验及正交实验确定替代氮源及其浓度,采用Plackett-Burman实验分析各因素的主效应,选出对响应值影响较大的3个因素,即花生粉、K2HPO4和玉米浆.对这些因素做爬坡实验,确定三个重要因素的中心点浓...     

Medium optimization for the production of thermal stable beta-glucanase by Bacillus subtilis ZJF-1A5 using response surface methodology

Polysaccharides, such as barley flour, dextrin and soluble starch, were better carbon sources than monosaccharides and disaccharides, such as glucose and maltose, for cell growth of Bacillus subtilis ZJF-1A5 and beta-glucanase production. beta-Glucanase produced by B. subtilis ZJF-1A5 was associated partially with cell growth and increased significantly when cells entered stationary phase; yeast extract was the best nitrogen source, followed by soybean flour. All inorganic nitrogen sources chosen in the experiments were not favorable for cell growth and enzyme production. A fractional factorial design (2(6-2)) was applied to elucidate medium components that significantly affect beta-glucanase production. The concentration of barley flour, corn flour and soybean flour in medium were significant factors. The steepest ascent method was used to locate the optimal domain and a central composite design was used to estimate the quadratic response surface from which the factor levels for maximum production of beta-glucanase were determined. The composition of fermentation medium optimized with response surface methodology was (g/l): barley flour, 63.5; corn flour, 44.8; KH2PO4, 1.0; MgSO4 x 7H2O, 0.1; CaCl2, 0.1. beta-Glucanase activity was 251 U/ml at 48 h using optimized medium, 1.4 times higher than that in original medium.     

Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp. OU01

The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacterium sp. OU01 in shake-flask. A significant influence of (NH(4))(2)SO(4), MgSO(4).7H(2)O and initial pH on chitosanase production was noted with Plackett-Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0g/L (NH(4))(2)SO(4), 1.3g/L MgSO(4) and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6U/mL to 118U/mL.