Arrowroot (Marantha arundinacea) starch as a new low-cost substrate for alkaline protease production

The feasibility of arrowroot (Marantha arundinacea) starch for alkaline protease production using an alkalophilic Bacillus lentus isolate was evaluated in batch fermentations in shake flasks and in a bioreactor under a range of conditions. A new arrowroot starch-casein medium (pH 10.2) was formulated having a composition (%, w/v): arrowroot starch 1, casein 1, sodium succinate 0.25, NH₄Cl 0.05, NaCl 0.05, KH₂PO₄ 0.04, K₂HPO₄ 0.03, MgCl₂ 0.01, yeast extract 0.01 and Na₂CO₃ 1.05. The isolate produced a maximum protease yield (6754.7 U ml^–1) in this medium when grown for 72 h at 250 rev/min and 37 °C. Scaling-up studies in a bioreactor showed a 5-fold increase in alkaline protease yields (31899 U ml^–1) at a lower production time of 45 h, aeration of 1 v/v/m and agitation of 400 rev/min at 37 °C.     

Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53

Rice bran and yeast extract were found to be the best combination of carbon and nitrogen sources for the production of carboxymethycellulase (CMCase) by Bacillus subtilis subsp. subtlis A-53. Optimal concentrations of rice bran and yeast extract for the production of CMCase were 5.0% (w/v) and 0.10% (w/v), respectively. Optimal temperature and initial pH of medium for cell growth of B. subtilus subsp. subtilis A-53 were 35 °C and 7.3, whereas those for the production of CMCase by B. subtilus subsp. subtilis A-53 were 30 °C and 6.8. Optimal agitation speed and aeration rate in a 7 L bioreactor were 300 rpm and 1.0 vvm, respectively. The optimal agitation speed and aeration rate for the production of CMCase by B. subtilus subsp. subtilis A-53 were lower than those for cell growth. The highest productions of CMCase by B. subtilus subsp. subtilis A-53 in 7 and 100 L bioreactors were 150.3 and 196.8 U mL⁻¹, respectively.     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

Biosynthesis and properties of an extracellular thermostable serine alkaline protease from <Emphasis Type="Italic">Virgibacillus pantothenticus</Emphasis>

In this communication, we report the presence of a newly identified serine alkaline protease producing bacteria, Virgibacillus pantothenticus (MTCC 6729) in the fresh chicken meat samples and the factors affecting biosynthesis as well as characterization of protease. The strain produced only 14.3 U ml⁻¹ protease in the standard medium after 72 h of incubation, while in optimized culture conditions the production of protease was increased up to 18.2 U ml⁻¹. The strain was able to produce protease at 40°C at pH 9.0. The addition of dextrose and casein improved protease production. The protease was partially purified and characterized in terms of pH and temperature stability, effect of metal ions and inhibitors. The protease was found to be thermostable alkaline by retaining its 100% and 85% stability at pH 10.0 and at 50°C respectively. The protease was compatible with some of the commercial detergents tested, and was effective in removing protein stains from cotton fabrics. The V. pantothenticus, MTCC 6729 protease appears to be potentially useful as an additive in detergents as a stain remover and other bio-formulations.     

Screening,selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease

Bacillus subtilis microbe is commonly found in soil and produces proteases on nitrogen and carbon-containing sources and increases the fertility rate by degrading nitrogenous organic materials. The present study was aimed to develop hyper producing mutant strain of B. subtilis for the production of proteases, to improve the process variables by the response surface methodology (RSM) under central composite design (CCD) and the production of protease by the particular mutant strain in a liquid state fermentation media. The mutation of the strain was carried out using ethidium bromide. Pure B. subtilis strain was collected and screened for hyper-production of protease. The production of protease by mutant B. subtilis strain was optimized by varying temperature, inoculum size, pH and incubation time under liquid state fermentation. The CCD model were found to be reliable with r² of 0.999. The maximum enzyme activity of B. subtilis IBL-04 mutant with 3 mL/100 mL inoculum size, 72 h fermentation time, pH 8, and 45 °C temperature was developed with enzyme activity 631.09 U/mL, indicates 1–7-fold increase in enzyme activity than the parent strain having 82.32 U/mL activity. These characteristics render its potential use in industries for pharmaceutical and dairy formulation.     

Optimisation of physical factors on the production of active metabolites by Bacillus subtilis 355 against wood surface contaminant fungi

A Surface Response Model was used to study the effect of pH, temperature and agitation on growth, sporulation and production of antifungal metabolites by Bacillus subtilis CCMI 355.Strong agitation, temperature between 27 and 34 °C and pH 6 favoured cell growth. Alkaline pH, strong agitation and temperature between 28 and 34 °C favoured spore formation. No relationship was found between sporulation and the production of antifungal metabolites. According to the model, pH 8, 37 °C and the absence of agitation were the optimal conditions for the production of broad-spectrum antifungal metabolites against Botrytis cinerea, Penicillium expansum, Trichoderma sp, Trichoderma harzianum, Trichoderma koningii and Trichoderma virgatum.In situ assays using green wood impregnated with Bacillus subtilis CCMI 355 inoculated in Yeast Extract Glucose Broth medium in the conditions above, displayed an efficient protection against wood surface contaminant fungi.     

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.     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

Isolation,partial purification,biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis,Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37?°C for 48?h with a constant agitation of 120?rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1?M ice cold CaCl₂ and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60?s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.     

Effect of aeration and agitation on the protease production by S<Emphasis Type="Italic">taphylococcus aureus</Emphasis> mutant RC128 in a stirred tank bioreactor

The modified rotating simplex method has been successfully used to determine the best combination of agitation rate and aeration rate for maximum production of extracellular proteases by Staphylococcus aureus mutant RC128, in a stirred tank bioreactor operated in a discontinuous way. This mutant has shown altered exoprotein production, specially enhanced protease production. Maximum production of proteases (15.28 UP/ml), measured using azocasein as a substrate, was obtained at exponential growth phase when the bioreactor was operated at 300 rpm and at 2 vvm with a volumetric oxygen transfer coefficient (K _L a) of 175.75 h⁻¹. These conditions were found to be more suitable for protease production.     

Multifarious potential applications of keratinase of Bacillus subtilis K-5

Abstract

Bacillus subtilis K-5, an isolate from compost, utilized a wide range of keratinous wastes viz. diverse feather types, nails, hair, scales, etc. for growth and produced a thermostable alkaline protease (keratinase) with broad proteolytic activity. Optimization of cultural and environmental variables using a Plackett–Burman design and response surface methodology resulted in enhanced keratinase production (89%). Keratinase was partially purified (15-fold) by ammonium sulfate precipitation and carboxymethyl cellulose chromatography. The optimum pH and temperature for keratinase activity were 9.0 and 60°C, however, considerable activity and stability was observed over broad pH (5–10) and temperature range (50–90°C). B. subtilis K-5 keratinase exhibited excellent stability toward detergents (cetyl trimethylammonium bromide, Tween 80, and sodium dodecyl sulfate) and organic solvents (benzene, acetonitrile, phenylmethylsulfonyl fluoride); however, metal ions like Mn²⁺, Cu²⁺, Na⁺, Hg²⁺, K⁺, Ca²⁺, and Zn²⁺ inhibited the activity. B. subtilis K-5 protease showed remarkable potential for diverse applications like blood stain removal, gelatin hydrolysis from waste X-ray films and dehairing of animal hide.     

Thermostable Alkaline Protease from a Novel Marine Haloalkalophilic Bacillus Clausii Isolate

An oxidative and SDS-stable alkaline protease secreted by a marine haloalkalophilic Bacillus clausii isolated from the tidal mud flats of the Korean Yellow Sea near Inchon City was investigated in batch fermentation in shake flasks and in a bioreactor under a range of conditions. The isolate produced maximum protease yields (15,000 U ml⁻¹) under submerged fermentation conditions at 42 °C for 40 h with an aeration of 1.5 v/v/min and agitation of 400 rev/min in a formulated soybean—casein medium (pH 9.6) containing (w/v): soybean meal (2%), casein (1%), corn starch (0.5%), NH₄Cl (0.05%), NaCl (0.05%), KH₂PO₄(0.04%), K₂HPO₄(0.03%), MgSO₄(0.02%), yeast extract (0.01%) and Na₂CO₃(0.6%). The optimal pH and temperature of activity of the partially purified enzyme were 11.5 and 80 °C, respectively. The alkaline protease showed extreme stability towards SDS and oxidizing agents, retaining its activity above 96 and 75% on treatment for 72 h with 5% SDS and 5% H₂O₂, respectively. The inhibition profile exhibited by phenylmethanesulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases.     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

Stabilization of proteolytic enzymes in solution

The stability of the neutral and alkaline proteases in a Bacillus subtilis enzyme mixture was studied in aqueous solutions at room temperature. Stabilization of the proteases in solution for periods up to 25 days was achieved by the addition of various protein preparations including casein and soya protein. The degree of stabilization by casein was concentration dependent to about 2% protein. The instability of the neutral protease in solutions of the B. subtilis enzyme mixture was shown to be due primarily to proteolysis by the alkaline protease since the diisopropylfluorophosphate-treated enzyme was quite stable. Formulation of such enzyme solutions at low pH gave greater stability as did solutions containing an alkaline protease inhibitor from potatoes. A Conceptual approach to the formulation of enzyme solutions containing proteolytic enzyme to ensure maximum stability is proposed.     

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l⁻¹) in the fermentation medium further increased naringinase production (8.9 IU ml⁻¹) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.     

Enhancing the efficiency of the bioagent Bacillus subtilis JF419701 against soil-borne phytopathogens by increasing the productivity of fungal cell wall degrading enzymes

This study was devoted to increasing the production of fungal cell wall degrading enzymes by Bacillus subtilis JF419701 to enhance its efficiency in the biological control process. In dual culture, B. subtilis JF419701 showed the highest antagonistic effect of the 256 bacterial strains tested against six soil-borne pathogens, Alternaria alternata, Exserohilum rostratum, Fusarium oxysporum, Macrophomina phaseolina, Pythium ultimum and Rhizoctonia solani. The production potentiality of the enzymes α-1,3-glucanase, β-1,3-glucanase, chitinase and protease by B. subtilis JF419701 was studied in vitro. Results proved that the maximum production of enzymes by this bacterium was achieved after a two-day incubation period at a slightly alkaline pH (8). The addition of colloidal chitin or S-glucan to the growth media enhanced the production of all the enzymes except protease, which was stimulated by casein. This study therefore recommends that to obtain an efficient and strong bioagent culture of B. subtilis JF419701, it is necessary to grow this micro-organism on a specific medium containing either chitin or its derivatives at pH 8 for two days.     

Production of different proteases from fish gut microflora utilizing tannery fleshing

Three alkaline protease‐producing strains designated as ANFLR1, NPLR1, and PROLR15 were isolated from Labeo rohita fish gut. These strains are able to produce alkaline protease using tannery fleshing (TF) as the sole carbon and nitrogen source and were identified as Bacillus megaterium, Serratia marcescens, and novel Pontibacter sps. Proteases from these organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, and column chromatography. SDS‐PAGE revealed molecular weights of the proteases to be 46 kDa (ANFLR1), 52 kDa (NPLR1), and 58 kDa (PROLR15). The optimum pH and temperature for the protease activity of ANFLR1, NPLR1, and PROLR15 were found to be 10.5, 11.5, 9, and 70^°C, 60^°C, and 50^°C, respectively. The maximum protease activities at the optimum conditions were 420 U/mL (ANFLR1), 550 U/mL (NPLR1), and 530 U/mL (PROLR15). Inhibition of the NPLR1 protease by pepstatin confirmed aspartate‐type enzymatic activity. Fe³⁺ enhanced the activity of PROLR15 protease. Unlike all other microbial proteases known so far, the PROLR15 enzyme did not require Ca²⁺ for activity and thermal stability. SDS‐PAGE and scanning electron microscopy analyses confirmed the conversion of high molecular weight substrate (TF) to low molecular weight peptides by these proteases. The alkaline metalloprotease production by novel Pontibacter sps. and aspartate protease production by S. marcescens remain unexplored. Hence, TF with its relatively abundant availability can be beneficially utilized for alkaline protease production through the fish gut microbial fermentation processes.     

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.     

Effect of bioprocess conditions on growth and alkaline protease production by halotolerant Bacillus licheniformis 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.     

Statistical optimization of fermentation conditions and comparison of their influences on production of cellulases by a psychrophilic marine bacterium, <Emphasis Type="Italic">Psychrobacter aquimaris</Emphasis> LBH-10 using orthogonal array method

The optimal conditions for the production of cellulases by a marine bacterium, Psychrobacter aquimaris LBH-10, were established and their effects were compared using orthogonal array experiments based on the Taguchi method. The optimal conditions of rice bran, peptone and initial pH for the production of avicelase and CMCase by P. aquimaris LBH-10 were 50.0, 3.0, and 8.0 g/L, respectively, whereas those for filter paperase (FPase) were 100.0, 3.0, and 8.0 g/L, respectively. Rice bran was found to be the most important factor for the production of cellulases based on the calculated percentage of participation P (%) from an analysis of the variance (ANOVA). The optimal temperature for the cell growth of P. aquimaris LBH-10 was 25°C, whereas that for the production of avicelase, CMCase and FPase was 30°C. The optimal agitation speed and aeration rate for cell growth was 400 rpm and 1.5 vvm, respectively, whereas those for the production of CMCase were 300 rpm and 1.0 vvm, respectively. Aeration was found to be more important for cell growth and CMCase production than agitation. The maximum production of avicelase, CMCase and FPase in a 100 L bioreactor for 72 h under optimized conditions was 83.2, 388.7, and 75.4 U/mL, respectively.