Protease production by Aspergillus oryzae in solid‐state fermentation using agroindustrial substrates

BACKGROUND: An inexpensive and readily available agroindustrial substrate such as rice bran can be used to produce cheap commercial enzymes by solid‐state fermentation. This work investigates the production of food‐grade proteases by solid‐state fermentation using readily available Thai rice bran. RESULTS: A local strain of Aspergillus oryzae (Ozykat‐1) was used to produce proteases. Rice bran used alone proved to have poor substrate morphology (insufficient porosity) for satisfactory solid‐state fermentation. A certain amount of wheat bran was necessary to improve the morphology of the substrate. The following variables affected protease production: substrate composition, initial moisture content and initial pH. A high protease activity (∼1200 U g⁻¹ dry solids) was obtained on a substrate that had a wheat bran to rice bran ratio of 0.33 by dry weight, a moisture content of 50%, initial pH of 7.5, and incubation temperature of 30 °C. CONCLUSION: Nutritionally, rice bran used alone was as good a substrate as mixed bran for producing protease, but rice bran had poor morphological characteristics for consistent fermentation. A substrate that had a wheat bran to rice bran ratio of 0.33 by dry weight was best for producing protease. Copyright © 2008 Society of Chemical Industry     

Optimization of media constituents through response surface methodology for improved production of alkaline proteases by Serratia rubidaea

BACKGROUND: Response surface methodology is used to build a predictive model of the combined effects of independent variables (pH, salt concentration starch and casein). The model was validated in a laboratory‐scale bioreactor for extracellular protease production from a newly isolated Serratia rubidaea. RESULTS: Optimum medium conditions obtained from the optimization experiments after 48 h incubation were starch, 8 g L^?1; casein, 4 g L^?1; salt concentration 6.25 g L^?1; and initial pH, 8. A coefficient of determination (R²) value of 0.9305 shows the fitness of the second‐order model for the present studies. Results of model coefficients estimated by multiple linear regressions indicate that linear effects of casein concentration (P < 0.001308) and initial pH (P < 7.91 E‐07) are more significant than similarly interactive effects of starch and casein (P < 0.019153), casein and salt concentration (P < 0.016294), casein and pH (P < 0.039904) and salt concentration and pH (p < 0.017845). The P‐values of quadratic effects of casein, x2 × x2 (P < 0.000171); SC, x3 × x3 (P < 0.009134); initial pH, x4 × x4 (P < 0.000114) are more significant for maximal production. After optimization, protease production was enhanced experimentally by almost 65% in a shake flask and by almost 115% in a bioreactor. CONCLUSION: The alkaline proteases secreted by S. rubidaea were significant from an industrial perspective because of their stability against surfactants, oxidants and solvents. The statistical design is useful in economic protease production in a cost‐effective medium for potential use on an industrial scale. Copyright © 2007 Society of Chemical Industry     

Optimization of alkaline protease production from Shewanella oneidensis MR‐1 by response surface methodology

BACKGROUND: The proteases are among the most important groups of enzymes. Therefore, it is important to produce inexpensive and optimized media for large‐scale commercial production. In the present work, three different Shewanella species were screened on skim milk agar medium for their ability to produce alkaline protease. The effects of different culture conditions were optimized for alkaline protease production by S. oneidensis MR‐1 using a Box–Behnken design combined with response surface methodology (RSM). RESULTS: Highest yield (112.90 U mL^?1) of protease production was obtained at pH 9.0, a temperature of 30 °C, glucose (12.5 g L^?1), tryptone (12.5 g L^?1) and an incubation period of 36 h. A second‐order polynomial regression model was used for analysis of the experiment. The experimental values were in good agreement with predicted values, with correlation coefficient 0.9996. CONCLUSION: Carbon and nitrogen, pH, temperature and incubation period were chosen as the main factors to be used in an experimental design for optimization to produce low‐cost enzymes, potentially for use on an industrial scale. A 60% increase in enzyme activity was achieved in the optimized medium compared with the original medium. Copyright © 2008 Society of Chemical Industry     

Detergent‐Compatible Robust Alkaline Protease from Newly Isolated Halotolerant Salinicoccus sp. UN‐12

Currently, enzyme‐containing blended detergent preparations are favored in the detergent industry. The detergent compatibility of an enzyme depends on its robustness to withstand harsh operating conditions and maintain efficient functioning in the presence of various surfactants and detergents. Alkaline proteases from halotolerant microbes having the capability to work in demanding environments are desirable for their application in the detergent industry. The protease secreted by a halotolerant soil isolate Salinicoccus sp. UN‐12 was evaluated for its suitability in detergent formulations. The studied enzyme is an alkaline serine protease (63 kDa) with an optimum temperature of 55 °C and optimum pH of 8.5. The protease was purified 17‐fold with a 4% yield. The protease was active in NaCl (up to 4 mol) and showed enhanced activity in the presence of CaCl₂, KCl, and MnCl₂. In addition, the proteolytic activity was sustained in the presence of various organic solvents, cyclodextrin, disodium cocoamphodiacetate, ionic liquids, hydrotropes, and ultrasound. This report is the first to document the multifarious robustness and novel attributes of a protease secreted by the newly isolated Salinicoccus sp. UN‐12. The protease‐detergent formulation efficiently removes blood, egg, grass, and tomato sauce stains. This protease is now available for efficient and eco‐friendly applications in the detergent industry.     

Protease stabilization by highly concentrated anionic surfactant mixtures

We have found that anionic surfactants such as linear alkylbenzene sulfonate (LAS) can solubilize proteases in a substantially nonaqueous environment without loss of proteolytic activity. Moreover, in mixtures of anionic and nonionic surfactants with a moderate amount of water (water less than 30 wt%), controlled levels of LAS and water solubilize proteases; yet, in these concentrated surfactant mixtures, enzymes maintain their activity for extended periods. Experimental design techniques have been used to delineate the relationship between protease stability and the water, pH and anionic surfactant levels in these surfactant concentrates. As the sum of water and LAS levels is increased, maximum enzyme stability is observed, after which stability falls off. At low water and LAS levels (sum of both <20%), protease solubility is low, while at high levels of water and LAS (sum of LAS and water >45%), denaturation predominates. Additionally, we have developed a new and simple method to predict protease stability by which a synthetic peptide is used to measure protease activity directly in the surfactant concentrate. From the application of this new technique to our system and to commercial liquid detergent formulations, it is apparent that water facilitates the loss of activity of proteases in surfactant concentrates by increasing the rate of autolysis.     

Bioprocess development to add value to canola cake used as substrate for proteolytic enzyme production

Microbial proteases are one of the largest groups of industrial enzymes. This important market has led to a need for technically and economically efficient bioprocesses for protease production that could be exploited commercially. The aim here was to develop a complete bioprocess for protease production, from microbial fermentation up to dried product formulation. Evaluation was made of the effects of operational conditions on the production of protease by a selected strain of Aspergillus oryzae, cultivated under solid-state fermentation (SSF) with canola cake as a sole carbon source in an instrumented lab-scale bioreactor. Statistical experimental design revealed that the air flow rate, inlet air relative humidity, and initial substrate moisture content had significant effects on the efficiency of protease production. The highest protease production by A. oryzae was achieved at a fixed air flow rate of 12 mL/min, with inlet air relative humidity within the range 66–94% and initial substrate moisture content between 30% and 40%. The enzymatic extract produced under the selected conditions was spray dried using different concentrations of additives (glucose, maltodextrin, and CMC). The stability of the dried enzymatic powder during shelf storage was evaluated over a period of 90 days. There was a positive effect of CMC and a negative effect of glucose on protease activity and stability, while the influence of maltodextrin was negative in enzyme recovery at time zero, but it was positive on protease stability over a longer period. The spray dried proteolytic enzymatic formulation obtained from SSF of canola cake using A. oryzae has potential for applications in the industrial sector.     

Partition of alkaline protease in aqueous two-phase systems of polyethylene glycol 1000 and potassium phosphate

This article presents a study of polyethylene glycol 1000 (PEG1000)/potassium phosphate aqueous two-phase systems (ATPSs) forBacillus subtilis NS99 alkaline protease extraction. The objectives were to evaluate effects of system pH (7.5, 8.5,9.5, and 10.5), and NaCl concentration (0,4,7, and 10% (w/w)) on ATPS binodal curves, effects of system pH, NaCl concentration, and tie-line length (TLL) on alkaline protease partition coefficient (K) and yield (Y%) at room temperature (30±2 ‡C). Casein hydrolysis was used for determination of alkaline protease activity. It was revealed that system pH had the slightest effect on locations of binodal curves (except at pH 10.5). In contrast, addition of NaCl appeared to have a significant effect on phase characteristics since binodal curves of systems with NaCl (4-10% (w/w)) shifted significantly towards the origin in comparison to the ones without NaCl. Increased NaCl concentration from 4 to 10% (w/w), however, showed trivial influence on locations of the binodal curves. Changes of system compositions due to variation in system pH, TLL, and NaCl concentrations obviously resulted in varied obtainable K and Y% of alkaline proteases. Longer TLL and higher pH generally resulted in higher K. In contrast, the lower NaCl concentration, the higher K. Since the same phase volume ration (1:1) was used throughout the experiments, Y% depended solely on K. The most suitable PEG1000/potassium phosphate ATPS was determined at pH 9.5, and comprised PEG1000, potassium phosphate, and NaCl 18.0,13.0, and 0% (w/w), respectively. This system resulted in considerably high K, and Y% of 20.0, and 95.1%, respectively. Information from this study will be important for further development of an ATPS extraction unit for alkaline protease recovery.     

A Taguchi design approach for the enhancement of a detergent-biocompatible alkaline thermostable protease production by Streptomyces mutabilis strain TN-X30

The ability of microorganisms to grow at high temperature, alkaline pH, and high salinity makes them an attractive target for enzyme-production with several industrial applications. One strain TN-X30 has been selected as protease producer and identified as Streptomyces mutabilis after a phenotypic and molecular study. Its production of protease was improved using Taguchi L27 design. The strategy was carried out to identify the optimum levels and the interaction of the screened factors. Following this step, maximum protease activity (10,895 U/ml) was achieved after 6-days of incubation. The TN-X30 protease activity had an optimum of pH and temperature of 10 and 65°C, respectively. Thermodynamic parameters at 60°C were enthalpy 14.26 kJ/mol, entropy −220 J/mol/K, and Gibbs free energy 90.53 kJ/mol. TN-X30 protease production displayed a 16-fold increase reaching 175,000 U/ml in a 100-L fermentor. Furthermore, the lyophilization in presence of sorbitol enhanced the stability of the TN-X30 protease which remained active at 75% after 24-months of storage. The lyophilized TN-X30 protease exhibited exceptional stability indexes in presence of some known commercialized detergent components as NEODOL® 25-7, Dehydol® LT 7, Na₂ CMC, Galaxy LAS, Galaxy LES 70, Galaxy 110, Galaxy CAPB Plus, and Sulfacid K. The lyophilized enzyme also displayed high stability with respect to both solid and liquid detergents. Finally, TN-X30 protease exhibited remarkable destaining of blood, egg, and chocolate stained cloth pieces. These findings may promote TN-X30 protease for use as bioadditive in detergent formulation, thereby reducing environmental chemical threat.     

Versatile Substrates and Probes for IgA1 Protease Activity

Bacterial meningitis is a severe infectious disease with high mortality. Gram‐positive and Gram‐negative bacteria that cause meningitis secrete immunoglobulin A1 (IgA1) proteases to assist in mucosal colonization, invasion, and immune evasion. IgA1 proteases have unique selectivity, with few reported substrates other than IgA1 from human tissue. Here we describe the design, characterization, and application of peptide substrates for diverse IgA1 proteases from Neisseria, Haemophilus, and Streptococcus bacteria. IgA1 proteases from diverse strains showed unexpected selectivity profiles among peptide substrates derived from autoproteolytic sites. A fluorescence probe derived from one of these peptides was used to quantitate IgA1 protease activity in buffer and in human cerebrospinal fluid; it was able to detect recombinant Haemophilus influenzae type 1 IgA1 protease at less than 1 μg mL^?1. We also used the probe to establish the first high‐throughput screen for IgA1 protease inhibitors. This work provides tools that will help investigate the roles of IgA1 proteases in bacterial colonization, immune evasion, and infection.     

Purification and characterization of a cold-active protease from psychrotrophic Serratia marcescens AP3801

Protease activity was detected in the culture medium of Serratia marcescens AP3801 grown at 10°C, which was isolated from soil collected from the top of a mountain. The enzyme, designated as CP-58 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographies. The molecular mass of the protease was 58 kDa, and its isoelectric point was close to 6.0. Maximal activity toward azocasein was observed at 40°C and from pH 6.5 to 8.0. The activity was strongly inhibited by 1,10-phenanthroline, suggesting that the enzyme is a metalloprotease. The N-terminal amino acid sequence was Ser-Leu-Asn-Gly-Lys-Thr-Asn-Gly-Trp-Asp-Ser-Val-Asn-Asp-Leu-Leu-Asn-Tyr-His-Asn-Arg-Gly-Asn (or Asp)-Gly-Thr-Ile-Asn-Asn-Lys-Pro-Ser-Phe-Asp-Ile-Ala. A search through databases for sequence homology aligned CP-58 protease with metalloprotease. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-58 protease has a broader specificity than other proteases against the peptide substrate.     

Mechanistic studies of proteases and lipases for the detergent industry

The wash performance of three specific proteases (asp, lys/arg and glu specific, respectively) is demonstrated to be comparable to that of commercial unspecific detergent proteases (Alcalase® and Savinase®). A hypothesis for the high performance of these specific proteases is proposed. The influence of pH and ionic strength on protease performance is discussed. The removal of oily stains by lipases in detergent solution is improved only slightly under acidic conditions, whereas pH values >> pK_a of the fatty acids formed results in strongly enhanced removal. Wash trials with Lipolase^TM show very little effect of the lipase after the first wash, but high effect after the second and subsequent washes. This is explained by lipolytic activity under drying. Maximum activity is found when the water content of the cotton swatches is between 10 and 40% (w/w).     

Structure-Based Macrocyclization of Substrate Analogue NS2B-NS3 Protease Inhibitors of Zika,West Nile and Dengue viruses

A series of cyclic active-site-directed inhibitors of the NS2B-NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue-4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d -lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α-amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with K_i values <5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin-like serine proteases and furin-like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure-activity relationships were observed for these enzymes, thus suggesting their potential application as pan-flaviviral protease inhibitors.     

Optimization and Characterization of Biosurfactant Rhamnolipid Production by Pseudomonas aeruginosa Isolated from an Artificially Contaminated Soil

Biosurfactants are surfactants biologically produced by microorganisms, presenting several advantages when compared to synthetic surfactants. Pseudomonas aeruginosa is known for producing rhamnolipids, considered one of the most interesting types of biosurfactants due to their high yields, when compared to other types. In this work, the production of rhamnolipid from P. aeruginosa was optimized. At first, the Plackett–Burman design was used to select most significant variables affecting the biosurfactant production yield among nine variables—carbon–nitrogen ratio, carbon concentration, nitrogen source, pH, cultivation time, potassium and magnesium concentrations, agitation, and temperature. Then, using main variables, a central point experimental design aiming to optimize rhamnolipid production was performed. The maximum biosurfactant concentration obtained was 0.877 mg L⁻¹. The rhamnolipid also displayed a great emulsification rate, reaching approximately 67%, and the ability to reduce water surface tension from 72.02 to 35.26 mN m⁻¹ at a critical micelle concentration (CMC) of 127 mg L⁻¹, in addition to presenting a good stability when exposed to wide pH and salinity ranges. The results suggest that rhamnolipids are promising substitutes for synthetic surfactants, especially due to lower impacts on the environment.     

Optimization of Serine Protease Purification from Mango (Mangifera indica cv. Chokanan) Peel in Polyethylene Glycol/Dextran Aqueous Two Phase System

Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.     

Protease immobilization onto porous chitosan beads

Water-insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate, N-benzyl-L -arginine ethyl ester (BAEE), but rather low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constant K_m and the maximum reaction velocity V_m for free and immobilized proteases on the porous chitosan beads are estimated. The apparent K_m values were larger for immobilized proteases than for the free ones, while V_m values were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Activity‐Based Probes for Monitoring Postproline Protease Activity

Postproline proteases constitute a subset of serine proteases involved in the regulation of many signaling events and are emerging as promising therapeutic targets for prevalent diseases, such as diabetes and cancer. Therefore, monitoring their activity in different tissues and diverse physiological states would certainly facilitate the elucidation of their physiological role and the establishment of new therapeutic targets. Here, we have synthesized a dipeptidyl phosphonate activity‐based probe that has proved to be highly selective for a specific postproline protease, prolyl oligopeptidase (POP). Its high sensitivity allows the detection of the endogenous activity of POP both by in‐gel analysis and mass spectrometry. The evidence provided by mass spectrometry for the high selectivity of the synthesized probe opens the possibility of using dipeptidyl phosphonates not only for activity‐based profiling (ABP), but also for other ABP applications like substrate‐based protease identification.     

KINETIC MODELING OF CELL GROWTH AND PRODUCT FORMATION IN SUBMERGED CULTURE OF RECOMBINANT ASPERGILLUS NIGER

The kinetics of cell growth and protein production for the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP]#11 were investigated. An unstructured kinetic model was developed to describe the cell growth and product formation mathematically. The dynamics of glucose consumption and biomass production can be well-described by the model. The assumption that the degradation of GFP by proteases is a first-order reaction can express the trend of the GFP profile reasonably well. The dynamics of protease production is also described reasonably well by considering the inhibition effect of glucose on protease production. Because of the high initial glucose concentration and keeping the pH value of the broth at 6, the protease activity could be kept low most of the time during the fermentation. The increase of protease activity near the end of the culture might be caused by cell lysis because the intracellular protease activity was much higher than the extracellular activity.     

发酵工业中毕赤酵母表达的影响因素

毕赤酵母是目前应用最广泛的外源蛋白表达系统。综合论述了外源基因、启动子、拷贝数、诱导温度、pH值、通气量、甲醇诱导、蛋白酶等对毕赤酵母表达系统的影响,为毕赤酵母在工业生产中的实际应用提供参考。     

Effectiveness of ozonation and catalytic ozonation (iron oxide) in the degradation of sunset yellow dye

Azo dyes present in industrial effluents represent a hurdle that regular treatments cannot overcome. In this study, the application of ozone and a catalytic (iron oxide) ozone treatment were proposed as a means of degrading aqueous sunset yellow dye. In order to understand the factors involved, a rotatable central composite design was applied using the variables time, initial dye concentration (C₀), pH, ozone inlet concentration (O₃), and mass of catalyst, which varied in each case. All variables were significant in colour removal. Extremes in pH, lower C₀, and higher ozone concentrations are conditions that favour dye degradation. A complete colour loss occurred for certain combinations of these parameters. The application of iron oxide as a catalyst did not present a satisfactory improvement in the reaction rate. Chemical oxygen demand and total organic carbon showed minimum values of 80% and 78%, respectively, for the worst experimental conditions (pH 7.0, C₀ of 45 mg · L⁻¹, and 5 g O₃ · m⁻³), while their values were 88% and 83% for the best conditions applied. There was no immobilization of Artemia salina nauplii, even for the experimental run where the maximum concentration of dye of the set was used (45 mg dye · L⁻¹). Ozonation is a promising alternative in the degradation of aqueous sunset yellow dye, being favoured in acidic or basic media, which is especially important since food effluents usually present low pH and show low toxicity. The mathematical model proposed can be useful in the design of wastewater treatment processes.     

Effect of dissolved oxygen tension and ph on the production of extracellular protease from a new isolate of bacillus subtilis K2, for use in leather processing

Scale‐up of production of an alkaline protease, previously characterised from a new isolate of Bacillus subtilis for use as a bating enzyme in leather processing, is described. Before large‐scale commercial production of the protease is possible, characteristics of the growth of the bacterium and enzyme production in fermenters must be defined. In 2 dm³ fermenters an optimal specific activity of 296×10³ U g ⁻¹ cell dry weight was obtained after 60 h with the dissolved oxygen tension (DOT) kept above 10% and pH left uncontrolled. Culture pH was 6 on inoculation, falling to 5.3 after 12 h before rising steadily to ∽8 at the end of fermentation. DOT was maintained above 10% by agitation in the range 300 to 500 rpm. The same criteria were adopted for scale‐up to 20 dm³ but the increase in activity occurred 24 h later. © 1999 Society of Chemical Industry