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The main purpose of this study was to discover new tyrosinase inhibitory peptides from eel bone hydrolysates. After enzyme screening, alkaline protease was utilized, and optimal conditions were achieved through response surface optimization: solid-liquid ratio (1:25), time (5 h), and enzymolysis base ratio (3500 U/g). Inhibitory peptides of 1–3 kDa were isolated (IC50 = 2.162 mg/mL), and the inhibition was determined to be non-competitive and reversible. Subsequent LC-MS/MS and molecular docking revealed that the peptide with the lowest binding energy exhibited hydrogen bonding, hydrophobic interactions, and provided a potential explanation for the mechanism of inhibition by the peptides. [ABSTRACT FROM AUTHOR]

An extracellular alkaline protease from Scytalidium thermophilum was produced in a glucose-containing medium supplemented with 5 mM NaCl for 3 days at pH 8.0 and 45 °C. The enzyme was 10-fold purified using ammonium sulfate precipitation followed by ion-exchange chromatography, and its molecular weight was calculated as 80 kDa from SDS-PAGE. The enzyme exhibited optimum activity at pH 8.0 and 60 °C. It was stable at pH and temperature range of 6.0–10.0 and 30–80 °C, respectively. Its half time was 30 h at pH 6.0, 7.0 and 8.0, while those were 22, 16, 8, and 3 h at 50 °C, 60 °C, 70 °C, and 80 °C, respectively. Kinetic parameters including Km (2 ± 0.02 mg/ml), Vmax (18.7 ± 1.5 µmole tyrosine ml−1 min−1), and kcat (2.5 x 103 min−1) were determined using casein. Ca2+ increased the enzyme activity, but it was slightly reduced by EDTA, Triton X-100, Tween 20, and Tween 80. It was active against reducing agents like β-mercaptoethanol but completely inhibited by phenyl methyl sulphonyl fluoride supporting the enzyme belonging to the serine protease family. Chloroform (143%), methanol (138%), and isopropanol (111%) increased the enzyme activity at 5% (v/v), while ethanol (71%) and acetone (81%) moderately reduced the proteolytic activity at the same concentration. Dimethyl sulfoxide (5%, v/v) did not significantly affect the enzyme. The enzyme was compatible with several detergents (1%, w/v), maintaining more than 90% of its original activity in almost all detergents tested. The stability of the enzyme presented against pH, temperature, organic solvents, and detergents indicates its potential use in various industrial applications, especially in peptide synthesis and the laundry industry. [ABSTRACT FROM AUTHOR]

Response surfaces methodology was established in order to optimize ultrasound-assisted aqueous alkaline protease extraction parameters of Pinus koraiensis nuts oil (PNO) in this short communication. On the oil yield, the impacts of single factors were studied. The solid–liquid ratio, enzyme concentration, enzyme hydrolysis temperature, and enzyme hydrolysis duration were chosen for further optimization of the extraction process utilizing a Box–Behnken design based on statistical significance analysis. Under ideal extraction conditions, a maximum oil recovery of 68.35% was achieved: solid–liquid ratio, enzyme concentration, enzyme hydrolysis temperature, and enzyme hydrolysis duration were 1:5 (g/mL), 3.23 mg/g, 44 °C, and 2.84 h, respectively. Furthermore, physicochemical properties testing revealed that the oil was of higher quality than other approaches. Meanwhile, the DPPH radical-scavenging activities increased with increased content compared to olive oil, with an IC50 value of 0.082 mg/mL. The method has a lot of potential when it comes to extracting oils from plants. [ABSTRACT FROM AUTHOR]

The effects of adding a probiotic mixture of Bacillus subtilis and B. licheniformis to an aquaponics system on water quality, fish growth and digestive enzyme activity in Mozambique tilapia, Oreochromis mossambicus, were tested. The fish (36.4 g fish−1 ± 0.10; 3.64 kg m−3 tank volume, 10 fish per tank) were kept in 386-L systems and fed 2% of body mass day−1 for ten weeks. The Bacillus product (Sanolife®PRO-W; 5.0 × 1010 CFU g−1) was added twice weekly. Each treatment was duplicated. The product reduced total dissolved solids and electrical conductivity levels and increased weight gain of Mozambique tilapia. There was no difference between treatment and control in the proximate analysis of the fish. In the Bacillus treatment the activity of the digestive enzymes α-amylase, alkaline protease and alkaline phosphatase was higher than in the control. The probiotic used in this study improved water quality and increased fish growth. [ABSTRACT FROM AUTHOR]

This study aimed to analyze in vitro growth of Lactobacillus spp. with crude extract from yellow yam (Dioscorea cayennensis; CEYY) at different concentrations (0%, 2%, 4%, 6%, and 8%) and the growth performance and metabolic responses of tambaqui juveniles fed with diets containing different CEYY concentrations (0, 20, 40, 60, and 80 g kg–1). In the in vitro assay, there was no effect on the Lactobacillus spp. growth. The inclusion of 80 g CEYY kg diet–1 increased growth performance, plasma lysozyme activity and intestinal lactate levels. Hepatic glycogen levels were higher with 60 g CEYY kg diet–1. The lipase and nonspecific alkaline protease enzymes displayed the highest activity in the control group. There was no influence on the hematologic parameters, plasma glucose and amylase enzyme. In conclusion, the supplementation of 80 g CEYY kg diet–1 could be indicated for tambaqui, as it improved growth performance, plasma lysozyme and intestinal lactate. [ABSTRACT FROM AUTHOR]

The increased commercial use and spread of nanoceria raises concerns about the risks associated with its effects on living organisms. Although Pseudomonas aeruginosa may be ubiquitous in nature, it is largely found in locations closely linked with human activity. P. aeruginosa san ai was used as a model organism for a deeper understanding of the interaction between biomolecules of the bacteria with this intriguing nanomaterial. A comprehensive proteomics approach along with analysis of altered respiration and production of targeted/specific secondary metabolites was conducted to study the response of P. aeruginosa san ai to nanoceria. Quantitative proteomics found that proteins associated with redox homeostasis, biosynthesis of amino acids, and lipid catabolism were upregulated. Proteins from outer cellular structures were downregulated, including transporters responsible for peptides, sugars, amino acids and polyamines, and the crucial TolB protein of the Tol-Pal system, required for the structural formation of the outer membrane layer. In accordance with the altered redox homeostasis proteins, an increased amount of pyocyanin, a key redox shuttle, and the upregulation of the siderophore, pyoverdine, responsible for iron homeostasis, were found. Production of extracellular molecules, e.g. pyocyanin, pyoverdine, exopolysaccharides, lipase, and alkaline protease, was significantly increased in P. aeruginosa san ai exposed to nanoceria. Overall, nanoceria at sublethal concentrations induces profound metabolic changes in P. aeruginosa san ai and provokes increased secretion of extracellular virulence factors, revealing the powerful influence this nanomaterial has on the vital functions of the microorganism. [ABSTRACT FROM AUTHOR]

Organic selenium has been widely studied for its ability to better fulfill the physiological functions of selenium. In this study, a novel organic selenium (wheat protein hydrolyzate chelated selenium, WPH-Se) was prepared by chelating the hydrolyzate of wheat protein hydrolyzed by alkaline protease (WPH) with selenium, its preparation process was optimized; and structural and functional properties were investigated. The results showed that the highest selenium content of 10.97 mg/g was obtained in the chelate under the optimal conditions (peptide/selenium mass ratio 2:1, temperature 80°C, pH 8, time 60 min). UV-Vis, FTIR, DSC and SEM provided more information for the characterization of WPH-Se. During the chelating process, selenium ions might be effectively bound to WPH through carboxyl, carbonyl and amino ligands, and the C=O bond and -NH, -OH groups might be the sites. At different pHs, the solubility of WPH-Se was generally lower than that of WPH, which reached a maximum of 32.69% at pH 6. The EAI of WPH-Se was higher than that of WPH, which reached a maximum of 0.84 m2/g, and the ESI reached a maximum of 47.3 min at pH 10. The free sulfhydryl content of WPH-Se was greater than that of WPH, which reached a maximum of 12.78 μmol/g at pH 8. WPH-Se was superior to WPH in terms of foaming ability and emulsification properties. WPH-Se contained a certain amount of aromatic amino acids (7.962%) and a relatively high amount of hydrophobic amino acids (38.490%), and had high nutritional value. Wheat protein peptide chelated selenium would be a useful functional additive, this study would provide data to support further research and application of selenium dietary supplements. [ABSTRACT FROM AUTHOR]

Conventional fermentation processes need to be upgraded to produce a wide array of biomolecules to overcome lower product yield. The cost of production of biomolecules using the fermentation method could be reduced by increasing the product yield by various process enhancement methods. In this study, different innovative process enhancement methods were evaluated to increase the co-production of uricase and alkaline protease at the bioreactor level. Ultrasound-assisted fermentation (UAF), Extractive fermentation (ATPS), and Ultrasound-assisted extractive fermentation (UATPS) are the three innovative methods used for process enhancement. Maximum enzyme production was obtained in a combinatorial approach of ultrasound and extractive fermentation, i.e., ultrasound-assisted extractive fermentation where uricase and protease production enhanced by 2.5 fold and 1.9 fold, respectively, as compared to conventional fermentation. [ABSTRACT FROM AUTHOR]

Felting shrinkage of wool is a major problem which has negative impacts on its performance attributes. Traditional chemical methods used for shrink-resist finishing are suffering from some drawbacks. Therefore, this work aimed at the utilization of microbial protease in wet processing of wool. The bacterial strain Bacillus licheniformis ALW1 produced 52.1 U/mL enzyme activity that was thermophilic with optimum activity at pH 9. Additionally, the enzyme possessed a keratinolytic activity of 4.1 U/mL, suggesting its applicability in wool processing. Therefore, the effects of different treatment conditions; Viz. concentration of the enzyme, temperature, treatment period and pH on some of the physical and chemical characteristics of wool were studied. The felting shrinkage of wool fibers was determined using the Aachener 3-D felting machine. The influence of the enzymatic modification of wool on its dyeability with anionic dye was assessed. Chemical, physical, and mechanical properties of treated samples were evaluated using amino acid analysis, alkali solubility, Fourier Transform Infrared spectroscopy, whiteness index, and tensile properties. Scanning electron microscopy was applied for the examination of wool fibers' surface. The results proved that the modification of wool fibers with the produced enzyme highly improved their felting resistance and dyeability with acid dye without adverse effects on its inherent properties. [ABSTRACT FROM AUTHOR]

The vital state variables in marine alkaline protease (MP) fermentation are difficult to measure in real-time online, hardly is the optimal control either. In this article, a dynamic soft sensor modeling method which combined just-in-time learning (JITL) technique and ensemble learning is proposed. First, the local weighted partial least squares algorithm (LWPLS) with JITL strategy is used as the basic modeling method. For further improving the prediction accuracy, the moving window (MW) is used to divide sub-dataset. Then the MW-LWPLS sub-model is built by selecting the diverse sub-datasets according to the cumulative similarity. Finally, stacking ensemble-learning method is utilized to fuse each MW-LWPLS sub-models. The proposed method is applied to predict the vital state variables in the MP fermentation process. The experiments and simulations results show that the prediction accuracy is better compared to other methods. [ABSTRACT FROM AUTHOR]

Alkaline proteases having activity and stability at alkaline pH possess a large variety of applications in many industries. Growing renewed interest urges the need to find a single alkaline protease with promising properties to be used in different industrial processes. Herein, alkaline proteases produced through fermentation of cheap and easily available organic municipal solid wastes by Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 were purified to investigate their kinetic and thermodynamic parameters, detergent compatibility, dehairing and feather-degrading capability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the purified protease from B. subtilis and E. indicum had molecular mass of ∼45 and 75 kDa, respectively. The protease from B. subtilis and E. indicum showed highest activity at 55 and 50 °C having low Km 1.17 and 0.567 mg/mL and high Vmax 416.67 and 333.33 µmole/min, respectively. The activation energy and temperature quotient of protease from B. subtilis and E. indicum were 26.52 and 65.75 kJ/mole, and 1.0004 and 1.0003 at 20–55 and 20–50 °C, respectively. Thermodynamics analysis revealed the formation of more ordered enzyme–substrate complexes along with spontenity of enzyme reaction. The protease from E. indicum exhibited better compatibility at higher concentration of detergents compared to that from B. subtilis. However, both proteases could retain more than 80% of the activity in the presence of 0.1% commercial laundry detergents. The purified protease from the both sources could degrade almost 90% of barbs and 40% of dry weight of the native feather and that from E. indicum could dehair cow skin. Results reported herein suggest that the alkaline protease from B. subtilis AKAL7 and E. indicum AKAL11 has biotechnological implications in detergent, leather and poultry feather processing industries. [ABSTRACT FROM AUTHOR]

Proteolytic enzymes are one of the significant commercially manufactured enzymes. The manufacture of extracellular alkaline protease by Aspergillus tamarii MTCC5152 was explored using several agricultural by-products as substrates viz., cottonseed meal, wheat bran, skimmed milk and soya flour in submerged fermentation, were found to be efficient for enzyme production and commercially significant. Response surface methodology (RSM) is a statistics-based experimental design, sourced to explore the impact of physical parameters on the manufacture of protease from A. tamarii in a batch stirred tank bioreactor (STBR). The four substantial variables (pH, temperature, inoculum size, and agitation) were carefully chosen for optimization analyses and the statistical pattern was created using a central composite design and the quadratic model has been developed. The optimum conditions for protease production (1.51 U mL−1) where: pH 6.4, temperature 27 °C, inoculum size 2.6%, and agitation 327 rpm. The analysis revealed that the anticipated values were in accord with trial data with a correlation coefficient of 0.969. [ABSTRACT FROM AUTHOR]

The demand for keratinolytic proteases has increased in recent years, in view of their applications in the feed, detergent, fertilizer, leather and textile industries. Recently, studies have focussed on exploration of new and inexpensive carbon sources for their production. Among keratin wastes, dog hair presents no utility and is an environmental concern. In this study, we evaluate the feasibility of using white and melanized dog hair (WDH and MDH, respectively) as alternative substrates for protease production by a Fusarium oxysporum strain. The effects of dog hair concentration, cultivation period, and medium pH on alkaline protease production were investigated using a central composite rotary design (CCRD) and response surface methodology (RSM). The optimization process increased protease activity 14.85- and 7.19-fold, using WDH and MDH, respectively. The alkaline proteases produced from WDH and MDH showed distinct biochemical characteristics. To our knowledge, this is the first report on biotechnological use of this problematic, waste residue. Our results open new avenues for conversion of dog hair into other valuable products, especially feed or fertilizer. [ABSTRACT FROM AUTHOR]

This study evaluated the growth and metabolic activity of consortium and pure cultures Fusarium lateritium LP7 and Trichoderma viride LP5 in response to the presence of 0.5% ethoxylated oleyl-cetyl alcohol (EOCA) in the liquid Czapek-Dox medium. The effectiveness of mentioned cultures was monitored according to the following parameters: biomass dry weight (BDW), pH, quantity of free and total organic acids, proteolytic activity and the qualitative composition of carbohydrates, during 19 days. The biodegrading efficiency was determined spectrophotometrically. The BDW of consortium was significantly stimulated by EOCA (16.59%) whereas biomass of LP7 was significantly inhibited (30.61%). The EOCA had influence on decrease in pH value of the media of LP5 and consortium, and pH changes were correlated with the amount of excreted organic acids. The alkaline protease activities of consortium, LP7 and LP5 retained 73%, 62.2% and 49.5% activity respectively in the presence of EOCA. Consortium has shown the best biodegradation capacity up to 82% of EOCA. The pure cultures were less effective in biodegradation and removed approximately 65% (LP7) and 60% (LP5) of EOCA after 19 days. In brief, the synergistic interaction between pure cultures enhances capacity to reduce EOCA in environment and influences production of some biotechnological useful metabolites. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Discovery of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerization with potent liver cancer inhibitory activity." The authors apologize for errors in Figure 5A ∼ C of the article and provide the correct version of the figure. The figure shows that compound MY-1121 binds to β-tubulin directly on the colchicine binding site, and its affinity with the binding site is negatively correlated with the level of the tubulin adduct band. Additionally, the compound is able to inhibit the hydrolysis of β-tubulin by Alkaline Protease. [Extracted from the article]

To overcome the problem that soft-sensing model cannot be updated with the bioprocess changes, this article proposed a soft-sensing modeling method which combined fuzzy c-means clustering (FCM) algorithm with least squares support vector machine theory (LS-SVM). FCM is used for separating a whole training data set into several clusters with different centers, each subset is trained by LS-SVM and sub-models are developed to fit different hierarchical property of the process. The new sample data that bring new operation information is introduced in the model, and the fuzzy membership function of the sample to each clustering is first calculated by the FCM algorithm. Then, a corresponding LS-SVM sub-model of the clustering with the largest fuzzy membership function is used for performing dynamic learning so that the model can update online. The proposed method is applied to predict the key biological parameters in the marine alkaline protease MP process. The simulation result indicates that the soft-sensing modeling method increases the model's adaptive abilities in various operation conditions and can improve its generalization ability. [ABSTRACT FROM AUTHOR]

In this study, 6 isolates out of 30, forming comparatively zone as a result of casein and skim milk hydrolysis were selected and further studied for selection of the isolate producing maximum alkaline protease activity. Isolate No. F19 was the most potential alkaline protease producing fungal strain. Upon 18S rRNA analysis, it displayed maximum similarity with Alternaria alternata. Agricultural residue, cauliflower leaves were used as main substrate for production of alkaline protease under solid state fermentation. Supplementation of different nutritional components parameters such as carbon, nitrogen and metals were statistically optimized for maximum enzyme production. The enzyme secretion improved 1.55 fold, when the variables were optimized by Response surface methodology. Under the optimized conditions, the protease experimental yield (930 U/gds) closely matched the yield predicted by the statistical model (931 U/gds) with R2 value of 0.9965. [ABSTRACT FROM AUTHOR]

Conventionally, degumming is carried out at 90°C—110°C temperature by boiling the raw silk with Marseilles soap and sodium bicarbonate which eventually requires a lot of water and energy. In this study, degumming of Chinese bivoltine raw silk fibres with alkaline protease produced by Beauveria sp. (MTCC 5184) is studied. Complete degumming was obtained in 45 min with 75 units of enzyme per gram of silk. Degumming was found to be optimal at 50°C and pH 9.0. Scanning electron micrographs showed that the sericin deposits were removed and the obtained fibres were clean, separated, had smooth feel with shine as compared to untreated fibres. Sericin isolated from silk cocoon (by-product which goes waste) was hydrolyzed with the same alkaline protease obtained from Beauveria sp. to get small molecular weight peptides. These peptides can be utilized further for cosmetic, pharmaceutical, and various industrial applications. [ABSTRACT FROM AUTHOR]

This study explores a novel concept of coproduction of uricase and alkaline protease byBacillus licheniformisusing single substrate in single step. Seven local bacterial strains were screened for uricase production, amongst whichB. licheniformisis found to produce highest uricase along with alkaline protease. Optimization of various factors influencing maximum enzyme coproduction byB. licheniformisis performed. Maximum enzyme productivity of 0.386 U/mL uricase and 0.507 U/mL alkaline protease is obtained at 8 hr of incubation period, 1% (v/v) inoculum, and at 0.2% (w/v) uric acid when the organism is cultivated at 25°C, 180 rpm, in a media containing xylose as a carbon source, urea as a nitrogen source, and initial pH of 9.5. The statistical experimental design method of Box–Behnken was further applied to obtain optimal concentration of significant parameters such as pH (9.5), uric acid concentration (0.1%), and urea concentration (0.05%). The maximum uricase and alkaline protease production byB. licheniformisusing Box–Behnken design was 0.616 and 0.582 U/mL, respectively, with 1.6- and 1.13-fold increase as compared to one factor at a time optimized media. This study will be useful to develop an economic, commercially viable, and scalable process for simultaneous production of uricase and protease enzymes. [ABSTRACT FROM PUBLISHER]

Cashew nut protein powder was hydrolysed by alkaline protease to prepare ACE inhibitory peptides. Results showed that the optimum conditions were: substrate concentration, 7 g/100 mL; amount of enzyme, 3%; pH, 10.5; hydrolysis time, 6 h; and temperature, 45°C. Under these conditions, ACE inhibitory rate was 57.45%. ACE inhibitory peptides were filtered using an ultrafiltration membrane of different molecular weights and Sephadex G-15 gel column. We found that the molecular weight was <3000 Da, and the ACE inhibitory rate concentration was 100 mg/mL. The M2 component, which was the most active component in ACE inhibitory peptides, yielded an ACE inhibitory rate of 77.58%. The M2 component contained 18 types of amino acids, including seven types of essential amino acids with the highest proline content reaching 23.56% (P < 0.01). The molecular weight of the M2 constituent was observed as 389 Da, with an amino acid sequence of Glu- Ser - Arg. [ABSTRACT FROM AUTHOR]

Cashew nut protein powder was hydrolysed by alkaline protease to prepare ACE inhibitory peptides. Results showed that the optimum conditions were: substrate concentration, 7 g/100 mL; amount of enzyme, 3%; pH, 10.5; hydrolysis time, 6 h; and temperature, 45°C. Under these conditions, ACE inhibitory rate was 57.45%. ACE inhibitory peptides were filtered using an ultrafiltration membrane of different molecular weights and Sephadex G-15 gel column. We found that the molecular weight was <3000 Da, and the ACE inhibitory rate concentration was 100 mg/mL. The M2component, which was the most active component in ACE inhibitory peptides, yielded an ACE inhibitory rate of 77.58%. The M2component contained 18 types of amino acids, including seven types of essential amino acids with the highest proline content reaching 23.56% (P< 0.01). The molecular weight of the M2constituent was observed as 389 Da, with an amino acid sequence of Glu- Ser - Arg. [ABSTRACT FROM AUTHOR]

In this study, the extracellular thermostable alkaline protease out of A10 strain was purified 1.38-fold with 9.44% efficiency through the ammonium sulfate precipitation-dialysis and DE52 anion exchange chromatography methods. The molecular weight of the enzyme in question along with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be approximately 40.55 kDa, whereas the optimum pH and temperature ratings were identified as 9.0 and 70 °C, respectively. It was seen that the enzyme had remained stable between pH 7.5–10.5 range, protecting more than 90% of its activity in the wake of 1 h incubation at 60–70 °C. It was also observed that the enzyme enhanced its activity in the presence of Mg2+, Mn2+, K+, while Fe2+, Ni2+, Zn2+, Ag+ and Co2+ decreased the activity. Ca2+, however, did not cause any change in the activity. The enzyme was seen to have been totally inhibited by phenylmethylsulfonyl fluoride, therefore, proved to be a serine alkaline protease. [ABSTRACT FROM PUBLISHER]

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at −20 °C and −80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at −20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at −20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at −80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay. [ABSTRACT FROM PUBLISHER]

Proteases are now recognized as the most indispensable industrial biocatalyst owing to their diverse microbial sources and innovative applications. In the present investigation, a thermostable, organic solvent-tolerant, alkaline serine protease fromBacillus circulansMTCC 7942, was purified and characterized. The protease was purified to 37-fold by a three-step purification scheme with 39% recovery. The optimum pH and temperature for protease was 10 and 60°C, respectively. The apparent molecular mass of the purified enzyme was 43 kD as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Kmand Vmaxvalues using casein-substrate were 3.1 mg/mL and 1.8 µmol/min, respectively. The protease remained stable in the presence of organic solvents with higher (>3.2) logPvalue (cyclohexane,n-octane,n-hexadecane,n-decane, andn-dodecane), as compared to organic solvents with lower (<3.2) logPvalue (acetone, butanol, benzene, chloroform, toluene). Remarkably, the protease showed profound stability even in the presence of organic solvents with less logPvalues (glycerol, dimethyl sulfate [DMSO],p-xylene), indicating the possibility of nonaqueous enzymatic applications. Also, protease activity was improved in the presence of metal ions (Ca2+, Mg2+, Mn2+); enhanced by biosurfactants; hardly affected by bleaching agents, oxidizing agents, and chemical surfactants; and stable in commercial detergents. In addition, a protease–detergent formulation effectively washed out egg and blood stains as compared to detergent alone. The protease was suitable for various commercial applications like processing of gelatinous film and as a compatible additive to detergent formulation with its operative utility in hard water. [ABSTRACT FROM AUTHOR]

Fish bone is a good source of gelatin. In this study, gelatins were prepared from tilapia bone after the bone was pretreated with alkali protease, desalted immediately by 0.6 mol L−1HCl, and hydrolyzed by papain or limed by Ca(OH)2. Gelatins extracted from papain-treated tilapia bone exhibited space structures similar to those of alkali-treated tilapia bone. Despite this similarity, many differences were observed between these gelatin samples. Compared with alkali-treated gelatin, papain-treated gelatins showed higher values for imino residue content, molecular weight proportion, bloom strength, and viscosity. The bloom strengths of the second and third papain-treated gelatins were 163 and 94 bloom, respectively, which were lower than the bloom strength of the first papain-treated gelatin (189 bloom). The viscosities of the three papain-treated gelatin samples were 4.18, 2.81, and 0.51 mPa.s−1. The first papain-treated gelatin achieved the highest gelling (16 °C) and melting points (23.9 °C). The yields of the first (5.40%) and second (6.71%) papain-treated gelatins were higher than those of the alkali-treated gelatins (3.33 and 5.76%, respectively). However, the yield of the third papain-treated gelatin (2.27%) was lower than that of the third alkali-treated gelatin (5.42%). More importantly, papain hydrolysis can prevent destruction by Ca(OH)2in the bone structure and effectively reduce the denaturation temperature of tilapia bone collagen. Moreover, papain hydrolysis can dramatically reduce the time required for liming (0.8% of traditional liming process spent). Papain hydrolysis is a clean production method that can replace traditional liming. [ABSTRACT FROM PUBLISHER]

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 asPenicillium citrinumby 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 byP. citrinumYL-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 byP. citrinumYL-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. [ABSTRACT FROM PUBLISHER]

Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods. [ABSTRACT FROM PUBLISHER]

A fungal alkaline protease ofScopulariopsisspp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0–12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co2+and Mn2+at 10 mMbut was unaffected by Ba2+, Mg2+, Cu2+, Na+, K+, and Fe2+. Ca2+and Fe3+moderately reduced the activity (∼18%); however, a reduction of about 40% was seen for Zn2+and Hg2+. The enzyme activity was completely inhibited by 5 mMphenylmethylsulfonyl fluoride (PMSF) and partially byN-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H2O2and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (−20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel–enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations. [ABSTRACT FROM AUTHOR]

An efficient alkaline protease producer was isolated from the water of the hyperalkaline-saline Lonar soda lake and identified as Bacillus alcalophilus LW8 by culture-dependent techniques by its morphological, microscopic, biochemical, physiological and molecular characteristics. The 16S rRNA gene sequence was submitted to the GenBank nucleotide repository under accession number KC689353 . Alkaline protease production was optimized by adopting a one-variable-at-a-time approach in a submerged fermentation system in modified fermentation medium (MFM). The optimized components of MFM were (w/w) casein (1%), sugarcane molasses (1%), NaCl (1%), ammonium sulphate (0.5%), KH 2 PO 4 (0.05%), K 2 HPO 4 (0.05%) and Na 2 CO 3 (1%). Optimized culture conditions were used for alkaline protease production. The final yield of partially purified alkaline protease after dialysis was 53.35%. The molecular mass of the dialysed alkaline protease was 27 kDa. On a Lineweaver–Burk plot, the calculated K m and V max values were 24 mg/mL and 1000 U/mg, respectively. The enzyme was remarkably stable in the pH range 7.0–12.0, with optimum activity at pH 10.0. LW8 alkaline protease was completely inhibited by phenylmethylsulphonyl fluoride at 10 mmol/L, indicating that the enzyme belongs to the serine protease class. The metal ions Ca 2+ , Ba 2+ , Mg 2+ , Zn 2+ , Fe 3+ , Cu 2+ and Mn 2+ increased the catalytic activity of partially purified alkaline protease. The protease effectively decomposed the gelatinous coating on an X-ray film, hydrolysed blood clot, a blood-stain from a piece of cotton fabric and hairs from a piece of goat skin. [ABSTRACT FROM AUTHOR]

Response surface methodology (RSM) was employed to enhance the production of a thermostable alkaline protease from Bacillus circulans. Significant influences of peptone, yeast extract, and glucose on protease production were noted with a one-variable-at-a-time optimization strategy. Then, a full factorial central composite design (CCD) was applied to study the effects of glucose, peptone, and yeast extract to determine the optimal concentrations of these compounds for protease production by B. circulans under shake flask fermentation conditions. The statistical reliability and significance of the model was validated by an F-test for analysis of variance (ANOVA); enzyme production was improved significantly under optimized conditions. The enzyme was purified by ammonium sulphate fractionation, and gel filtration chromatography. Maximum enzyme activity was observed at 60°C temperature, and at pH 10. Alkaline protease from B. circulans showed excellent compatibility and stability in the presence of commercial detergents like Ariel, Surf Excel, Tide, Rin, Nirma, Wheel, and Doctor and showed excellent blood destaining effectiveness with commercial detergents. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Degumming of Chinese bivoltine silk with alkaline proteases from various microbial sources was investigated and compared with commercial enzymes. Among the proteases tested, two fungal and two actinomycete proteases were promising, which showed weight loss similar to conventional method (19.58% to 21.78%). Conidiobolus brefeldianus and BOA-2 proteases were best enzymes, which showed weight loss similar to conventional method with low enzyme concentrations and in shorter time. No significant differences were found in tensile strength or elongation at break by enzymatic degumming indicating that there was no damage to the fiber. Scanning electron micrographs showed the sericin deposits were removed and the fibers were separated. [ABSTRACT FROM AUTHOR]

A new hyperactive alkalophilic bacterial strain (Bacillussp. BGS) was isolated from samples collected from soil that received the effluent of a milk processing industry located in Madurai, Tamilnadu, India, and this bacterial strain was used for the production of alkaline protease. Four out of eight variables, such as molasses, peptone, pH, and inoculum size, have been identified through Plackett–Burman (PB) design and used for the alkaline protease production. These significant variables were further optimized through a hybrid system of response surface methodology (RSM) followed by genetic algorithm (GA). The optimal combination of media components and culture conditions for maximal protease production was found to be 16.827 g/L of peptone, 1.128% (v/v) of molasses, pH value of 11, and 2% (v/v) of inoculum size. A 6.36-fold increase in protease production was achieved through the RSM–GA hybrid system. The protease activity increased significantly with an optimized medium (2,992.75 U/mL) as opposed to an unoptimized basal medium (470.35 U/mL). [ABSTRACT FROM PUBLISHER]

The precursor tripeptide of thymopentin was synthesized by a combination of chemical and enzymatic methods. First, Val-Tyr-OH dipeptide was synthesized by a novel chemical method in two steps involving preparation of NCA-Val. Second, the linkage of the third amino acid Z-Asp-OMe to Val-Tyr-OH was completed by an enzymatic method under kinetic control. An industrial alkaline protease alcalase was used in water–organic cosolvent systems. The synthesis reaction conditions were optimized by examining the effects of several factors including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-Tyr-OH. The optimum condition is of pH 10.0, 35°C, acetonitrile/Na2CO3-NaHCO3 buffer system (85:15, v/v), and reaction time of 2.5 hr, which achieves tripeptide yield of more than 70%. [ABSTRACT FROM AUTHOR]

BLAP (alkaline protease from Bacillus licheniformis) was used as a biocatalyst in the Knoevenagel condensations of aromatic, hetero-aromatic and α;β-unsaturated aldehydes with less reactive acetylacetone or ethyl acetoacetate. The reactions were performed in organic solvent in the presence of water. The functionalized trisubstituted alkenes and α,β,γ,δ-unsaturated carbonyl compounds could be obtained in moderate to good yields with E/Z selectivities up to >99:1. This biocatalytic reaction provided an alternative pathway for Knoevenagel condensation, which also demonstrates a novel case of unnatural activity of existing enzymes. [ABSTRACT FROM AUTHOR]

The effect of different feeding strategies, involving constant rate and linear feeding with negative and positive slopes, on protease production of an indigenous Bacillus clausii was investigated. The results indicated that alkaline protease was produced at high levels soon after glucose was completely consumed. Alkaline protease activity was at a maximum during the constant feeding rate and in the absence of glucose and presence of mineral salts and yeast extract in the feed medium. Maximum protease production in the fed-batch culture using an optimized level of feeding composition was 2430 ± 67 U/mL, which increased by up to 35% when compared to the 1800 ± 14 U/mL produced during batch culture. During batch fermentation, the protease yield and productivity obtained were 90000 U/g and 64285 U/L · h; however, under fed-batch conditions, these were 121500 U/g and 71470 /L · h, respectively. Hence, the suggested strategy has the potential to be applied to industrial production of protease used in detergent products. [ABSTRACT FROM AUTHOR]

Wool is commonly treated with alkaline protease enzyme to improve handle and shrink resistance. The limitations associated with the usage of this enzyme on wool are excessive weight and strength losses and pH sensitive enzyme activity, etc. Wool is generally stable if processed in acidic conditions rather than alkaline conditions. This study explores the possibility of using acid protease enzyme to improve handle and shrink resistance of wool. The acid protease enzyme is applied on acid and alkaline peroxide bleached wool fabrics using optimized process parameters, and the results are compared with alkaline protease enzyme treated wool fabric. The acid protease treated fabrics show 30% higher softness than alkaline protease treated fabrics with 72% and 62% lower weight and strength losses, respectively. The alkaline peroxide bleaching prior to acid protease treatment provides 100% and 67% higher whiteness and shrinkage resistance, respectively, to wool than the acid peroxide bleaching. The action of the enzymes on wool and shrink resistance and handle of the treated fabrics is characterized using SEM, FT-IR and KES-F method. [ABSTRACT FROM AUTHOR]

A 33 kDa protein present in Aspergillus flavus–infected maize embryo was identified as a fungal alkaline protease (ALP). This protein became one of the major extracellular proteins of A. flavus in potato dextrose broth medium culture filtrate after 3 days, but was expressed at low levels or undetectable in A+M medium containing gelatin or starch, respectively. The activity of purified ALP was significantly inhibited in the presence of 0.5 mM phenylmethylsulfonyl fluoride (PMSF), or 1.43 μμ (200 μg/ml) maize 14 kDa trypsin inhibitor. Further study demonstrated that reduction of this ALP by PMSF also significantly reduced the level of aflatoxin accumulation in A. flavus culture, suggesting an important role for ALP in the infection of maize kernels and subsequent aflatoxin accumulation. [ABSTRACT FROM AUTHOR]

An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60°C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The Km and kcat values for hydrolysis of this substrate are 0.347 mM and 1141 min-1 respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30°C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus clausii reported in the literature. [ABSTRACT FROM AUTHOR]

Purpose: To measure the specific virulence contributions of two Pseudomonas aeruginosa proteases, elastase B and alkaline protease, when expressed separately by Pseudomonas putida in a rabbit model of bacterial keratitis. Methods: P. putida KT2440 was transformed with plasmids that enabled the extracellular production of either elastase or alkaline protease. Protease expression was confirmed by zymography and immunoblotting. P. putida expressing elastase, alkaline protease, or vector alone was injected intrastromally (103 colony forming units [CFU]) into rabbit corneas (n = 6). Infected eyes were graded by slit-lamp examination (SLE) at 20, 24, 28, and 32 hr postinfection (PI). Rabbits were sacrificed at 33 hr PI, and the log CFU (±SEM) per cornea was determined. Results: SLE scores for eyes infected with P. putida producing elastase were significantly higher than those infected with vector alone at all time points (p ≤ 0.008). SLE scores for eyes infected with P. putida producing alkaline protease were not significantly higher than the control (p ≥ 0.1), but small erosions formed in 33% of corneas. At both 24 and 28 hr PI, the SLE scores for corneas infected with P. putida producing elastase were significantly higher than those infected with P. putida producing alkaline protease (p ≤ 0.002). Conclusions: Elastase production by P. putida caused significant increases in SLE scores whereas expression of alkaline protease caused limited corneal erosions. This suggests that the production of elastase during P. aeruginosa keratitis enhances ocular pathology, whereas alkaline protease production contributes to limited corneal erosion. [ABSTRACT FROM AUTHOR]

Chicken feather is mainly keratin which is not normally degraded by common proteolytic enzymes. It is generated in large quantities as a by-product of poultry processing industry and little is known about its recycling. We reported the biodegradation of chicken feather waste (CFW) by a recombinant Bacillus subtilis strain harboring a multicopy alkaline protease (aprE) gene which acted as a keratinase. In present work, degradation of CFW was greatly enhanced by optimizing several factors. Pretreatment of CFW with NaOH or two times autoclaving enhanced the process. Cultures supplemented, separately, with 0.1% yeast extract or 0.5% corn oil enhanced the degradation of CFW. Biodegradation was optimized when the cultures contained 2% (w/v) CFW, 5% inoculum size, and incubated at 45°C for 3–4 days. Biodegradation was evaluated by monitoring soluble proteins and NH2-free amino groups that were obtained as a result of the enzymatic hydrolysis of feather. Biodegradation of feather waste using these recombinant cells represents an alternative way to improve the nutritional value of feather. Moreover, the release of soluble proteins and amino acids from feather as well as the secreted keratinase enzyme would promote several industries based on feather waste. [ABSTRACT FROM AUTHOR]

PURPOSE. Alkaline protease has been associated with virulence in Pseudomonas aeruginosa corneal infections. To define the role of this enzyme in such infections, isogenic mutants of P. aeruginosa deficient in alkaline protease production were constructed. This study examines the ability of these mutants to adhere to scarified corneal tissue in vitro and to establish corneal infections in vivo. METHODS. Mutants were constructed by allelic exchange in two phenotypically different wild type strains, PAO1 (invasive) and ATCC 19660 (cytotoxic). Alkaline protease-deficient mutants were characterized by zymography and western blot analysis of bacterial culture supernatants. Allelic exchange was confirmed by PCR analysis of the disrupted aprA gene of the mutants. Adherence of wild type and mutant strains to scarified corneal epithelium was assessed by an in vitro organ culture assay, while ocular virulence of the strains was determined in vivo using a mouse scarification model of bacterial keratitis. RESULTS. Being isogenic, phenotypes of mutants were identical to their respective parents with the exception of the loss of alkaline protease production. The absence of alkaline protease did not alter corneal adherence or ocular virulence of the organisms when compared to similar wild type strains. CONCLUSIONS. These data provide evidence that alkaline protease produced by P. aeruginosa is not essential in the pathogenesis of P. aeruginosa keratitis. [ABSTRACT FROM AUTHOR]