Purification and characterization of an organic solvent and detergent‐tolerant novel protease produced by Bacillus sp. RKY3

BACKGROUND: Purification and characterization of a novel protease produced by Bacillus sp. RKY3, has been investigated, with special emphasis on the stability of the enzyme in the presence of different oxidizing and reducing agents as well as organic solvents. The enzyme was purified in two steps through concentration of the crude enzyme by ammonium sulfate precipitation, followed by anion exchange chromatography. RESULTS: The purified protease had a molecular mass of approximately 38 kDa, which was highly active over a broad range of pH between 7.0 and 9.0 and was also stable over a wide pH range from 5.0 to 11.0. Although the optimum temperature for enzyme activity was found to be 60 °C, it was rapidly deactivated at temperatures above 60 °C. It also showed good stability at 50 °C, with a 70 min half‐life. Ca²⁺ ions did not greatly enhance the activity or the stability of the enzyme. PMSF (1 mmol L^?1) completely inhibited the protease activity, and thus the purified protease was considered to be serine protease. The purified protease was stable with oxidants (H₂O₂, 2%), reducing agents (sodium dodecyl sulfate, 2%), and organic solvents (25%) such as benzene, hexane, and toluene. CONCLUSION: The purified enzyme, protease, seems to possess potential applications in protease‐based detergent and bleaching industries. The enzymatic activity against a wide variety of substrates suggests that the purified enzyme should be investigated for a range of commercial applications, especially for soy protein and gelatin hydrolysis in the food processing industry. Copyright © 2008 Society of Chemical Industry     

Cold-active enzymes from cold-adapted bacteria

The properties of amylase, lipase and protease, excreted by newly isolated bacteria from snow-covered soil, salmon intestine and crab intestine, have been investigated. One amylase, one lipase, and three proteases have been characterized by shifts in their apparent optimal activities toward low temperatures and by reductions in their activation energy values. The discovered enzymes were rapidly inactivated at temperatures above the optimum (30 to 40°C). These results suggest that the enzymes are cold-active. The best cold-active protease producer, isolated from salmon intestine, has been identified as Flavobacterium balustinum by the analysis of 16S rRNA. The optimal growth temperature of this bacterium was 20°C, but a higher amount of protease activity was present at 10°C.     

Effects of Spray Drying Temperature and Additives on the Stability of Serine Alkaline Protease Powders

In this study, after production by recombinant Bacillus subtilis (BGSC-1A751), carrying pHV1431::subc gene in the complex medium and separation of solids from the fermentation broth, serine alkaline protease (SAP) was dried in order to investigate the stabilization during spray drying and subsequent storage. The effect of air inlet temperature of the spray dryer between T = 70 and 130°C and the effect of protective additives, glucose and maltodextrin, at 0–2% (w/v) on SAP activity during spray drying and storage stability of obtained SAP powders at 4°C for a long period (6 months) were evaluated. Increasing drying air inlet temperature generally resulted in an increase in activity loss; moreover, higher absorbance peaks observed at wave number 1061 cm^?1 of the IR spectrums when drying temperature is increased indicates the structural change in the SAP molecule. In most cases presence of additives provided higher activities both after drying and during storage period compared to no additive case. Drying the enzyme with 1% (w/v) glucose at T = 110°C resulted in the highest enzyme activity after drying and storage processes.     

Kinetics of aspartame precursor synthesis catalysed by Pseudomonas aeruginosa elastase

Elastase isolated from Pseudomonas aeruginosa IFO 3455 was found to be an efficient protease to catalyse the synthesis of N-benzyloxycarbonyl-aspartyl-phenylalanine methyl ester, the precursor of the dipeptide sweetener, aspartame. The influence of methanol as a cosolvent in this synthetic reaction was investigated. It was found that the synthesis of the dipeptide precursor was most efficient in 25% (v/v) methanol, pH 7·0 at about 25°C for a reaction time of about 3 h. However, the activity of the enzyme was greatly reduced in 90% methanol. The values of K and k₂ for N-benzyloxycarbonyl-aspartic acid were 0·17 mol dm^?3 and 11·9 mol dm^?3 s^?1 respectively.     

Cyclopinolenic acids: Synthesis,derivatives and thermal properties

The two isomeric cyclopinolenic acids (CP-1 and CP-2), components of distilled tall oil, have been synthesized by means of an intramolecular Diels-Alder reaction of isomers of 5,10,12-octadecatrienoic esters, themselves synthesized in a stereocontrolled manner. The 5cis,10-trans,12trans isomer cyclizes at 200°C to a 1:3 mixture of esters of CP-1 and CP-2. At 200°C, the 5cis,10trans,12cis isomer is unreactive, but at 240°C it gives the same CP-1 + CP-2 ester mixture, presumably by way of prior isomerization to the 5cis,10trans,12trans isomer. A subambient thermal study of CP-1 and CP-2 and their derivatives shows that the compounds, excluding CP-1, lack crystalline structures or melting points, and display glass transitions only, below −80°C for esters and below −50°C for the carboxylic acids.     

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.     

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.     

Purification and characterization of thermophilic and alkalophilic tributyrin esterase fromBacillus strain A30-1 (ATCC 53841)

An extracellular esterase (EC 3.1.1.1) from a thermophilicBacillus A30-1 (ATCC 53841) was purified 139-fold to homogeneity by sodium chloride (6 M) treatment, ammonium sulfate fractionation (30–80%) and phenyl-Sepharose CL-6B column chromatography. The native enzyme was a single polypeptide chain with a molecular weight of about 65,000 and an isoelectric point at pH 4.8. The optimum pH for esterase activity was 9.0, and its pH stability range was 5.0–10.5. The optimum temperature for its activity was 60°C. The esterase had a half-life of 28 h at 50°C, 20 h at 60°C and 16 h at 65°C. It showed the highest activity on tributyrin, with little or no activity toward long-chain (12–20 carbon) fatty acid esters. The enzyme displayed K_m and K_cat values of 0.357 mM and 8365/min, respectively, for tributyrin hydrolysis at pH 9.0 and 60°C. Cyclodextrin (α, β, and γ), Ca²⁺, Co²⁺, Mg²⁺ and Mn²⁺ enhanced the esterase activity, and Zn²⁺ and Fe²⁺ acted as inhibitors of the enzyme activity. The enzyme activity was not affected by ethylenediaminetetraacetic acid, p-chloromercuribenzoate andN-bromosuccinimide. This paper was presented in part at the 82nd Annual Meeting and Exposition of the American Oil Chemists’ Society, held May 12–15, 1991, in Chicago, Illinois.     

Immobilization of proteolytic enzyme on highly porous activated carbon derived from rice bran

Highly porous activated carbon (HPAC) was used as carrier matrix for immobilization of acid protease (AP). Immobilization of acid protease on mesoporous activated carbon (AP-HPAC) performs as best enzyme carrier. At pH 6.0, 250 mg acid protease g⁻¹ HPAC was immobilized. The optimum temperature for both free and immobilized AP activities were 50 °C. After incubation at 50 °C, the immobilized AP maintained about 50% of its initial activity, while the free enzyme was completely inactivated. When testing the reusability of AP-HPAC combination immobilized system, a significant catalytic efficiency was maintained along more than five consecutive reaction cycles. The highly porous nature of the carbon permits significant higher loadings of enzyme, which results in a higher enzyme-support strength and increased stability. The changes in the AP, HPAC and AP-HPAC were confirmed by Fourier Transform Infrared spectroscopy (FT-IR). Furthermore, scanning electron microscopy (SEM) allowed us to observe that the morphology of the surface of HPAC and the AP-HPAC.     

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     

Optimizing reaction parameters for the enzymatic synthesis of epoxidized oleic acid with oat seed peroxygenase

Peroxygenase is a plant enzyme that catalyzes the oxidation of a double bond to an epoxide in a stereospecific and enantiofacially selective manner. A microsomal fraction containing peroxygenase was prepared from oat (Avena sativa) seeds and the enzyme immobilized onto a hydrophobic membrane. The enzymatic activity of the immobilized preparation was assayed in 1 h by measuring epoxidation of sodium oleate (5 mg) in buffer-surfactant mixtures. The pH optimum of the reaction was 7.5 when t-butyl hydroperoxide was the oxidant and 5.5 when hydrogen peroxide was the oxidant. With t-butyl hydroperoxide as oxidant the immobilized enzyme showed increasing activity to 65°C. The temperature profile with hydrogen peroxide was flatter, although activity was also retained to 65°C. In 1 h reactions at 25°C at their respective optimal pH values, t-butyl hydroperoxide and hydrogen peroxide promoted epoxide formation at the same rate. Larger-scale reactions were conducted using a 20-fold increase in sodium oleate (to 100 mg). Reaction time was lengthened to 24 h. At optimized levels of t-butyl hydroperoxide 80% conversion to epoxide was achieved. With hydrogen peroxide only a 33% yield of epoxide was obtained, which indicates that hydrogen peroxide may deactivate peroxygenase.     

Chemical modification and immobilization of papain

Papain, an endolytic cysteine protease (EC: 3.4.22.2), from Carica papaya latex has been chemically modified using succinic anhydride. This reagent reacts with the amino group of the lysine residues in the enzyme, thereby changing its net charge from positive to negative. The resultant enzyme had its optimum pH shifted from pH 6 to 8 and there was no change in the temperature optima of 70 °C. The modified papain had a specific activity of about 62.8 IU mg^?1 of protein at pH 8.0 at 30 °C, whereas for the native enzyme it was 46.57 IU mg^?1 under same conditions. Stability of the modified papain was further increased by entrapping in alginate/starch beads. The immobilized papain retained its activity even after six cycles of hydrolysis. The wet beads, when dried at 50 ± 2 °C, increased the storage stability of the immobilized enzyme. The succinylated papain is active in various organic solvents and hence can be successfully used in biotransformations as well as being used as a proteolytic component in detergents. Copyright © 2004 Society of Chemical Industry     

Production,thermal stability and immobilisation of inulinase from Fmarium oxysporum

Fusarium oxysporum produced maximum extracellular inulinase after 9 days of its growth at 25°C on a medium (pH 5.5) containing 3% fructan and 0.2% sodium nitrate. The level of this enzyme decreased on the addition of either glucose, fructose, galactose or sucrose to F. oxysporum already growing on a fructan-containing medium. A significant increase in invertase production which resulted in an increase of the invertase/inulinase (S/I) ratio, was observed on addition of inulin to this fungus growing on other carbon sources. Glycerol (10%) gave better protection to inulinase against thermal denaturation at 50°C compared to ethylene glycol and sorbitol. Inulinase immobilised in polyacrylamide gel retained 45% of its original activity. The immobilised enzyme showed a higher optimum temperature (45°C) compared to free enzyme (37°C). The immobilised enzyme after storage at 25°C for 96 h showed 58% activity. Thermal stability of entrapped inulinase increased in the presence of inulin.     

Separation of the protease enzymes of Bacillus licheniformis from the fermentation medium by crossflow ultrafiltration

The separation from fermentation medium of extracellular serine alkaline protease (SAP) enzyme produced by Bacillus licheniformis was investigated using a crossflow ultrafiltration system. SAP was separated from the high molecular weight neutral protease (NP) and amylase (AMY) enzymes and from the low molecular weight organic acids and amino acids in a crossflow ultrafiltration system with 30 000 Da and 10 000 Da MWCO polysulfone membranes, respectively. The effects of transmembrane pressure (TMP), recirculation velocity (v), and initial enzyme concentration (C_E) on the permeate flux, on the activities of SAP, NP and AMY enzymes, and on the recovery of SAP were investigated. High permeate flux was obtained at high recirculation velocity and TMP, but at low initial enzyme concentration. SAP enzyme recovery and activity measured in the system also showed alterations with hydrodynamic conditions. The best operation conditions for the separation of SAP from NP and AMY were TMP = 20 kPa, v = 0.50 ms⁻¹ and C_E = 0.28 gdm⁻³. The separation of SAP from the organic and amino acids was best performed at TMP = 100 kPa, v = 0.33 ms⁻¹ and C_E = 0.40 gdm⁻³. © 2000 Society of Chemical Industry     

Characterization of membrane-bound lipase from a thermophilic Rhizopus oryzae isolated from palm oil mill effluent

The characteristics of the membrane-bound lipase from a thermophilic Rhizopus oryzae were studied. The pH and temperature optima for lipase activity were at 7.0 and 37°C, respectively. The enzyme was stable and acidic conditions, retaining more than 80% of its initial activity at pH 4.0 after 30 min incubation. It was stable up to 50°C with 70% of initial activity retained after 3 h incubation. The enzyme is 1,3 specific and exhibits substrate preference. Monoacid triglyceride substrates were hydrolyzed better than methyl esters, polyoxysorbitan and sorbitan substrates.     

Partial purification and characterization of acylester hydrolase from Lupinus mutabilis

An alkaline acylester hydrolase was partially purified from germinated seeds of Lupinus mutabilis. Hydrolytic activity was absent in the crude extract of ungerminated lupine seed, but it increased and peaked at the fourth day in the germinating seedling. The purification scheme involved homogenization, centrifugation, acetone precipitation, anion exchange chromatography, pH precipitation, and hydrophobic interaction chromatography. The acylester hydrolase was purified 126-fold, and the overall activity yield was 10%. The molecular weight estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis was 60 kDa. The enzyme had an isoelectric point of 6.2 and showed maximal activity at pH 8.0. The enzyme showed good stability between pH 5.0 and 9.0. In the pH range 7.0–7.5, enzyme precipitation was observed. The enzyme was stable from 0 to 25°C for 5 h and at 45°C lost 50% of its activity in the same period of time. At higher temperatures, the enzyme showed low thermal stability. However, the highest initial activity was found to be at 45°C. Nonionic surfactants and cholic acid enhanced the activity of the enzyme. The activity was reduced by the addition of toluene and isooctane and increased by the addition of diethyl ether, acetonitrile, methanol, and pyridine. The activity was reduced by 37% in the presence of 1 mM Cu²⁺ ions. The enzyme-hydrolyzed triolein showing no positional specificity.     

Immobilization of <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> on developed support using microbial transglutaminase

α-amylase from Bacillus licheniformis was successfully immobilized on developed support, which was prepared by coating a chitosan-casein film on silica, at 20 °C, pH 6.0 for 5 hr with microbial transglutaminase (MTG) as the cross-linking factor. The optimal support was obtained when 1% chitosan and 1% casein were used in the coating mixture. The optimal condition for immobilization catalyzed by MTG was confined to be at MTG concentration of 15 U/mL, pH 6.0, reacting for 6 hr at 20 °C. The highest specific activity of immobilized α-amylase was achieved as 236 U/g. After immobilization, the obtained enzyme showed broader pH profile and maintained more than 70% of the original activity after 20 reuses.     

Some Biochemical Properties of Lipase from Bay Laurel (<Emphasis Type="Italic">Laurus nobilis</Emphasis> L.) Seeds

Lipase was isolated from bay laurel (Laurus nobilis L.) seeds, some biochemical properties were determined. The bay laurel oil was used as the substrate in all experiments. The pH optimum was found to be 8.0 in the presence of this substrate. The temperature optimum was 50 °C. The specific activity of the lipase was found to be 296 U mg protein⁻¹ in optimal conditions. The enzyme activity is quite stable in the range of pH 7.0–10. The enzyme was stable for 1 h at its optimum temperature, and retained about 68% of activity at 60 °C during this time. K _m and V _max values were determined as 0.975 g and 1.298 U mg protein⁻¹, respectively. Also, storage stability and metal effect on lipolytic activity were investigated. Enzyme activity was maintained for 9, 12, and 42 days at room temperature, 4 and −20 °C, respectively. Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, and Mg²⁺ lightly enhanced bay laurel lipase activity.     

Immobilization of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil

Immobilization of lipase AY from Candida rugosa by entrapment within a chemically inert hydrophobic sol-gel support was studied. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and isobutyltrimethoxysilane. Certain modifications were incorporated into the conventional immobilization procedure, including the use of glucose as additive and the application of vacuum during the drying and aging stages. The activity and thermostability of immobilized enzyme were subsequently determined in hydrolyzing soybean oil. Hydrolysis results showed more than 95 mol% of the theoretical yield for the formation of FF after 1 h of reaction at 40°C. The level of FFA was 3.3 times greater than that seen when an immobilized enzyme was prepared by the conventional sol-gel process. The immobilized enzyme retained most of its hydrolytic activity compared to the free enzyme and kept more than 95% activity after 120 h of incubation at 40°C, whereas the free enzyme lost 67% of its activity after 24 h of incubation and almost all of its activity after 96 h of incubation at 40°C. The immobilized enzyme also proved to be very stable, as it retained more than 90% of the initial activity after 16 one-hour reactions. Surface characterization studies suggested that the enzyme-containing sol-gel particles have amorphous morphology and are void of micro/meso pores.     

Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

The milk-clotting enzyme from Bacillus amyloliquefaciens D4 was purified to 17.2-fold with 20% recovery by precipitation in ammonium sulfate and ion-exchange chromatography. The molecular mass of the enzyme was 58.2 kDa as determined by SDS-PAGE, and it was proved to be a metalloprotease by EDTA inhibition. The enzyme was active in the pH range 5.5–7.0 and was inactivated completely by heating at 55 °C for 20 min. The highest level of enzyme activity was obtained at 65 °C, pH 5.5, in the presence of 25mM CaCl₂. The milk-clotting activity was inhibited only slightly by Na⁺ and K⁺ and significantly by Cu²⁺, Zn²⁺ and Sn²⁺. The K _m value of this enzyme was 0.471 mg/mL. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from B. amyloliquefaciens D4 should be considered as a potential substitute for calf rennet.