Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems

The equilibrium yields of the peptide Cbz-Arg-Leu-NH2 synthesized from Cbz-Arg and Leu-NH2 using the PST-01 protease in the presence of organic solvents were investigated under various conditions. The equilibrium yields depended little on the concentration of the carboxyl component, but significantly on the concentration of the nucleophile. The optimum temperature and pH for a high equilibrium yield were 30 degrees C and greater than 5.0, respectively. Under optimum conditions the equilibrium yields were 71.6% and 87.7% in the presence of 50% (v/v) DMF and 60% (v/v) DMSO, respectively. Furthermore, the PST-01 protease also catalyzed the syntheses of the dipeptides Cbz-Lys-Leu-NH2, Cbz-Ala-Leu-NH2, Cbz-Ala-Phe-NH2, Cbz-Arg-Leu-NH2, and Cbz-Lys-Phe-NH2 with equilibrium yields of more than 60% in the presence of 50% (v/v) DMF and 50 mM sodium phosphate buffer (pH 7.0).     

Purification and characterization of a novel serine protease compositain from compositae (Scorzonera hispanica L.)

In this research, a protease from the compositae (Scorzonera hispanica L.) was extracted and purified through (NH₄)₂SO₄ precipitation, CM-Sephadex and Sephacryl S200. At the end of purification by gel filtration on a Sephacryl S-200 column, 87.11-fold purification was achieved. It was shown that purified enzyme was homogeneous in terms of SDS-PAGE with molecular mass estimate of 30?kDa. The enzyme named compositain depicted an optimal pH of 8.0 and was stable at pH 7.0–9.0, and its optimal temperature was at 50?°C. While Tween 80 (0.2?%) was activated to the purified protease enzyme, it was partially inhibited by 5?mM concentration of some metal salts and EDTA, PMSF, dithiothreitol, H₂O₂ and β-mercaptoethanol. The enzyme activity was stable even in the presence of detergents and organic solvents. In addition, it was investigated whether the purified and characterized protease enzyme would cause to congeal milk. As a result, it was determined that the compositain could congeal milk and it would be used for cheese production. The compositain had potential application in food processing.     

Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil

The Pseudomonas aeruginosa san-ai strain was isolated from water-soluble cutting oil used for cooling and lubrication during industrial metal-working processes. This strain, which is grown in a high alkaline (pH 10) mixture of surfactants and mineral oil, produces an extracellular proteolytic enzyme. We have purified and characterized this 18 kDa protease. The P. aeruginosa san-ai protease functions optimally at pH 9.0 and 60 degrees C. Additionally, it is a Zn-containing metalloenzyme, and its monomeric structure contains at least one disulfide bond. Because the enzyme is stable in the presence of organic solvents, it is suitable for peptide synthesis. Furthermore, the P. aeruginosa san-ai protease could be used in an intelligent drug delivery system (DDS) designed for applications in the metal industry for prevention of putrefaction of cutting oil.     

Wheat-bug damage in New Zealand wheats: Some properties of a glutenin hydrolysing enzyme in bug-damaged wheat

Some properties of a glutenin hydrolysing enzyme present in bug (Nysius huttoni) damaged wheat (Triticum aestivum) were examined using a modified SDS sedimentation test reported previously. The enzyme appears to be a water-soluble alkaline protease with an activity optimum at pH 9.0. It is relatively heat stable, but the temperature optimum for activity is quite low (35–40°C). The enzyme is not inhibited by EDTA or N-ethylmaleimide, but is inhibited by the metal ions Co²⁺, Mn²⁺ and Fe²⁺.     

Purification and characterization of organic solvent-stable lipase from organic solvent-tolerant Pseudomonas aeruginosa LST-03

An organic solvent-stable lipase (LST-03 lipase) secreted into the culture broth of the organic solvent-tolerant Pseudomonas aeruginosa LST-03 was purified by ion-exchange and hydrophobic interaction chromatography in the presence of 2-propanol. The purified enzyme was homogeneous as determined by SDS-PAGE. The molecular mass of the lipase was estimated to be 27.1 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum pH and temperature were 6.0 and 37 degrees C. LST-03 lipase was stable at pH 5-8 and below 40 degrees C. Its hydrolytic activity was highest against tricaproin (C6), methyl octanoate (C8), and coconut oil respectively among the triacylglycerols, fatty acid methyl esters, and natural oils investigated. The enzyme cleaved not only the 1,3-positioned ester bonds, but also the 2-positioned ester bond of triolein. It exhibited high levels of activity in the presence of n-decane, n-octane, DMSO, and DMF as well as in the absence of an organic solvent. In addition, LST-03 lipase was stabler in the presence of n-decane, ethyleneglycol, DMSO, n-octane, n-heptane, isooctane, and cyclohexane than in the absence of an organic solvent.     

Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.     

Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature

A haloalkaliphilic bacterium, isolated from Coastal Gujarat (India) was identified as Oceanobacillus sp. (GQ162111) based on 16S rRNA gene sequence. The organism grew and secreted extra cellular protease in presence of various organic solvents. At 30% (v/v) concentration of hexane, heptane, isooctane, dodecane and decane, significant growth and protease production was evident. The alkaline protease was purified in a single step on phenyl sepharose 6 FF with 28% yield. The molecular mass as judged by SDS-PAGE was 30?kDa. The temperature optimum of protease was 50°C and the enzyme retained 70% activity in 10% (v/v) isooctane. Effect of salt and pH was investigated in combination to assess the effect of isooctane. In organic solvents, the enzyme was considerably active at pH 8-11, with optimum activity at pH 10. Salt at 2?M was optimum for activity and enzyme maintained significant stability up to 18?h even at 3?M salt concentration. Patters of growth, protease production, catalysis and stability of the enzyme are presented. The study resumes significance as limited information is available on the interaction of haloalkaliphilic bacteria and their enzymes with organic solvents.     

Purification and characterization of an alkaline lipase from Pseudomonas aeruginosa isolated from putrid mineral cutting oil as component of metalworking fluid

Extracellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa, an extremophile which naturally grows in water-soluble mineral cutting oil (pH 10) used as metalworking fluid (MWF) for cooling and lubrication in industrial metalworking processes. The molecular mass of the purified lipase was estimated by SDS-PAGE to be 54 kDa. The optimum pH and temperature were 11 and 70 degrees C, respectively. The enzyme is stabile over a broad pH range (pH 4-11.5). The lipase preferably acted on triacylglycerols with medium-chain fatty acids. The lipase was inhibited strongly by Zn(2+), Hg(2+), Cu(2+) and slightly by Ca(2+) and Mg(2+). Non-ionic detergents and sodiumdeoxycholate enhanced lipase activity. Alkaline lipase from P. aeruginosa, capable of growing in a water-restricted medium has excellent properties and good potential for biotechnological applications in the metal industry. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool in a water-restricted medium with a variety of applications including organosynthetic reactions and the control and prevention of MWF putrification in the metal industry.     

毛霉亮氨酸氨肽酶的纯化及性质研究

采用硫酸铵盐析、离子交换层析、疏水层析、凝胶层析及超滤方法对毛霉胞外蛋白酶组分进行分离纯化,从毛霉的发酵麸曲中分离纯化得到氨肽酶组分,并对其性质进行探讨。结果表明：纯化的毛霉氨肽酶是亮氨酸氨肽酶,其对小肽N端的亮氨酸有非常强的水解活性;该氨肽酶在40℃、pH6.5有最大催化活性,在40℃以内,pH5.0～8.0有很好的稳定性;在所实验的几种蛋白酶抑制剂(PMSF、EDTA、E-64、Pepstatin A)中,仅EDTA可以完全抑制该氨肽酶的活性,由此说明,纯化的毛霉氨肽酶是一种金属蛋白酶;常见的金属离子中,Ca2+对该氨肽酶有激活作用,而Zn2+、Cu2+及Mn2+对其有强的抑制作用;该氨肽酶对大豆多肽的苦味有明显的去除效果,大豆多肽脱苦处理4h后其苦味基本消除。     

嗜热芽孢杆菌高温蛋白酶HS08的化学修饰研究

分离纯化了嗜热芽孢杆菌产生的耐高温蛋白酶HS08,以SDS-PAGE凝胶电泳和Superdax75预装柱凝胶过滤,测定蛋白酶HS08的分子质量为30.6ku,同时研究了酶专一性抑制剂对酶活性的影响。结果表明,蛋白酶HS08的活性被酶活性中心丝氨酸残基专一性抑制剂PMSF以及金属离子螯合剂EDTA强烈抑制,而NBS、WRK、TNBS、Phenylgloxalhydrate等修饰剂对酶活性无明显抑制作用。K+、Na+、Ca2+、Mg2+、Zn2+等金属离子对酶活性影响实验结果表明,Zn2+可使该酶活性明显提高,Cu2+对该酶活性有抑制作用。因此,该酶属于金属离子激活的丝氨酸族蛋白酶。蛋白酶HS08的酶动力学实验表明,以BSA为底物,PMSF可使蛋白酶HS08最大反应速率Vmax下降50%,而对其米氏常数Km影响不大。     

纳豆芽孢杆菌蛋白酶的制备及性质研究

纳豆芽孢杆菌是纳豆发酵生产的主要菌种,对其胞外蛋白酶催化特性的研究有助于了解纳豆的发酵生产过程,并有利于从中发掘有开发应用潜力的蛋白酶。实验从不同纳豆产品中分离筛选得到1株高产蛋白酶的纳豆芽孢杆菌,对其胞外蛋白酶的催化特性及活性影响因子进行了分析。结果显示,纳豆芽孢杆菌蛋白酶是一典型的金属蛋白酶,EDTA几乎可完全抑制其活性,Ca2+是其活性中心的辅基;该蛋白酶在60℃、pH 7.0有最大催化活性,在pH 6～9和低于45℃条件下具有很好的稳定性;50℃时,该蛋白酶会缓慢失活,其热失活规律符合一级指数衰减模型:a=0.124+0.887e(-0.45t);该蛋白酶对大豆蛋白有很强的水解能力,其水解效率与Alcalase蛋白酶相当,可以实现底物蛋白的深度水解,具有一定的开发应用价值。     

Purification and characteristics of serine protease from the head of pacific white shrimp

Serine protease from the head of Pacific white shrimp was purified by the following techniques: ammonium sulfate fractionation, Q-Sepharose HP ion exchange chromatography, and Sephadex G-100 gel filtration. The molecular weight was estimated as 32.8 kDa using SDSPAGE. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 10.0 and 40°C. It was stable at pH range from 8.0 to 11.0 and had good thermal stability. Pb²⁺, Ca²⁺, Mg²⁺, Cu²⁺, and Mn²⁺ could active the enzyme certainly when Zn²⁺ and Hg²⁺ strongly inhibited the activity. The enzyme was inhibited by the general serine protease inhibitor (PMSF) and the specific trypsin inhibitors (TLCK, SBTI). The modification of various amino acid modifiers for the purified enzyme determined that the enzyme active center included tryptophan, histidine, and serine, moreover, arginine had a certain relationship with the enzyme activity.     

Purification and characterization of an extracellular lipase from Mucor hiemalis f. corticola isolated from soil

We have screened 39 microfungi isolates originated from soil in terms of lipolytic activity. Out of all screened, a novel strain of Mucor hiemalis f. corticola was determined to have the highest lipase activity. The extracellular lipase was produced in response to 2% glucose and 2.1% peptone. The lipase was purified 12.63-folds with a final yield of 27.7% through following purification steps; ammonium sulfate precipitation, dialysis, gel filtration column chromatography and ion exchange chromatography, respectively. MALDI-TOF MS analysis revealed 31% amino-acid identity to a known lipase from Rhizomucor miehei species. The molecular weight of the lipase was determined as 46kDa using SDS-PAGE and analytical gel filtration. Optimal pH and temperature of the lipase were determined as 7.0 and 40°C, respectively. The enzyme activity was observed to be stable at the pH range of 7.0-9.0. Thermostability assays demonstrated that the lipase was stable up to 50°C for 60min. The lipase was more stable in ethanol and methanol than other organic solvents tested. Furthermore, the activity of the lipase was slightly enhanced by SDS and PMSF. In the presence of p-NPP as substrate, K(m) and V(max) values of the lipase were calculated by Hanes-Woolf plot as 1.327mM and 91.11μmol/min, respectively.     

Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719

An alkaline protease was purified to apparent homogeneity from culture supernatants of Bacillus sp. PS719, a novel alkaliphilic, thermophilic bacterium isolated from a thermal spring soil sample, by ammonium sulfate precipitation followed by DEAE-cellulose and alpha-casein agarose column chromatographies. The purified enzyme migrated as a single protein band of 42 kDa during both denaturing and nondenaturing gel electrophoresis, suggesting that it consists of a single polypeptide chain. Its isoelectric point was approximately 4.8. The protease exhibited maximum activity towards azocasein at pH 9.0 and at 75 degrees C. The enzyme activity was stimulated by Ca2+, but was inhibited in the presence of Fe2+ or Cu2+. The enzyme was stable in the pH range 8.0 to 10.0 and up to 80 degrees C in the absence of Ca2+. Since phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin (DCI) in addition to N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) completely inhibited the activity, this enzyme appears to be a trypsin-like serine protease. Among the various oligopeptidyl-p-nitroanilides tested, the protease showed a preference for cleavage at arginine residues on the carboxylic side of the scissile bond of the substrate, liberating p-nitroaniline from N-carbobenzoxy (CBZ)-L-arginine-p-nitroanilide with the K(m) and V(max) values of 0.6 mM and 1.0 micromol.min(-1).mg protein(-1), respectively.     

Purification and Characterization of a Trypsin‐Like Protease from Flatfish (Paralichthys olivaceus) Intestine

A trypsin‐like protease was purified from the intestine of flatfish (Paralichthys olivaceus) by gel filtration and anion‐exchange chromatography. The molecular weight was estimated to be 29.6 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Flatfish protease had maximal activity at 70C and pH 7.5 using N‐α‐benzoyl‐dl ‐arginine‐ρ‐nitroanilide as substrate. It was stable to heat treatment up to 50C and to pH ranges between 7.0 and 10.0. It was activated by calcium ion and completely inhibited by mercury ion and known serine‐protease inhibitors, such as phenylmethylsulfonyl fluoride, tosyl lysine chloromethyl ketone and benzamidine.     

Purification and characterisation of a polyphenol oxidase from Boletus erythropus and investigation of its catalytic efficiency in selected organic solvents

Polyphenol oxidase (PPO) was purified from Boletus erythropus using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. Optimum pH and temperature were found to be 8.0 and 20 °C, respectively, using 4-methylcatechol as a substrate. The enzyme was extremely stable between pH 3.0 and 9.0 after 24 h incubation at 4 °C. B. erythropus PPO was also quite stable between 10 and 30 °C after 4 h incubation. The K_m and V_max values were calculated as 2.8 mM and 1430 U/mg protein by Lineweaver–Burk curve, respectively. The enzyme activity was inhibited by sodium metabisulfite, ascorbic acid, sodium azide and benzoic acid. It was seen that the mushroom PPO was an effective biocatalyst in selected organic solvents, such as dichloromethane, dichloroethane and toluene, when catechin was used as a substrate. All data support that B. erythropus has a highly active PPO, possessing similar biochemical and kinetic characteristics to other plant PPOs.     

嗜热芽孢杆菌蛋白酶HS08的分离纯化研究

从高温土壤中分离得到的一株产耐热中性蛋白酶的嗜热芽孢杆菌,该菌分泌的胞外蛋白酶粗酶液经80%饱和度硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换层析和Superdax75凝胶层析分离纯化后,纯化倍数提高4．36倍,得率为5．3%,经SDS-PAGE电泳测得其分子量为30．6kDa。酶的最适温度与pH实验表明,该酶最适温度为65℃、最适pH为7．5,并且该酶在50℃时表现出1h以上的稳定性。该蛋白酶的活性受到丝氨酸族蛋白酶专一性抑制PMSF和金属离子螯合剂EDTA强烈抑制,Zn2+能提高酶活性,因此该蛋白酶为Zn2+激活的丝氨酸族蛋白酶。     

Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste

A novel salt-tolerant acid protease was produced from Aspergillus oryzae LK-101 (AOLK-101). The AOLK-101 protease was purified to homogeneity by ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatographies in order. The specific activity and the purification ratio of the purified protease were 2,301 unit/mg and 11.6 fold, respectively, with 25 kDa of molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrpphoresis (SDS-PAGE). Its optimal pH and temperature were pH 6.5 and 50°C, respectively. This protease was relatively stable at pH 4.5–7.5, below 40°C, and up to 10% salt concentration. The protease was moderately inhibited by Ag²⁺ and Zn²⁺, and strongly by ethylenediamide tetraacetic acid (EDTA) and phenylmethysulfonyl fluoride (PMSF), but activated by Cu²⁺ and Mn²⁺. Therefore, the AOLK-101 protease was a serine protease based on the influence of metal ions and inhibitors. K _m , V _max , k _cat , and k _cat /K _m values of AOLK-101 protease for hammastein milk casein were 1.04 mg/mL, 124.84 unit/L, 163.5/sec, and 3.9×10⁶/m·sec, respectively.     

A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation

A novel aspartic protease was extracted from the defatted viscera of sardinelle (Sardinella aurita) and purified, with a 9.5-fold increase in specific activity and 23.3% recovery. The molecular weight of the purified enzyme was estimated to be 17 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme appeared as a single band on native-PAGE. The optimum pH and temperature for protease activity were around 3.0 and 40 °C, respectively. The enzyme showed pH stability between 2.0 and 5.0 and retained more than 50% of its activity after heating for 30 min at 50 °C. The enzyme lost 90% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride. Its K_m value was determined to be 0.73 × 10⁻⁴ M using haemoglobin as a substrate. The N-terminal 12 amino acid sequence of the purified acidic protease was R V I I E D X D Q F C T. This sequence showed low homology with aspartic peptidases of several other species of fish, suggesting that the enzyme is a new aspartic protease.     

Inhibition of β-carotene degradation in oil-in-water nanoemulsions: Influence of oil-soluble and water-soluble antioxidants

The utilisation of carotenoids as functional ingredients (pigments and nutraceuticals) in many food and beverage products is currently limited because of their poor water-solubility, high melting point, chemical instability, and low bioavailability. This study examined the impact of antioxidants on the chemical degradation of β-carotene encapsulated within nanoemulsions suitable for oral ingestion. β-Carotene was incorporated into oil-in-water nanoemulsions stabilized by either a globular protein (β-lactoglobulin) or a non-ionic surfactant (Tween 20). Nanoemulsions were then stored at neutral pH and their physical and chemical stability were monitored under accelerated stress storage conditions (55 °C). β-Carotene degradation was monitored non-destructively using colour reflectance measurements. The rate of β-carotene degradation decreased upon addition of water-soluble (EDTA and ascorbic acid) or oil-soluble (vitamin E acetate or Coenzyme Q10) antioxidants. EDTA was more effective than ascorbic acid, and Coenzyme Q10 was more effective than vitamin E acetate. The utilisation of water-soluble and oil-soluble antioxidants in combination (EDTA and vitamin E acetate) was less effective than using them individually. Emulsions stabilized by β-lactoglobulin were more stable to colour fading than those stabilized by Tween 20. These results provide useful information for designing effective nanoemulsion-based delivery systems that retard the chemical degradation of encapsulated carotenoids during long term storage.