Purification and characterisation of proteases A and D from Debaryomyces hansenii

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.     

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.     

ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

Low molecular weight serine protease from the viscera of sardinelle (Sardinella aurita) with collagenolytic activity: Purification and characterisation

A new low molecular weight (LMW) serine-protease from sardinelle (Sardinella aurita) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 3.82-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 14.2 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60 °C, respectively. The purified protease was strongly inhibited by phenylmethylsulphonyl fluoride, a serine-protease inhibitor, and soybean trypsin inhibitor. The N-terminal amino acid sequence of the first 10 amino acids of the purified protease was APVQPCVVVI. This sequence showed low homology with several peptidases, suggesting that the enzyme is a new protease. Interestingly, the protease was found to cleave collagen type I and hydrolyze succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin. Our findings indicate that the S. aurita protease is a new LMW enzyme with collagenolytic activity.     

Purification and Partial Characterization of Trypsin from the Viscera of Tropical Sierra (Scomberomorus Sierra) from the Gulf of California

Trypsin from the viscera of sierra (Scomberomorus sierra) was purified by affinity chromatography on Sepharose‐4B coupled to soybean trypsin inhibitor and characterized with respect to its purity, sensitivity to temperature, pH and inhibition. Trypsin was purified from sierra viscera with 11.9‐fold and 29.7% yield. The enzyme had a molecular weight of 25.4 kDa estimated by SDS‐PAGE and two possible trypsin isoforms were observed in activity gels. Trypsin activity was strongly inhibited by soybean trypsin inhibitor and porcine trypsin inhibitor, showing a partial inhibition by a serine protease inhibitor. The optimal activity of the enzyme was observed at pH 9 and 60C with n‐α‐benzoyl‐dl‐arginine‐p‐nitroanilide as a substrate. The enzyme maintained more than 50% of its activity in temperatures up to 50C and within the pH range of 8–10 for a period of up to 2 h.     

Purification and properties of an arginyl aminopeptidase from Debaryomyces hansenii

A metallo arginyl aminopeptidase (EC 3.4.11.6) activated by Co(2+) was isolated from Debaryomyces hansenii CECT 12487. The enzyme was purified after precipitation with protamine sulphate, followed by a weak anion exchange chromatography, gel filtration chromatography and a strong anion exchange chromatography. The arginyl aminopeptidase (AAP) was purified 337 folds, with a 18% recovery. The AAP appeared to be a dimer with a molecular mass of 101 kDa. The enzyme was active in the pH range from 6 to 9. The optimal activity was detected at pH 7.0 and at 37 degrees C. AAP activity was inhibited by typical aminopeptidase inhibitors (puromycin and bestatin), reducing agents (DTT), chelating agents (EDTA, EGTA and phenantroline) and sulphydryl groups reagents (iodoacetate). Ca(2+), Mn(2+) and Co(2+) activated the enzyme, while Cu(2+), Cd(2+), Hg(2+) and Mg(2+) inhibited it. The K(m) values calculated for Arg-AMC (7-amido-4-methylcoumarin) and Leu-AMC were 0.071 and 0.094 mM, respectively. The enzyme showed maximum specificity for basic amino acids (Arg and Lys), but was also able to hydrolyze non-charged amino acids (Leu, Met and Ala) and, at a minor rate, aromatic amino acids (Phe and Tyr). AAP showed higher activity when an acid residue was located at the C-terminal position of dipeptides.The described purification of an arginyl aminopeptidase from the yeast D. hansenii can contribute to the lack of knowledge about the exopeptidase activity in one of the yeasts more frequently isolated in sausage and to understand its role during the ripening of a fermented sausage.     

Alkaline protease from Bacillus cereus VITSN04: Potential application as a dehairing agent

The objective of this work is to use protease enzyme as an ecofriendly alternative to chemicals in dehairing. An alkaline protease producing bacterium was isolated from protein-rich soil sample. The bacterium was identified as Bacillus cereus VITSN04 by 16S rRNA gene sequencing method. Growth characteristics and protease activity were studied in yeast, malt, beef, nutrient broth and soybean casein digest media and the enzyme secretion was found to correspond with growth. Maximum protease production was obtained in soybean casein digest medium at 16h with the activity of 200.1±0.68U/ml and a correlation coefficient of 0.965 between growth and enzyme production. The crude enzyme was found to have maximum activity at 30°C and pH 8.0. The protease was purified by ammonium sulphate precipitation, Sephadex G-50 and G-100 gel filtration chromatography. The purified protease was homogeneous on non-denaturing PAGE and its molecular weight was estimated to be 32kDa. The purified protease was of the serine type as it was inhibited by phenylmethylsulphonyl fluoride. The crude enzyme preparation was found to be effective in dehairing goat skins in leather processing.     

荧光假单胞菌胞外蛋白酶的纯化及特性研究

采用硫酸铵沉淀、DEAE-Sepharose FF离子交换层析、Sephacryl S-100 HR凝胶过滤层析方法对从原料乳中分离出一株荧光假单胞菌胞外耐热蛋白酶进行纯化及特性研究。将纯化后的单一蛋白酶进行SDS-PAGE分子质量测定,并考察最适温度、最适pH值、热稳定性、金属离子对其酶活性的影响及对该蛋白酶的氨基酸种类分析。纯化后蛋白酶的分子质量为47kD,经纯化后蛋白酶比活力提高了近61.38倍;最适温度和pH值分别为30℃和7.0;二硫苏糖醇对蛋白酶活性具有一定的抑制作用,Fe2+可以促进蛋白酶活性的提高;该酶具有较强耐热性,原酶液经130℃热处理3min后,残留酶活仍超过原酶液的47.67%;该蛋白酶氨基酸组成中甘氨酸(Gly)、丙氨酸(Ala)和谷氨酸(Glu)的含量占明显优势,其中Gly含量最高,物质的量分数高达42%。     

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61–63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg²⁺, Mn²⁺, Ba²⁺ and iodacetic acid and stimulated by Li⁺, Ca²⁺, Cu²⁺, β‐mercaptoethanol and dithiothreitol, while Zn²⁺, L ‐cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K_m of 0.562 mg mL^?1 with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease. Copyright © 2011 Society of Chemical Industry     

Protease (PrA and PrB) and prolyl and arginyl aminopeptidase activities from Debaryomyces hansenii as a function of growth phase and nutrient sources

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE>10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE>10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.     

南极磷虾蛋白酶分离纯化及部分性质研究

对在南极磷虾自溶过程中起主要作用的蛋白酶的分离纯化条件和主要生化性质进行了研究。经过30%～70%硫酸铵分级沉淀,DEAE-Sepharose Fast Flow阴离子交换层析,Sephacryl S-200 HR凝胶层析后得到电泳纯的蛋白酶,该酶纯化倍数为12.59,产率为43%,分子质量约为28 ku;最适pH和最适温度为pH 8.0和50℃;该酶pH稳定范围为pH 7.0～9.5,并在45℃以下较稳定;苯甲基磺酰氟(PMSF)、大豆胰蛋白酶抑制剂(SBTI)、对甲苯磺酰基赖氨酸氯甲基酮(TLCK)对该酶活性有较强的抑制作用。     

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.     

Extracellular protease from Mucor pusillus: purification and characterization

Extracellular protease from Mucor pusillus was purified 18-fold with 7.56% recovery by ion-exchange chromatography and gel filtration. The enzyme was found to be monomeric in nature, having a molecular mass of 49 kDa. The enzyme acted optimally at 50°C and was stable in the temperature range 30–50°C. It was completely inactivated by heating for 30 min at 65°C. The optimum of activity for the purified extract was observed at milk CaCl₂ concentration of 0.02  m and at milk pH of 5. These properties, except for temperature, were similar to those of rennet.     

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。     

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.     

毛霉AS3.2778脯氨酸氨肽酶的部分纯化及性质研究

毛霉蛋白酶对大豆蛋白有较高的水解效率并对蛋白水解物有良好的脱苦效果,因此在大豆多肽的制备方面显示出很好的应用前景。为了开发这一蛋白酶系,实验中P采用硫酸铵盐析、离子交换层析、疏水层析及凝胶层析等方法对其进行了分离纯化,从雅致放射毛霉AS3.2778的发酵麸曲中部分纯化得到一氨肽酶组分,并对其性质进行了探讨。纯化的毛霉氨肽酶是一典型的脯氨酸氨肽酶,它对小肽N端的脯氨酸有非常强的水解能力;该氨肽酶在40～45℃、pH6.5有最大催化活性,在30℃以内,pH5.0～8.0有很好的稳定性;在所试验的几种蛋白酶抑制剂中,仅1 mmol/L的苯甲基磺酰氟(PMSF)对毛霉氨肽酶有抑制作用,由此说明纯化的毛霉氨肽酶可能是一种丝氨酸蛋白酶;常见金属离子对该氨肽酶活性的影响不明显;脱苦实验结果表明,纯化的毛霉氨肽酶对于大豆蛋白水解物的苦味有明显的去除效果。     

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.     

DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.     

CHARACTERIZATION OF AFFINITY-PURIFIED TRYPSIN FROM HYBRID TILAPIA (TILAPIA NILOTICA/AUREA)

Trypsin was extracted from the intestines of tilapia and purified 136-fold on the basis of amidase activity by a sequence of ammonium sulfate fractionation, acetone precipitation and affinity chromatography on soybean trypsin inhibitor bound to 4.0% beaded agarose. The purified trypsin exhibited higher esterase than amidase activity at its optimum pH of 9.0. The enzyme was stable in the range of pH 7–9. The optimum temperature for enzyme activity was 40C and the Q₁₀ for enzyme activity was 1.7 from 20C–30C and 30C–40C. The Arrhenius energy of activation was 8.9 and 8.7 kcal./mol respectively, for the ranges of 20C–30C and 30C–40C.     

PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.