Identification of heat stable protease of Klebsiella oxytoca isolated from raw milk

AIMS: The aim of this study was to identify and characterize heat stable proteinases of psychrotrophic proteolytic bacteria isolated from raw milk. METHODS AND RESULTS: A strain of Klebsiella oxytoca producing a high proteolytic activity when cultured on milk was isolated. Maximum proteolytic activity was observed at the stationary phase during growth on milk or casein-peptone broth. The bacterium demonstrated the capability to grow at 7 degrees C, classified as psychrotrophic. The crude enzyme showed optimum activity at 37 degrees C, and pH 5.0 and 7.0. The proteinase was very resistant to heat, maintaining 74% of initial activity after incubation at 142 degrees C. CONCLUSIONS: A heat stable protease of a psychrotrophic strain of K. oxytoca was identified and partially characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: Thermal stable proteases may constitute a serious problem to ultra-high temperature (UHT) processed milk, leading to undesirable physical and sensory alterations.     

Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A

An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37 degrees C and 8.0, respectively, was completely inactivated by heating for 1 min at 80 degrees C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7.5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheese and may contribute to proteolysis of these cheeses during ripening.     

Candida guilliermondii isolated from HIV-infected human secretes a 50 kDa serine proteinase that cleaves a broad spectrum of proteinaceous substrates

Non-albicans Candida species cause 35-65% of all candidemias in the general population, especially in immunosuppressed individuals. Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism. This clinical yeast isolate was identified as Candida guilliermondii through mycological tests. C. guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 degrees C. After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS-PAGE. A 50 kDa proteolytic enzyme was detected with activity at physiological pH. This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class. E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase. Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C. guilliermondii by approximately 50%. Proteinases are a well-known class of enzymes that participate in a vast context of yeast-host interactions. In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components. We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides. Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non-albicans Candida.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682.

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatographies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25,000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37 degrees C for 60 min. The optimum pH was pH 11.5-13.0 at 37 degrees C and the optimum temperature was 70 degrees C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80 degrees C and stability from pH 4-12.5 at 60 degrees C and below 75 degrees C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH2-terminal amino acid was deleted.     

Haemophilus paragallinarum secretes metalloproteases

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH(4))(2)SO(4)) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4-9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 degrees C, but its activity diminished at 70 degrees C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.     

Purification and properties of a proteolytic enzyme from French beans.

1. A proteolytic enzyme with some features of a carboxypeptidase has been purified some 1180-fold from the sap of French beans (Phaseolus vulgaris var. Prince). A bright blue protein, plastocyanin, was separated from the enzyme by DEAE-cellulose chromatography. 2. Unlike carboxypeptidase A or B of animal origin, there is no evidence that the enzyme is a metalloprotein. There was no stimulation of activity by a number of metal ions, reducing agents or 2-mercapto-ethanol. Neither EDTA nor 1,10-o-phenanthroline inhibited the enzyme. 3. The proteolytic enzyme from beans, readily soluble at neutral or slightly acidic pH values, has a pH optimum of pH5.6 for the hydrolysis of leucine from benzyloxy-carbonylglycyl-l-leucine. Solutions of the enzyme in 0.1m-sodium acetate, pH5.5, lose about 2% of their activity/week at 4 degrees . Virtually no loss of activity results after prolonged storage at -15 degrees . 4. Incubation of the bean enzyme with peptides indicates that the enzyme will release acidic, neutral and basic amino acid residues as well as proline, although adjacent acidic residues in a peptide appear to inhibit the enzyme. The possibility of endopeptidase activity in the purified preparation requires further examination.     

Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2

Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.     

Production, purification, and characterization of a novel thermostable serine protease from soil isolate, Streptomyces tendae

An isolate of Streptomyces tendae produced a extracellular protease which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 21 kDa. Optimum activity was at 70 degrees C and pH 6. It was stable at 55 degrees C for 30 min and between pH 4 and 9. It was resistant to neutral detergents and organic solvents such as Triton X-100, Tween 80, methanol, ethanol, acetone, and 2-propanol at 5% (v/v). The enzyme was completely inhibited by 5 mM PMSF, indicating it to be a serine protease. N-terminal amino acid sequence did not show any homology with other known proteolytic enzymes. The protease may therefore be a novel neutral serine protease, which is stable at high temperature and over a broad range of pH.     

Proteolytic activity of the enzyme complex of the mammalian pancreas in comparison with pancreatin

The proteolytic activity and thermal stability of the enzyme complex of cell suspension from pig and bovine pancreas glands was compared with those of pancreatin. The enzyme complex displayed the highest thermal stability and activity at 50 degrees C. The kinetic constants, energies of activation and inactivation of the enzyme complex, and pH optimum (7.0 +/- 0.1) at which this complex had the maximum proteolytic activity were determined. Pancreatin had a pH optimum of 8.0 +/- 0.1.     

节杆菌β-半乳糖苷酶的分离纯化及酶学性质研究

分离纯化了由节杆菌所产的fI-半乳糖苷酶(EC 3.2.1.23)并研究了其酶学性质.以乳糖发酵培养基培养,离心收集茵体,超声破碎细胞得到粗酶液;采用硫酸铵沉淀、Phenyl-Sepharose疏水、DEAE-Sepharose离子交换和p-aminobelazyl-1-thio-β-galactopyranoside...     

Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro.

Proteinase Ak.1 was produced during the stationary phase of Bacillus sp. Ak.1 cultures. It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 60 and 70 degrees C, with half-lives of 13 h and 19 min at 80 and 90 degrees C, respectively. Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro-Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide). Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln-4-His-5, and Glu-13-Ala-14. The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75 degrees C. The enzyme is expressed in E. coli as an inactive proproteinase at 37 degrees C and is converted to the mature enzyme by heating the cell-free media to 60 degrees C or above. The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa. The NH2-terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point. Heating the proenzyme resulted in the production of active proteinase with an NH2-terminal sequence identical to that of the native enzyme. The characteristics of the cloned proteinase were identical to those of the native enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Assessment of Proteolytic Enzymes in Tetrahymena thermophila

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures ≥60° C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichloroisocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.     

球孢白僵菌Bb174固态发酵产几丁质酶产酶及酶学特征研究

对球孢白僵菌(Beauveria bassiana)Bb174产几丁质酶进行了固态发酵条件及酶学特征的研究．结果表明,以4：1麸皮：蚕蛹粉、蛋白胨1g·L^-1作为产酶最适培养基,在7．5g培养基中接种3ml液态种子,自然pH下28℃培养2d,酶活可达最高,为126U·g^-1(干培养基)．粗酶液的最适反应温度为40℃,最适反应pH5．0,在30-70℃保温1h,得半失活温度48℃．在30--40℃、pH4～6范围内,酶的性质最稳定．根据Lineweaver-Burk作图法,得到该酶的动力学参数Km为0．52mg·ml^-1,Vm为0．7△E680·h^-1．     

Purification and preliminary characterization of a cold-adapted extracellular proteinase from Trichoderma atroviride

Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 degrees C. Based on the activity profiles determined with paranitroanilide substrates at 5 degrees C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 degrees C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on N-benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 degrees C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin.     

Separation and comparative characterization of the cationic protease and anionic protease from the culture medium of Thermoactinomyces vulgaris

The anionic protease component which frequently contaminates preparations of routinely isolated cationic protease (thermitase) from Thermoactinomyces vulgaris was purified, virtually to homogeneity, by rechromatography on controlled pore glass (CPG-10). Starting materials were column eluates with anionic protease, contaminated with residual thermitase activity. The purified anionic enzyme shares several properties with thermitase, such as size, sensitivity against phenylmethanesulfonyl fluoride and Hg2+, UV-spectral, immunological and pH behavior. On the other hand, the isoelectric point (at pH 6.5), temperature dependence (more heat stable) and enzymatic activity (less active) of anionic protease differ significantly from thermitase. At pH 8 or 6 and 25 degrees or 4 degrees C anionic protease is hydrolysed completely by thermitase. Like other protein substrates, anionic protease simultaneously acts as a stabilizer for thermitase. In contrast to thermitase, the anionic enzyme partially changes spontaneously during long-term storage at 4 degrees C and pH 6 to a cationic protein species endowed with proteolytic activity.     

温和气单孢菌YH311硫酸软骨素裂解酶的分离纯化与固定化

通过硫酸铵沉淀、QAESephadex-A50柱层析及Sephadex-G150凝胶过滤等纯化步骤,对源自温和气单孢菌YH311的ChSase进行了分离纯化。结果表明,ChSase经上述纯化步骤后被纯化了55倍,其最终纯度可达95%以上,比活为31.86u/mg。经SDSPAGE及IFE测定可知该酶的分子量约为80kD,等电点为4.3～4.8。将纯化后的ChSase用海藻酸钠或纤维素固定化后,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高：固定化酶用80℃水浴处理120min或于4℃冰箱放置30d后仍可保留50%以上的相对活力;但固定化酶的收率较低,仅为18.56%和18.86%。