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1.
Summary The action of the cell-envelope proteinase (PIII-type) from Lactococcus lactis ssp. cremoris AM1 on bovine -casein was studied. The results were compared with those obtained earlier with (PI-type) proteinases from the cell envelope of other L. lactis strains. From a 4-h digest (pH 6.2; 15°C) of -casein made with the PIII-type proteinase, 24 peptides were isolated and purified by selective precipitation followed by semi-preparative reversed-phase HPLC. Altogether, these peptides accounted for the preferential splitting of 16 peptide bonds in -casein by the PIII-type proteinase. In nine cases the primary cleavage site (P1-P1) was a Glx-X or X-Glx peptide bond. In ten cases at least one large hydrophobic residue (Met, Leu, Tyr, Phe) formed part of the cleavable bond. The P2-P3 and/or P2-P3 regions of the substrate consisted of hydrophobic and/or negatively charged side chains or of side chains potentially involved in hydrogen bonds. Nine of the peptide bonds split were reported previously to be also susceptible to cleavage by PI-type proteinases, although the kinetics may be different. The PIII-type proteinase shows a broader specificity in its initial cleavage of -casein than does the PI-type. Offprint requests to: S. Visser  相似文献   

2.
The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine -casein was studied. A 4-h digest (pH 6.2, 15°C) of -casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1–160, 1–151, 1–95 and 1–79 of -casein, whereas the main LMM products found were the 161–169 and 152–160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160–161 and 151–152 peptide bonds. Two minor LMM products were identified as the fragments 96–104 and 103–106, indicating additional cleavage at positions 102–103, 104–105 and 106–107 of the sequence. Also several peptide bonds within the 161–169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococaal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79–80 and 95–96. However, the C-terminal region of the -casein sequence is the exclusive target of the CEPIII-aand, to variable extents, of the mixed-type enzymes.  相似文献   

3.
Two intracellular oligopeptide-preferring endopeptidases have been detected in Lactococcus lactis. A neutral thermolysin-like oligoendopeptidase (NOP) has been purified to homogeneity and an alkaline oligoendopeptidase has been partially purified. The specificity of the oligoendopeptidases towards important intermediary cheese peptides, produced by chymosin action on the caseins, clearly differs from that of the cell-envelope proteinase (CEP). NOP is active under conditions prevailing in cheese and contributes to initial proteolysis in a young cheese. It probably plays a crucial role in the degradation of an important bitter peptide in cheese, the -casein 193–209 fragment. The relatively low activity of the alkaline endopeptidase is further suppressed in cheese by the highly competitive actions of NOP and CEP.  相似文献   

4.
Cathepsins L and L-like (58 kDa) proteinase from mackerel were purified to electrophoretical homogeneity by Concanavalin A-Sepharose and Econo-Pac S chromatographies. The molecular weights of cathepsins L and L-like proteinase were 30,000 and 58,000, and the optimal pH for the hydrolysis of Z-Phe-Arg-MCA (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-[4-methyl] coumarylamide) were 5.0 and 5.5, respectively. The stability of both purified proteinases at various pHs was low, when the pH was above 7.0. According to the substrate specificity analysis, these proteinases hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, but did not hydrolyze Z-Arg-MCA and L-Arg-MCA. The activities of these two proteinases were effectively activated by cysteine and dithiothreitol. Their thiol-dependent proteolytic activity against Z-Phe-Arg-MCA was strongly inhibited by E-64 (trans-epoxysuccinyl-L-leucylamido[4-guanidino]butane), antipain, chymostatin, iodoacetic acid, and leupeptin, but not inhibited by pepstatin or phenylmethane sulfonyl floride. The inactivation rate constants (KD) of cathepsins L and L-like proteinases at 50°C were 5.1 × 10?5 and 6.9 × 10?4 s?1, respectively. K+, Na+, Mg+, and Sr+ did not affect them, while Zn2+, Cd2+, Co2+, Ni2+, Cu2+, Hg2+, Fe2+, and Fe3+ inhibited the activity of the purified cathepsins L and L-like proteinase.  相似文献   

5.
The cell-envelope proteinase from Lactobacillus helveticus CRL 1062 was detected in the cell membrane fraction. The enzyme remained associated with the cells even after treatment with lysozyme and was not released from washed cells in absence of calcium. The proteinase was maximally active at pH 6.5–7.0 and 42°C and hydrolysed - and -caseins at different rates. Activity was inhibited (98%) by 1 mM PMSF, suggesting it was a serine-type protease.  相似文献   

6.
Summary The bacterial lacZ gene was introduced into two sibling strains of Saccharomyces cerevisiae, one a wild-type strain with normal proteinase activity and the other a pep4-3 mutant strain. The pep4-3 mutation resulted in 90% reduced activity of the four major vacuolar proteinases. By comparing the activity of the lacZ gene product (-galactosidase) in both strains the degradative effect of the major vacuolar proteinases on a heterologous protein was estimated. The mutant strain with reduced proteinase activity had higher -galactosidase activity under all the test conditions. In the most productive case the pep4-3 mutant had 55% higher -galactosidase activity than the wild-type. Batch cultures of the two strains were evaluated for growth characteristics. The strain with reduced proteinase activity grew to higher optical densities than the wild-type. Upon further examination it was found that not only were the optical densities of pep4-3 cultures greater but the cell numbers were much greater than expected due to the smaller size of pep4-3 cells. It is concluded that the strain lacking vacuolar proteinases maintained increased levels of -galactosidase and is physiologically as healthy as the wild-type.Offprint requests to: J. M. Wingfield  相似文献   

7.
Summary The action of the cell-wall-associated proteinases from Lactococcus lactis subsp. cremoris strains H2 and SK112 on bovine -casein was compared. The proteinase from the H2 strain was characterised as a PI-type proteinase since it did not hydrolyse s1-casein and the initial trifluoroacetic acid-soluble products of -casein hydrolysis were identical to those previously identified as hydrolysis products of PI-type lactococcal proteinase action. The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr193-Gln194 and Gln182-Arg183 were the first to be hydrolysed. Cleavage of the bonds Gln175-Lys176, Ser168-Lys169, Ser166-Gln167 and Leu163-Ser164 was also very rapid. Four of the five bonds in -casein most susceptible to hydrolysis by the PIII-type proteinase from strain SK112 were different from those cleaved by the PI-type proteinase, initial hydrolysis being at the sites Tyr193-Gln194, Leu192-Tyr193, Asp43-Glu44, Gln46-Asp47 and Phe52-Ala53. Early hydrolysis at the three sites in the N-terminal region of -casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of PI-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly. Offprint requests to: G. G. Pritchard  相似文献   

8.
A new serine alkaline proteinase (ALPase II) was purified from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight of ALPase II was estimated to be 32,000 by SDS-polyacrylamide gel electrophoresis. The enzyme had a very low isoelectric point (pI), which was determined to be 2.8. An optimum pH of this enzyme was 10.2. The specific activity was 0.28 katal/kg of protein for milk casein, 0.34 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and 8.5 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA).The substrate specificity of the alkaline proteinase was studied with the synthetic fluorogenic and chromogenic substrates. It was most favorable for the enzyme that the P1 site of the substrate might be hydrophobic and bulky amino residue (Phe or Tyr). When the substrate contained four amino residues, the proteinase efficiently expressed its activity. The alkaline proteinase had higher specificity than those of the bacterial serine proteinases, subtilisins Carlsberg and BPN, and lower specificity than that of serine alkaline proteinase with pI 8.2 (ALPase I) obtained from the same bacteria NKS-21. ALPase II did not react with the anti-ALPase I antiserum.  相似文献   

9.
Strains of Saccharomyces cerevisiae with reduced activity of the four major vacuolar proteinases were constructed and used as an expression system for a model heterologous gene product (-galactosidase from Escherichia coli). The vacuolar proteinases were inactivated by mutation within the structural genes encoding proteinase A (PRA1), proteinase B (PRB1), carboxypeptidase Y (PRC1) and carboxypeptidase S (CPS1). Strains were constructed with mutations in one or more of these structural genes. Having constructed the strains, the E. coli -galactosidase (lacZ) gene was introduced by transformation. Batch cultures of each strain were grown and the activity of -galactosidase measured. An assessment of the effect of the loss of specific proteinases on the heterologous gene product was then made. The results indicated that strains with reduced vacuolar proteinase activity showed as much as 173% higher -galactosidase activity than a strain with wild-type proteinase activity carrying the lacZ gene. The most productive strains of all were those with reduced carboxypeptidase activity and/or reduced proteinase A activity. At first sight the inclusion of a pra1 mutation and/or the pra1 and cps1 mutations would appear wortwhile for significantly enhanced expression of a heterologous gene product in yeast. However this conclusion is too simplistic: each heterologous protein will require a host specifically tailored to ensure optimum expression. Correspondence to: J. R. Dickinson  相似文献   

10.
Empirical approach was adopted to examine the interaction of a little and finger millet inhibitors with digestive proteinases ofdifferent field and storage pests. Accordingly, gut proteinases of four storage and phytophagous pests were characterized andtheir inhibition by finger millet (Eluesine coracana Gaertneri) and little millet (Panicum sumatrense Roth) inhibitors has beeninvestigated. The Callosobruchus sp showed acid proteinase with pH optimum of 3.5 and Sitophilus oryzae showed pHoptimum of 4.5 apart from their alkaline proteinases with pH 8.5. The proteinases of Tribolium castaneum had wider pHoptimum from 5.5 to 8.5. However, all lepidopteran insect proteinases had pH optima ranging from 8.5 to 10.5. The optimumtemperature was found to be 30 to 40 °C. The inhibitory activities of little and finger millet inhibitors towards the insectproteinases are very low except notable level inhibition of proteinases of some insects. The gut proteinase zymogram ofdifferent insects revealed 2 – 6 isozymes and the inhibitors moderately inhibited all the isozymes of insects tested.  相似文献   

11.
An exocellular proteinase synthesized by the geophilic dermatophyte Trichophyton vanbreuseghemii has been purified and characterized. The fungus obtained from soil in Iran was cultivated in modified Czapek–Dox liquid medium containing 0.1% bacteriological peptone and 1% glucose as the nitrogen and carbon sources. Partial purification of the proteinase was accomplished by (NH4)2SO4 precipitation, followed by ion exchange chromatography. Analysis of the enzyme by SDS-PAGE revealed a single polypeptide chain with an apparent molecular mass of 37 kDa. Proteinase activity was optimum at pH 8, but remained high in the range of pH 7–11. Moreover, the partially purified enzyme presented a keratinolytic activity as evidenced by the keratin azure test. The inhibition profile and the good activity of the enzyme towards the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide suggested that it belonged to the chymotrypsin/subtilisin group of serine proteinases. The keratinolytic properties of T. vanbreuseghemii suggest that this fungus may be an alternative for the recycling of industrial keratinic wastes.  相似文献   

12.
Summary Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast.Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD + yeast cells after a dose of 50 Jm-2 of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD + strains studied.The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts.A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented.Abbreviations UV Ultraviolet - BRIJ-35 Polyoxyethylene-23-lauryl ether - EDTA Ethylene diamine tetraacetic acid - EGTA Ethylene glycol bis (-aminoethyl ether) tetraacetic acid - MOPS 3-[N-morpholine]propansulfonic acid - HEPES N-2-Hydroxyethylpiperazine-N-2-ethansulfonic acid - Tris Tris(hydroxy methyl)amino methane - BTPNA N-benzoyl-L-tyrosine-p-nitroanilide - CP.Y Carboxypeptidase Y - Leu.AP Leucin amino peptidase - Lys.AP Lysin amino peptidase - DMFA Dimethyl formamide - CHX Cycloheximide - PMSF Phenylmethyl sulfonyl fluoride - TCA Trichloroacetic acid Code Number of Enzymes EC. 3.4.23.8 Proteinase A - EC. 3.4.22.9 Proteinase B - EC. 3.4.12.8 Carboxypeptidase Y - EC. 3.4.24.4 Thermolysin - EC. 3.4.23.1 Pepsin A  相似文献   

13.
Two Ca2+-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, Mr = 150,000 and Mr = 200, 000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca2+-requiring proteinases by incubation with Ca2+ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA81, 53–56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mm Ca2+ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca2+ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.  相似文献   

14.
A new protease (araujiain h l) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl2, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 M. When assayed on N--CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.  相似文献   

15.
16.
Chickpea (Cicer arietinum L.) seeds contain Bowman–Birk proteinase inhibitors, which are ineffective against the digestive proteinases of larvae of the insect pest Helicoverpa armigera. We have identified and purified a low expressing proteinase inhibitor (PI), distinct from the Bowman–Birk Inhibitors and active against H. armigera gut proteinases (HGP), from chickpea seeds. N-terminal sequencing of this HGP inhibitor revealed a sequence similar to reported pea (Pisum sativum) and chickpea -l-fucosidases and also homologous to legume Kunitz inhibitors. The identity was confirmed by matrix assisted laser desorption ionization – time of flight analysis of tryptic peptides and isolation of DNA sequence coding for the mature protein. Available sequence data showed that this protein forms a distinct phylogenetic cluster with Kunitz inhibitors from Glycine max, Medicago truncatula, P. sativum and Canavalia lineata. The isolated coding sequence was cloned into a yeast expression vector and produced as a recombinant protein in Pichia pastoris. -l-fucosidase activity was not detectable in purified or recombinant protein, by solution assays. The recombinant protein did not inhibit chymotrypsin or subtilisin activity but did exhibit stoichiometric inhibition of trypsin, comparable to soybean Kunitz trypsin inhibitor. The recombinant protein exhibited higher inhibition of total HGP activity as compared to soybean kunitz inhibitor, even though it preferentially inhibited HGP-trypsins. H. armigera larvae fed on inhibitor-incorporated artificial diet showed significant reduction in average larval weight after 18 days of feeding demonstrating potent antimetabolic activity. The over-expression of this gene in chickpea could act as an endogenous source of resistance to H. armigera.  相似文献   

17.
Potato proteinase inhibitor II (PI2) is a serine proteinase inhibitor composed of two domains that are thought to bind independently to proteinases. To determine the activities of each domain separately, various inactive and active domain combinations were constructed by substituting amino acid residues in the active domains by alanines. These derivatives were expressed as soluble protein inEscherichia coli and exposed on M13 phage as fusions to gene 3 in a phagemid system for monovalent phage display. Inactivation of both active domains by Ala residues reduced binding of phage to trypsin and chymotrypsin by 95%. Ten times more phage were bound to proteinases by domain II compared to domain I, while a point mutation (Leu5 Arg) altered the binding specificity of domain I of PI2 phage from chymotrypsin to trypsin. The mutants were used to show that functional PI2 phage mixed with nonfunctional PI2 phage could be enriched 323 000-fold after three rounds of panning. Thus, these results open up the possibility to use phage display for the selection of engineered PI2 derivatives with improved binding characteristics towards digestive proteinases of plants pests.The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number L37519 (p303.51).  相似文献   

18.
The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday  相似文献   

19.
Factors associated with the production of extracellular lipase and proteinase by Pseudomonas fluorescens B52 during the late-log, early-stationary phase of grown were examined. Active lipase production by resting cell suspensions was observed when cells were harvested during the log phase (A600 of 0.3–0.9) Resting suspensions of younger cells (A600<0.1) synthesized lipase after a significant lag. Addition of cells of the proteinase-and lipasedeficient mutant P. fluorescens RM14 to B52 cells at low density resulted in stimulation of lipase and proteinase production. Similar results were found using cell-free culture fluid of RM14. Gel filtration on Biogel P2 revealed that the stimulatory factor co-chromatographed with the iron(III) siderophore, pyoverdine. Partially purified pyoverdine stimulated enzyme synthesis at a concentration of 6 M while having no effect on activity of preformed enzyme. Production of pyoverdine and extracellular enzymes was also stimulated by transferrin, a strong iron(III) binding protein. Growth of B52 in deferrated media was limited to 27% of that found with untreated media. Maximum pyoverdine, proteinase and lipase synthesis was obtained at a final iron(III) concentration of 5.75 M. Growth was maximal in 8.75 M iron(III) while synthesis of pyoverdine, proteinase and lipase was reduced to 3.6, 6.6 and 30% respectively in 23.75 M iron(III). Lipase activity in cell-free culture fluid was slightly inhibited by the addition of up to 400 M iron(III) while proteinase activity was unaffected. In dilute cell suspensions, lipase synthesis was more sensitive to iron(III) than was proteinase (50% inhibition at 1.6 M and a maximum of 40% inhibition at 5.0 M, respectively). In the case of lipase, added pyoverdine was able to partially protect enzyme production from the effects of iron(III). The results are consistent with a role for iron(III) in the regulation of extracellular lipase and proteinase synthesis by P. fluorescens.Contribution No. 677 from the Food Research Centre  相似文献   

20.
Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V. sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (VPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.  相似文献   

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