The unique stability of Vibrio proteolyticus neutral protease under alkaline conditions affords a selective step for purification and use in amino acid-coupling reactions.

A procedure is described for the purification of a neutral protease from fermentation broths of Vibrio proteolyticus. The key feature of the purification scheme is the selective, irreversible inactivation of a contaminating exoenzyme, aminopeptidase, by alkali treatment, rather than removal of this enzyme by conventional chromatographic methods. Fermentation broths or concentrates were brought to pH 11.5 to 11.7 by Na2CO3-NaOH addition and incubated at 25 degrees C until aminopeptidase activity was diminished. The alkali treatment resulted in greater than 99% reduction of aminopeptidase activity with minimal loss of neutral protease activity. The neutral protease could be further purified to apparent homogeneity by QA-52 cellulose chromatography. The alkali treatment of fermentation concentrates was also useful for preparation of V. proteolyticus neutral protease to effect the coupling of N-protected aspartic acid and phenylalanine methyl ester for the production of N-aspartylphenylalanine methyl ester, a precursor for the sweetener aspartame.     

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.     

An Aminopeptidase from Mouse Brain Cytosol That Cleaves N-Terminal Acidic Amino Acid Residues

An aminopeptidase with specificity directed toward peptides with acidic N-terminal amino acid residues has been isolated from mouse brain cytosol. Purification by ion-exchange chromatography and gel filtration resulted in an enzyme that hydrolyzed aspartyl-phenylala-nine methyl ester at a rate of 13.2 μu,mol/min/mg protein at pH 7.5, an increase in specific activity of 1000-fold over that of brain homogenate. Its apparent molecular weight, determined by gel filtration, is ?450,000. Dipeptides with N-terminal aspartyl residues are cleaved preferentially to glutamic-containing analogs, and a neutral amino acid (or histidine) is necessary in the adjacent position. For pep-tides of the form aspartyl-X, relative activity was 100, 81, 71, 66, 19, or 0, where X was alanine, serine, leucine, phenylalanine, histidine, or proline, respectively. Tripep-tides were more rapidly hydrolyzed than dipeptides; however, activity tended to decline with increasing chain length. The acidic aminopeptidase can account for almost all of the activity of brain cytosol toward the N-terminal aspartyl residue of angiotensin II, aspartyl-phenylalanine methyl ester or aspartyl-alanine, and the N-terminal glu-tamyl residue of adrenocorticotropin(5-10). The enzyme was unaffected by bestatin or amastatin. It was inhibited by o-phenanthroline and EDTA. The latter effect could be reversed completely by Zn²⁺ and partially by Mn²⁺ or Mg²⁺; Co²⁺ and Fe²⁺ had no effect; Ca²⁺ was inhibitory. These properties distinguish the brain acidic aminopeptidase from aminopeptidase A isolated from human serum or pig kidney and the aspartyl aminopeptidase of dog kidney.     

Studies on proteolytic activity in commercial myoglobin preparations

Commercial myoglobin preparations from horse skeletal muscle degraded casein. The maximum activity was at pH8-8.5. A muscle myofibril preparation was also attacked. The protease could be partly separated from the myoglobin by selective ultrafiltration through a membrane with an exclusion limit of mol.wt. 30000. A greater than 1000-fold purification of the proteolytic activity was achieved by affinity chromatography with soya-bean trypsin inhibitor bound to CM-cellulose. The enzyme preparation hydrolysed p-toluenesulphonyl-l-arginine methyl ester and N-benzyloxycarbonyl-l-tyrosine p-nitrophenyl ester. Its activity was inhibited strongly by soya-bean and ovomucoid trypsin inhibitors, serum and the soluble fraction of muscle homogenates. EDTA, p-chloromercuribenzoate and phenylmethylsulphonyl fluoride also caused some inhibition.     

Cloning and nucleotide sequence of the Vibrio proteolyticus aminopeptidase gene.

The gene encoding the Vibrio proteolyticus aminopeptidase was cloned and sequenced and its amino acid sequence was deduced. The gene encodes a 54 kDa protein, larger than the previously reported size of 30 kDa for the purified aminopeptidase. Sequence alignments revealed a 43-45% homology with two other Vibrio sp. extracellular proteinases.     

Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.     

Preparation and Purification of Microbial Proteases With Special Reference to The Bacilli

Methods for the examination of bacteria for protease production on semisolid media are described. The selection of media for production of small quantities of crude bacterial proteases from pure cultures of selected microorganisms in shake flasks is discussed. The most useful media have been found to be a grain-based medium, a soya fluff-starch-yeast extract medium and a fish meal-enzose-cerelose-cornsteep liquor medium. The alkaline proteases and neutral proteases can be identified and differentiated by specific assays and a purification procedure planned dependent upon the enzymes present in the fermentation beer. Crude enzyme can be precipitated from the fermentation beer by the addition of organic solvents such as acetone or isopropanol or by the addition of salts such as ammonium or sodium sulfate. The alkaline proteases typified by B. subtilis alkaline protease can be extensively purified by chromatography on Duolite C-10 cation exchange resin, whereas the neutral protease of 3 subtilis is best purified by chromatography on hydroxylapatite. Methods for purification of other proteases are discussed and the prechromatography steps for removal of pigment and other gross impurities are described.     

Purification and partial characterization of poliovirus protease 2A by means of a functional assay.

The purification of poliovirus protease 2A from infected cells by a functional assay is described. A small synthetic peptide was cleaved specifically by an esterase present in poliovirus-infected cells. Since the enzyme proved extremely unstable in crude extracts a rapid and efficient purification procedure had to be developed. By treatment with different detergents followed by high-speed centrifugation, the esterase activity was separated from inactivating cellular enzymes and was solubilized. Purification to more than 90% homogeneity could be achieved by a single chromatography step, namely, by gel filtration through Superose 12 under fast-protein liquid chromatography conditions. The esterase activity was associated with a protein of 17,000 daltons and copurified with poliovirus protein 2A. Furthermore, antibodies to 2A specifically precipitated the esterase activity. Thus, the esterase was identified as poliovirus protease 2A. Inhibition studies with known protease inhibitors revealed that 2A is probably a sulfhydryl protease. Of the metal ions tested, only zinc exerted significant inhibitory effects. The esterase activity was optimal near neutral pH and had an extremely short half-life at physiological temperatures.     

Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities

Bacillus proteolyticus CFR3001 isolated from fish processing wastes (both fresh water and marine) produced an alkaline protease. The optimum conditions for cell growth and protease production were 37 degrees C, 96 h, agitation speed of 100 rpm and medium pH 9. The partially purified protease obtained from had specific activity of 22.05 at 37 degrees C was active between 40 degrees C and 50 degrees C and lost >20% of its activity around 60 degrees C. Its molecular weight was approximately 29 kDa and it inhibited the growth of several pathogenic organisms such as Escherichia coli, Listeria monocytogenes, Bacillus cereus and Yersinia enterocolytica. The scanning electron microscopy (SEM) studies revealed that the protease produced by B. proteolyticus CFR3001 lysed the cells of these pathogenic bacteria.     

A new method for protease activity measurement.

A new method for protease activity measurement is described. In the presence of excess leucine aminopeptidase from Aspergillus japonica, action of protease on succinyl-casein results in the production of l-amino acids and their amino acids are simultaneously determined by l-amino acid oxidase-peroxidase system. Our proposed method is less time consuming and has a much higher sensitivity than the casein-Folin method. The present method is suggested to be suitable for the assay of neutral or alkaline proteases from animals and microorganisms.     

Schistosoma mansoni: purification and characterization of a membrane-associated leucine aminopeptidase

Membrane-associated leucine aminopeptidase (EC 3.4.11.1, LAP) has been purified to homogeneity from Schistosoma mansoni egg homogenates by a combination of ultracentrifugation, chromatofocusing, and molecular sieve chromatography. A 260-fold increase in specific activity was observed after purification. This is a metalloenzyme, containing carbohydrate moieties. Optimal enzyme activity was found at neutral pH. Enzyme activity was measured using L-leucine-7-amino-4-trifluoromethylcoumarin (L-Leu-AFC); in addition, schistosome egg LAP hydrolyzed a variety of other aminopeptidase substrates. Hydrolysis of L-Leu-AFC was inhibited by a number of aminopeptidase inhibitors, including 1,10-phenanthroline, bestatin, and amastatin.     

Specificity studies on enteropeptidase substrates related to the N-terminus of trypsinogen.

The specificity of the synthetic substrate Gly-[L-Asp]4-L-Lys 2-naphthylamide originally developed for the assay of enteropeptidase (EC 3.4.21.9), was investigated with partially purified aminopeptidase. Our results indicate that, not only enteropeptidase, but also the concerted action of the aminopeptidases of the rat small intestine, can rapidly release 2-naphthylamine from the substrate. A previously undescribed, highly active, dipeptidylaminopeptidase, which hydrolyses a Gly-Asp dipeptide from the N-terminus of the substrate, was detected in rat small intestine. The resulting [L-Asp]3-L-Lys 2-naphthylamide fragment is then degraded by a combination of aminopeptidase A and N to yield free 2-naphthylamine. Thus the present substrate cannot be regarded as being specific for enteropeptidase, and its use leads to an over-estimation of enteropeptidase activity in homogenates and extracts of intestinal tissue. In order to prevent this non-specific hydrolysis by aminopeptidases, stereoisomeric substrates with the sequence L-Ala-D-Asp-[L-Asp]3-L-Lys methyl ester, D-Ala-[L-Asp]4-L-Lys methyl ester and L-Ala-[Asp]4-L-Lys methyl ester were synthesized and tested as alternative substrates by their ability to inhibit the enteropeptidase-catalysed activation of trypsinogen.     

Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae

An assay for the detection of yeast (Saccharomyces cerevisiae) protease activity, using partially purified yeast-derived recombinant hepatitis B surface antigen (rHBsAg) as substrate, was developed to monitor proteolysis of rHBsAg that may occur through fermentation and isolation. The method consists of incubating small amounts of yeast lysate (protease source) with the substrate at 35 degrees C for about 16 h. Substrate proteolysis is assessed by subjecting the incubation mixtures to SDS-PAGE followed by silver-staining. The type of protease responsible for particular cleavages can be identified by treating the yeast lysates with specific protease inhibitors prior to incubation with substrate. The treatment of lysates with PMSF indicated that while many lysates possessed only serine protease activity (Protease B), some possessed proteolytic activity that could not be quenched with high levels of PMSF or other serine protease inhibitors. The use of the aspartyl protease inhibitor Pepstatin A in conjunction with PMSF virtually eliminated all proteolytic activity in these lysates, indicating that an aspartyl protease (Protease A) is expressed under some fermentation conditions. The relative amount of each protease in a lysate can be determined semiquantitatively by scanning the SDS gels densitometrically and plotting the ratio of degradates to intact antigen in the presence and absence of protease inhibitors. This method was used successfully to monitor the time-dependent expression of these proteases throughout production-scale fermentations. The impact of fermentation and purification changes on those proteases specifically responsible for the rHBsAg degradation can be easily evaluated.     

尖孢镰刀菌M1耐热蛋白酶纯化及酶学性质

【背景】前期工作中,从北大仓白酒大曲分离到一株真菌,经形态学和分子生物学方法,将其鉴定为尖孢镰刀菌(Fusarium oxysporim)M1,研究发现该菌能产中性蛋白酶。中性蛋白酶是应用于工业化生产的重要酶制剂。由于其作用条件温和、催化速率较高,被广泛应用于食品、医药、皮革、饲料、化工和废弃物处理行业。【目的】为了使该菌蛋白酶应用于相关工业生产,需要对该蛋白酶进行纯化和酶学特性研究。【方法】采用硫酸铵分级分离、疏水和离子交换层析对该菌蛋白酶进行纯化,通过SDS-PAGE测定酶的纯度和分子量,并研究其热稳定性和酸碱适应性。【结果】经各步层析,蛋白酶纯化倍数达26.1,得率为7.9%;经测定纯酶的分子量为62 kD;该酶最适温度为40℃,最适pH为7.0,属于中性蛋白酶;该酶对酸较敏感,对碱有较强的耐受性;耐热性较强,但酶活性不受乙二胺四乙酸二钠盐抑制。【结论】由于该中性蛋白酶具有较好的耐热性,因此,可作为工业生产上潜在的生物催化剂。     

Dipeptide Synthesis by an Aminopeptidase from Streptomyces septatus TH-2 and Its Application to Synthesis of Biologically Active Peptides

Dipeptide synthesis by aminopeptidase from Streptomyces septatus TH-2 (SSAP) was demonstrated using free amino acid as an acyl donor and aminoacyl methyl ester as an acyl acceptor in 98% methanol (MeOH). SSAP retained its activity after more than 100 h in 98% MeOH, and in the case of phenylalanyl-phenylalanine methyl ester synthesis, the enzyme reaction reached equilibrium when more than 50% of the free phenylalanine was converted to the product. In an investigation of the specificity of SSAP toward acyl donors and acyl acceptors, SSAP showed a broad specificity toward various free amino acids and aminoacyl methyl esters. Furthermore, we applied SSAP to the synthesis of several biologically active peptides, such as aspartyl-phenylalanine, alanyl-tyrosine, and valyl-tyrosine methyl esters.     

Dipeptide synthesis by an aminopeptidase from Streptomyces septatus TH-2 and its application to synthesis of biologically active peptides

Dipeptide synthesis by aminopeptidase from Streptomyces septatus TH-2 (SSAP) was demonstrated using free amino acid as an acyl donor and aminoacyl methyl ester as an acyl acceptor in 98% methanol (MeOH). SSAP retained its activity after more than 100 h in 98% MeOH, and in the case of phenylalanyl-phenylalanine methyl ester synthesis, the enzyme reaction reached equilibrium when more than 50% of the free phenylalanine was converted to the product. In an investigation of the specificity of SSAP toward acyl donors and acyl acceptors, SSAP showed a broad specificity toward various free amino acids and aminoacyl methyl esters. Furthermore, we applied SSAP to the synthesis of several biologically active peptides, such as aspartyl-phenylalanine, alanyl-tyrosine, and valyl-tyrosine methyl esters.     

Isolation of a neutral histone-hydrolyzing protease from bovine spleen]

Neutral histone-hydrolyzing protease has been isolated by fractionation of bovine spleen extract. The low level of the protease activity in the extract may be due to the presence of an inhibitor. The enzyme activity was increased 100--1200-fold during ammonium sulfate fractionations, gel filtration on Sephadex G-100 and G-75, chromatography on CM- and DEAE-celluloses. The protease was detected in the fraction with a molecular weight lower than 25000. The enzyme was markedly activated by dithiothreitol and EDTA and inhibited by p-chloromercuribenzoate and iodoacetic acid. It was also inhibited by N-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanyl chloromethylketone, bovine blood serum and partially by soybean trypsin inhibitor DFP, trasylol and epsilon-amino caproic acid had no effect. Beside histone, the neutral protease hydrolyzed casein and gamma-globulin and fibrinogen in a low extent. The enzyme had no activity toward N-benzoyl-D,L-arginine p-nitroanilide, N-benzoyl-L-arginine ethyl ester and N-acetyl-L-tyrosine ethyl ester, collagen, elastin and fibrin. Some properties of the enzyme were similar to those of neutral SH-dependent proteases described by Hayashi and Lo Spalluto et al.     

Protease from bovine spleen with kininogenase activity]

A protease with kininogenase activity at pH 7.5 was isolated from bovine spleen extract by gel filtration and ion exchange chromatography. The protease was found in the fraction with molecular weight lower than 25.000 and was separated from the other neutral SH-dependent protease by chromatography on KM-cellulose. The kininogenase activity was inhibited by DFP and trasylol; soybean trypsin inhibitor had no effect. The protease did not split N-benzoyl-L-arginine ethyl ester and N-benzoyl-D, L-arginine p-nitroanilide.     

A secreted aminopeptidase of Pseudomonas aeruginosa. Identification, primary structure, and relationship to other aminopeptidases

Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was approximately 56 kDa; hence, it was designated AP(56). Heating (70 degrees C) of the partially purified aminopeptidase preparations led to the conversion of AP(56) to a approximately 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP(56) and AP(28) were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, approximately 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.     

Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver.

The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.