Purification and properties of an extracellular protease from Myxococcus virescens.

An extracellular protease from Myxococcus virescens was purified by phosphate precipitation, gel exclusion, and ion-exchange chromatography. The enzyme appeared homogeneous upon disc electrophoresis. The molecular weight of the protease was estimated to be 26,000. The enzyme was rapidly inactivated by ethylenediaminetetraacetate, but the activity could be partially restored by divalent cations. Diisopropylphosphorofluoridate inhibited enzyme activity completely. Michaelis-Menten kinetics were obeyed with casein and hemoglobin as substrates. First-order kinetics were obtained with elastin as the substrate, provided trypsin was in excess. Petidolytic activity indicated that the peptide bonds hydrolyzed by the enzyme were mainly those involving amino acids with nonpolar side chains.     

Isolation and properties of an extracellular proteinase of Staphylococcus aureus

Serine proteinase splitting the peptide bonds which are formed by carboxyl groups of dicarboxylic amino acids was isolated from the supernatant of the Staphylococcus aureus culture liquid. It is similar to the enzyme isolated by Drapeau from Staphylococcus aureus strain V8 in its specifidity, molecular weight, amino acid composition, existence of two pH optima (pH 4.6 and 8.2). But there are some differences between the two proteinase in the content of dicarbonic amino acid residues. It was found that the enzyme can exist in two molecular forms.     

Physicochemical and biological properties of an extracellular serine protease of Aeromonas sobria

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature-labile and had an optimal pH of 7.5. Metallo-protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability-enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability-enhancing activity in skin. This suggests that a trypsin-like protease from skin mediates the activity of the protease to enhance its vascular permeability.     

Biochemical and physiological properties of an extracellular protease produced by Bacillus megaterium

A protease, excreted by a sporogeneous strain of B. megaterium, growing exponentially in a minimum glucose ammonium medium, was isolated. It is a neutral endopeptidase, stabilized by Ca⁺⁺, inhibited by o-phenanthroline, but not by di-isopropylfluorophosphate. The specificity, studied on insulin B-chain, glucagon, cytochrome c, and dipeptides substrates, indicated the need for a dipeptide backbone with both substituted amino and carboxyl groups. A requirement was observed for a nonpolar lateral chain in the amino acid whose amino group was involved in the peptide bond (Leu, Phe, Ala, He, Val). Rates of hydrolysis varied also with the amino acid whose carboxyl group was involved (e.g., His > Ser > Ala > Gly). In complex medium, supplemented with Yeast Extract, the biosynthesis of the protease was repressed during growth, but the same enzyme was excreted during sporulation. The repression was apparently of the same nature as that controlling sporulation during and after growth (e.g., repression by a mixture of amino acids or high concentration of glucose). An asporogeneous mutant showed a normal product ion of protease under all conditions, and a low intracellular protease turnover after growth. A mutant unable to produce protease showed a normal sporulation and a high protein turnover. This protease, here termed megapeptidase, seems to be a typical growth enzyme, not related to either the sporulation process or to the protein turnover after growth.     

Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile

An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351. Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration. It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx. 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5. It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin. No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed. A specificity for small aliphatic amino acids on either side of the splitting point was indicated. Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms. Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h). The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min). None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin. Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days. Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h. At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx. 1 h, 1 h and more than 5 h, respectively.     

Isolation and some properties of an acid protease from Fasciola hepatica.

A new protease, detected in an extract of Fasciola hepatica, was isolated and partly purified. The pH optimum for the cleavage of denaturated haemoglobin by the enzyme is pH 3.0. This proteolytic activity is inhibited by diazoacetylnorleucine methyl ester, pepstatin, the pepsin inhibitor from Ascaris suum, and phenylalanine. The cathepsin D inhibitor from potatoes, EDTA, mercaptoethanol and the inorganic salts tested have no inhibitory effect. The cleavage of the B-chain of oxidized insulin by enzyme was studied and compared with the digestion of the same substrate by chicken and pig pepsin. The protease from Fasciola hepatica belongs to the carboxyl group of proteases and probably plays an important role in helminth nutrition.     

一株产碱性蛋白酶菌株的筛选鉴定及酶学特性研究

【目的】从丝茅草中筛选得到产蛋白酶菌株并研究驯化过程中微生物群落结构,以及探究该菌株的生长特性和蛋白酶的酶学特性。【方法】通过高通量测序探究来源于丝茅草的菌株在不同培养条件下细菌种类及丰度,通过选择性培养基来筛选能够分解酪素并产生蛋白酶的菌株,通过单因素试验方法确定环境因子对菌株生长和蛋白酶活性的影响。【结果】微生物群落结构在基础培养基和牛肉膏蛋白胨培养基中不同。通过含酪素的选择性培养基里筛选到1株产蛋白酶菌株H-16,经生理生化试验和16S r DNA鉴定知该菌株属于Escherichia marmotae,菌株H-16能产生分子量为70 k Da左右的单亚基蛋白酶。胰蛋白胨、蔗糖、30°C或35°C、p H 7分别为菌株生长的最适氮源、碳源、温度和p H。菌株H-16分泌的蛋白酶最适p H为6–8,在50°C及6%盐度以下酶活性几乎不受影响。此外,Cu(II)和Ag(I)等金属离子能够抑制蛋白酶的活性。【结论】该菌株H-16为嗜中温菌株,能够产生碱性蛋白酶。     

Immunological Studies on Dermatophytes II. Serological Reactivities of Mannans Prepared from Galactomannans I and II of Microsporum quinckeanum, Trichophyton granulosum, Trichophyton interdigitale, Trichophyton rubrum, and Trichophyton schoenleinii

The contribution of terminal galactofuranose residues to the antigenic specificity and to cross-reactivity of galactomannans isolated from five species of dermatophytes, Microsporum quinckeanum, Trichophyton granulosum, T. interdigitale, T. rubrum, and T. schoenleinii, was investigated. Galactofuranose units were removed from galactomannans I and galactomannans II by mild acid hydrolysis. The resulting mannans were tested for serological reactivity with rabbit antiserum to M. quinckeanum by qualitative precipitation in gel and by quantitative complement-fixation analyses. Our results showed that, with this antiserum, the galactofuranose residues contributed greatly to the antigenic specificity and to cross-reactivity of the galactomannans II, but these residues were less significant as antigenic determinants in the galactomannans I. We have shown that mannans isolated from three Candida species reacted with rabbit antiserum to M. quinckeanum.     

Purification and some properties of the extracellular protease ofBacillus pumilus

Supernatant of a culture ofBacillus pumilus D 78 was precipitated with ethanol and chromatographed on DEAE- and CM-cellulose to isolate and purify a neutral protease with fibrinolytic and caseinolytic activity. Analysis by ultracentrifugation and immunoelectrophoresis indicate the homogeneity of the purified enzyme with the sedimentation constant s_20,w equal to 2.3. The fibrinolytic activity had a lower heat stability and was also more sensitive to pH higher than 8.0. The caseinolytic activity was stable over a wide range of pH (4.5 to 11.0). The enzyme binds acid dyes and is inhibited by Cu²⁺, Zn²⁺, Ca²⁺ and Fe³⁺, as well as byL-cysteine and KCN at a concentration of 10mM. Likewise, EDTA andp-chloromercuribenzoate show an inhibitory effect.     

Kinetic properties of extracellular alkaline protease of Rhizopus oryzae

Production of alkaline protease by Rhizopus species is an unusual phenomenon. However, a locally isolated species of Rhizopus oryzae was found to secrete alkaline serine protease of industrial importance. The kinetic parameter V (reaction velocity) of the purified fractionated enzyme was evaluated under different environmental conditions and substrate to enzyme ratios. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were also estimated.     

Isolation and properties of Serratia proteamaculans 94 cysteine protease

A new cysteine protease (SpCP) with a molecular mass of about 50 kDa and optimal functioning at pH 8.0 was isolated from the culture medium of a Serratia proteamaculans 94 psychrotolerant strain using affinity and gel permeation chromatography. The enzyme N terminal amino acid sequence (SPVEEAEGDGIVLDV-) exhibits a reliable similarity to N terminal sequences of gingipains R, cysteine proteases from Porphyromonas gingivalis. Unlike gingipains R, SpCP displays a double substrate specificity and cleaves bonds formed by carboxylic groups of Arg, hydrophobic amino acid residues (Val, Leu, Ala, Tyr, and Phe), Pro, and Gly. SpCP can also hydrolyze native collagen. The enzyme catalysis is effective in a wide range of temperatures. Kinetic studies of Z-Ala-Phe-Arg-pNA hydrolysis catalyzed by the protease at 4 and 37°C showed that a decrease in temperature by more than 30°C causes a 1.3-fold increase in the k _cat/K _m ratio. Thus, SpCP is an enzyme adapted to low positive temperatures. A protease displaying such properties was found in microorganisms of the Serratia genus for the first time and may serve as a virulent factor for these bacteria.     

Isolation and characterization of AStreptomyces C5 mutant strain hyperproductive of extracellular protease

Summary A pleiotropic streptomycete mutant was isolated that overproduced extracellular protease activity on azocasein, whereas the parent strain,Streptomyces C5, produced only negligible extracellular azocaseinase activity. Also unlike the parent strain, the mutant, designated C5-A13, was nonpigmented, and was unable to sporulate or produce antibiotics.     

Isolation of a psychrotrophic Azospirillum sp. and characterization of its extracellular protease

A novel psychrotrophic bacterium secreting a protease was isolated from a mountain soil in Korea. On the basis of a 16S rDNA sequence analysis and physiological properties, the isolate was identified as an Azospirillum sp. The protease purified from the culture supernatant was a monomer in its native form with an apparent molecular mass of 48.6 kDa on SDS-PAGE. The protease was active in a broad pH range around 8.5 and at temperatures up to 40 degrees C and stable at temperatures below 30 degrees C for 3 days. The proteolytic activity was inhibited by iodoacetamide and EDTA. The Mg2+ ion did not activate the enzyme much but reversed the inhibition by EDTA, suggesting that the protease belongs to a cysteine protease stabilized by the Mg2+ ion.     

Isolation and properties of extracellular proteinases of Penicillium marneffei

Penicillium marneffei is a dimorphic fungus native to Southeast Asia. Disease caused by this organism, until recently a very rare condition, has increased dramatically in parallel with the increase in the number of individuals in the region immunocompromised by AIDS and other conditions. While much research has been performed on the control of dimorphic switching in P. marneffei, there is a relative dearth of information regarding the proteinases secreted by this pathogen. Our laboratory has purified and characterized two proteinases produced by this organism in liquid culture and cloned the gene of a third. Both the recombinant enzyme expressed from the cloned gene and one of those purified from culture supernatants have been identified as members of the eqolisin family, a group of pepstatin-insensitive acid proteinases. The other enzyme purified from a culture supernatant is a serine proteinase with activity in the neutral pH range. These enzymes appear to be differentially expressed, depending on culture conditions.