Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

T sujibo , H., M iyamoto , K., H asegawa , T. & I namori , Y. 1990. Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete. Journal of Applied Bacteriology 69 , 520–529.
Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21000 and 36000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10–12 (protease I) and pH 10.5 (protease II). The optimum temperature for the activity of protease I was 70°C and that for protease II was 60°C. Protease I was stable in the range of pH 4.0–8.0 up to 60°C and protease II was stable in the range of pH 6.0–12.0 up to 50°C.     

Secreted fungal aspartic proteases: A review

The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.     

Partial characterization of digestive proteases in Totoaba (Totoaba macdonaldi)

ABSTRACT

The objective of this study was to characterize the digestive proteases of totoaba (Totoaba macdonaldi). Fish were sacrificed to obtain the multienzymatic extracts from the stomach and intestine, and determine the stability and optimum pH and temperature values. Residual activity and number of isoforms were determined with some inhibitors. Optimal pH of stomach proteases was 2, with stability above 100% at that same pH. Optimum pH of intestinal proteases was between 9 and 11, with stability above 100% between 8-12. Optimum temperature for stomach proteases was 35°C and remained highly stable, while optimum temperature for intestinal proteases was 45°C, with high stability between 35-55°C. Pepstatin A totally inhibited acid protease activity and revealed a single band. SDS-PAGE zymogram revealed 8 bands in the intestine, where phenanthroline inhibited 80% of the total activity. The digestive capacity of T. macdonaldi is characteristic of a strict carnivore, similar to other marine fish species.     

Production of different proteases from fish gut microflora utilizing tannery fleshing

Three alkaline protease‐producing strains designated as ANFLR1, NPLR1, and PROLR15 were isolated from Labeo rohita fish gut. These strains are able to produce alkaline protease using tannery fleshing (TF) as the sole carbon and nitrogen source and were identified as Bacillus megaterium, Serratia marcescens, and novel Pontibacter sps. Proteases from these organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, and column chromatography. SDS‐PAGE revealed molecular weights of the proteases to be 46 kDa (ANFLR1), 52 kDa (NPLR1), and 58 kDa (PROLR15). The optimum pH and temperature for the protease activity of ANFLR1, NPLR1, and PROLR15 were found to be 10.5, 11.5, 9, and 70^°C, 60^°C, and 50^°C, respectively. The maximum protease activities at the optimum conditions were 420 U/mL (ANFLR1), 550 U/mL (NPLR1), and 530 U/mL (PROLR15). Inhibition of the NPLR1 protease by pepstatin confirmed aspartate‐type enzymatic activity. Fe³⁺ enhanced the activity of PROLR15 protease. Unlike all other microbial proteases known so far, the PROLR15 enzyme did not require Ca²⁺ for activity and thermal stability. SDS‐PAGE and scanning electron microscopy analyses confirmed the conversion of high molecular weight substrate (TF) to low molecular weight peptides by these proteases. The alkaline metalloprotease production by novel Pontibacter sps. and aspartate protease production by S. marcescens remain unexplored. Hence, TF with its relatively abundant availability can be beneficially utilized for alkaline protease production through the fish gut microbial fermentation processes.     

Partial purification and characterization of aspartate aminotransferases from seedling oat leaves.

As relatively little information is available on the properties of aspartate aminotransferase from photosynthetic tissue, isolation and characterization of the two major electrophoretically distinct forms of this enzyme from seedling oat leaf homogenates were undertaken. These two forms are designated I for the more anionic form and II for the less anionic form. Form I, 80 to 90% of the total activity, has been purified to a specific activity of 120 mumol/min/mg of protein (1100-fold) and is estimated to be 90 to 95% homogeneous, as judged by analytical polyacrylamide gel electrophoresis. Form II, 10 to 20% of the total activity, has been purified to a specific activity of approximately 6 mumol/min/mg of protein (300-fold). Both forms exhibit optimal activity at pH 7.5. Michaelis constants do not differ greatly between forms I and II and are similar to those reported for the pig heart cytosolic enzyme as well as aspartate aminotransferase from other plant sources. A molecular weight of 130,000 for the purified aspartate aminotransferase I was estimated by sedimentation equilibrium centrifugation; molecular weights of the two forms are similar as estimated by sucrose density gradient centrifugation. No activation by pyridoxal phosphate has been observed during purification.     

Alanine Aminotransferase and Aspartate Aminotransferase in Leishmania tarentolae1

SYNOPSIS. Culture stages (promastigotes) of Leishmania tarentolae were tested for alanine aminotransferase (E.C.2.6.1.2) and aspartate aminotransferase (E.C.2.6.1.1.). Neither enzyme was detected in crude cell extracts. After starch block electrophoresis, however, both transaminase activities were found in proteins migrating toward the anode. Only one species of each enzyme was found. Using coupled enzyme assay systems, the following physical and kinetic properties were seen: 1) aspartate aminotransferase was inhibited by α-ketoglutarate concentrations above 1.68 × 10^?2 M and alanine aminotransferase was inhibited by concentrations higher than 1.34 × 10^?2 M; 2) the Michaelis constant (Km[α-ketoglutarate]) was 5.4 × 10^?4 M for aspartate aminotransferase and 3.0 × 10^?4 M for alanine aminotransferase; 3) maximum activity was found at ?pH 8.5 (broad range between pH 7.75–9.0) for aspartate aminotransferase whereas maximum activity for alanine aminotransferase was ?pH 7.2 (range between pH 7.0–7.5); 4) both enzymes lost half of their activity after 4 days at 8 C; 5) aspartate aminotransferase was most active at 35 C and completely inactivated at 59.5 C, alanine aminotransferase exhibited maximum activity at 29.5 C and was completely inactivated at 61 C; and 6) neither enzyme showed enhanced activity with added pyridoxal phosphate.     

Activities and partial purification of extracellular proteases of Bacteroides nodosus from virulent and benign footrot

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55 degrees C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2CO3 and thioglycollic acid, the total proteolytic activity and its stability at 55 degrees C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8.8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8.8, suggest that this property may be used to distinguish virulent and benign strains.     

Proteolytic components of alkaline proteases of Bacillus strains. Zymograms and electrophoretic isolation

Characterization of proteolytic activity in preparations or fermentation broth is accomplished by methods based on a combination of disc electrophoresis and visualization of the activity. The methods permit a direct, comparison with reference preparations. All alkaline proteases studied, including the three subtilisins, were found to consist of several proteolytic components. The zymograms of the subtilisin type preparations show an irregular pattern. At least 14 different components may be observed, belonging to 6 types of mobility pattern, 3 of which could be assigned to the subtilisins. None of the components belongs to the group of metalloproteases. A quite regular pattern is shown by the zymogram of protease preparations produced by the alkalophilic Bacillus strains. A few of the components of the subtilisin preparations Maxatase and protease A were isolated by preparative disc electrophoresis and by disc electrofococusing, allowing a further characterization. Special attention was directed to the determination of properties specific for the application of subtilisins as additives in household detergents. Thermostability in sodium tripolyhosphate solution was found to range from about 10% for one of the minor components to 80% for the main component of Maxatase. Three types of curves representing the effect of pH on the activity were observed. The curve of the main component of Maxatase shows a characteristic shape with a maximum at pH 10.3: with other components lower pH optima were observed. Isoelectric points of the component were found to range from pH 7 to 10.     

Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): sequence characterisation, expression pattern and characterisation of proteolytic activity

Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51-58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2.     

Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.     

Chromogenic substrate autography: a method for detection, characterization, and quantitative measurement of serine proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gels

A chromogenic substrate autography is described which allows characterization and quantification of serine proteases in crude systems after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. Separation of samples containing proteases by either method is followed by an overlay of the gels on an indicator film prepared by incorporation of a suitable paranitroanilide substrate into agarose. Positions of the proteases are revealed by the formation of yellow-colored zones which can be quantified by densitometry at 405 nm. The technique proved suitable for determination of molecular weights, isoelectric points, and quantitative measurements of amidolytic activities of urokinase, tissue plasminogen activator, tissue and plasma kallikrein, and thrombin in biological fluids and purified preparations.     

米黑毛霉(M.miehei)天冬氨酸蛋白酶的研究

用硫酸铵分部盐析及离子交换层析技术从米黑毛霉半固体培养物的浸提液中提纯了天冬氨酸蛋白酶,酶活力收率为19.5％,,比活力达3080SU/mg蛋白,提纯8.5倍。用聚丙烯酰胺凝胶电泳、SDS-凝胶电泳、等电聚焦、双向免疫扩散及免疫电泳等方法鉴定该酶均一。本文还报道了关于该酶生化性质、动力学性质及化学修饰的研究结果。该酶以天冬氨酸为其活性的必需基团,系一典型的天冬氨酸蛋白酶。     

Purification and characterization of lysine-sensitive aspartate kinase from maize cell cultures

Aspartate kinase is a feedback-regulated enzyme that controls the first step common to the biosynthesis of lysine, threonine, isoleucine, and methionine in plants. Aspartate kinase was purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures for physical and kinetic characterization studies. Partial purification and elution from an anion exchange column resolved two lysine-sensitive aspartate kinase isoforms. Both isoforms were purified >1,200-fold to a minimum specific activity of 18 units/milligram of protein. Both isoforms were sensitive to the lysine analogues S-2-aminoethyl-l-cysteine, l-lysine ethyl ester, and δ-hydroxylysine. No threonine-sensitive form of aspartate kinase was detected at any stage during the purification. Additional purification steps were combined with preparative gel electrophoresis to obtain apparently homogeneous lysine-sensitive aspartate kinase. Aspartate kinase appeared to be a tetramer with a holoenzyme molecular weight of 254,000 and to be composed of 49,000 and 60,000 subunits. The tetramer appeared to disassociate during native gel electrophoresis to 113,000 dalton species that retained aspartate kinase activity.     

Electrophoretic analysis of proteases in Echinostoma Caproni and Echinostoma trivolvis

Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.     

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7–8 and at temperature close to 35°C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40–45°C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.     

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L ‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.     

海洋细菌QD80低温碱性蛋白酶的基因克隆和性质

对海洋细菌QD80所产低温碱性蛋白酶进行了基因克隆和序列分析,对此酶的性质进行了初步研究.此酶基因开放阅读框架为1377bp,分子量为49.9kD.此序列上游-8bp处为该基因的SD序列,-10区和-35区分别有5′TAGAAT3′和5′TTGACC3′的保守序列.该酶最适pH为9.5,最适反应温度为30℃,在10℃酶活力仍能保持30%以上.该酶对氧化剂H2O2的抗氧化作用明显,浓度达到4gL时酶活仍保留85%.该蛋白酶的低温适应性和抗氧化特性将对其在低温洗涤领域的应用提供广泛的潜在应用价值.     

Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.     

Purification and Properties of Proteases from a Marine-psychrophilic Bacterium

Protease which was found in the culture fluid of Pseudomonas sp. No. 548 was fractionated into four components with protease activity by a two step chromatography using DEAE-cellulose. Each protease was further purified by gel filtration on Sephadex G-100 and/or G-75. The protease of Ia was obtained in crystalline form and was shown to be homogeneous by analysis with electrophoresis, while the other three enzymes were also highly purified. The enzymatic properties of the proteases were investigated. All of the four enzymes were inactivated by ethylene diamine tetraacetate. Proteases Ia, Ib, and IIb were inactivated by diisopropylfluorophosphate. The optimum activity of protease Ia was shown to be at pH 10.0, and that of the other enzymes were at pH 7.0 to 8.0. The proteases of Ia, Ib, and IIb were stabilized by calcium ion. The effect of temperature, pH, and metal ions on the activity of the enzyme were also investigated.