Fungal Proteolytic Enzymes

Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.     

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.     

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 characteristics of alkaline proteinase from alkalophylic Bacillus sp.]

Alkaline protease was purified from Bacillus sp. isolated from soil. The pH optimum was 11.5 at 37 degrees C. Calcium divalent cation was effective to stabilize the enzyme especially at higher temperatures. The proteolytic activity was inhibited by active site inhibitors of PMSF (Phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, Hg. The enzyme was stable in the presence of some detergents, such as Triton-X-100, Tween-80, SDS (sodium dodecyl sulfate) and EDTA (ethylendiaminetetraacetic acid), pH 11.5 and 37 degrees C for 30 min. The optimum pH was 11.5 at 37 degrees C and the optimum temperature was 62 degrees C at pH 11.5.     

Characterization and isolation of a trypsin-like serine protease from a long-term culture cytolytic T cell line and its expression by functionally distinct T cells

We describe the characterization and purification of a trypsin-like serine protease isolated from cloned long-term culture cytolytic T cell line (CTLL AK). High amounts of proteolytic activity were isolated from extracts of CTLL AK after either nitrogen cavitation or detergent lysis. Trypsin-like protease was detected by using either the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester or a panel of low molecular amide substrates. The latter compounds were preferentially cleaved at the carboxyl termini of lysine and arginine residues. The enzyme activity was completely inhibited by two serine esterase inhibitors, diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and by aprotinin and meta-aminobenzamidine, which are known to block trypsin-like proteases. The pH optimum for CTLL AK-derived protease activity is 8 to 9. Analysis of the enzyme by gel filtration revealed that the cell-bound proteolytic activity was associated with a complex that could not be dissociated by treatment with Triton X-100. The CTLL AK-derived protease activity was found to reside in two proteins with relative molecular masses (Mr) of 32,000 and 40,000 daltons as determined by affinity labeling with [3H]diisopropylfluorophosphate and sodium dodecyl sulfate gel electrophoresis. High levels of enzyme activity were found in a panel of H-Y-specific cloned T cell lines with either cytolytic/suppressor (CTLL) or helper potential (THL), indicating a lack of correlation between trypsin-like protease activity and a particular T cell function. High enzyme activity was also detected in tumorigenic variants of CTLL. Furthermore, it was excluded that the trypsin-like activity detected was attributable to plasminogen activator activity. In contrast to cloned T effector cells and their in vitro or in vivo derived variants, considerably less activity was found in normal nonactivated or activated lymphocyte populations. The possible role of the trypsin-like serine protease in the function of T effector cells is discussed.     

Studies on Protein Metabolism in Higher Plants

A leaf protease of tobacco whose activity was enhanced during curing was purified about 60 times with ammonium sulfate fractionation, ethanol precipitation, calcium phosphate gel treatment and Sephadex G-200 column chromatography, and some properties of the protease were examined. The purified enzyme showed the optimum pH at 5.5 and the optimum temperature at 60°C. The protease activity was stable between pH 4.5 and 5.5 at 50°G or at pH 5.5 below 40°C for 1 hr, but completely destroyed at 70°C during 1 hr. The protease activity was greatly activated by reducing agents such as cysteine, glutathione or mercaptoethanol and inhibited by p-chloromercuribenzoate, phenyl- mercuric acetate or silver ions. Metal ions except for silver ion and ethylenediamine tetraacetic acid did not affect the protease activity so far examined.     

Isolation,partial purification,and some properties of protease I from a thermophilic mold Thermoascus aurantiacus var. levisporus

When the thermophilic mold Thermoascus aurantiacus var. levisporus was grown in a modified Czapek Dox medium containing casein the filtrate was found to contain proteolytic activity. The maximum production of activity occurred at 50 ° C in a medium containing 8% casein. The filtrate was subjected to ammonium sulfate fractionation and chromatography on DEAE-cellulose. Two proteases were separated. No further work was done on protease II. Protease I was further purified by gel filtration on Sephadex G 100–200. It showed a 40-fold purification with a final recovery of approximately 25%. It is a neutral protease with a pH optimum at 7.0. The optimal activity of the enzyme occurred in 0.02 M phosphate buffer but was completely inhibited at a concentration of 0.1 M. The optimum temperature for casein hydrolysis was found to be 55 ° C. The enzyme was inhibited by Hg⁺⁺ but was greatly stimulated by Cu⁺⁺ and mercaptoethanol. Metallo and sulfhydryl agents had no significant effect on enzyme activity.     

Isolation,identification and some cultural conditionsof a protease-producing thermophilic Streptomyces straingrown on chicken feather as a substrate

Six strains of thermophilic actinomycetes were isolated from soil using an enrichmenttechnique with feathers as the sole carbon and nitrogen source. They showed clear proteolyticactivity on casein agar medium. The most active strain was tentatively identified as Streptomycesthermonitrificans. This isolate was grown in a basal medium with feathers and:or other carbon andnitrogen sources. Supernatant from centrifuged cultures was examined for protease activity andtemperature and pH optima were determined for enzyme activity. Optimum proteolytic activity onbasal liquid medium containing 1% chicken feather pieces was obtained at 50°C, in a mediumadjusted at pH8 and incubated for 72 h at 150 rpm. Proteolytic activity was further increased by1.5% feather pieces and the time required for maximal activity was 96 h. The keratinolytic activityof S. thermonitrificans was examined by incubation with native chicken feather pieces and it wasfound that it is significantly active. The degradation of whole intact feathers by S.thermonitrificans was obtained after 48 h of incubation at 50°C. The pH and temperature optimafor proteolytic activity were 9.0 and 50°C, respectively. The proteolytic activity was stable at40°C for 1 h. The proteolytic activity was inhibited by DFP but not by EDTA or pCMB. Theseresults inidicated that the enzyme(s) can be classified as an alkaline protease. 1999 ElsevierScience Ltd. All rights reserved.     

A cysteine protease of Dieffenbachia maculata.

Plants of the genus Dieffenbachia, very popular as indoor ornamental plants, are known for their toxic as well as therapeutic properties. Their toxic manifestations have been partly attributed to their proteolytic activity. The work described in the present paper shows that stem leaves and petiole of Dieffenbachia maculata Schott, a commonly grown species, contain significant proteolytic activity, different parts showing different types of protease activities. Stem showed the highest enzyme activity and this protease was purified about 55 fold by solvent precipitation, gel filtration and ion exchange chromatography. The enzyme has a relative molecular mass of 61 kDa as determined by SDS-PAGE and has an optimum pH of 8.0 and optimum temperature of 50 degrees C. Effects of various substrates, inhibitors and activators indicate that the enzyme is a cysteine protease with leucylpeptidase activity.     

Protease activity in cells ofBacillus megaterium during derepression

A proteolytic activity hydrolyzing denatured proteins of Bacillus megaterium labelled with 35S or 14C amino acids was detected in cells of the asporogenic strain of Bacillus megaterium. The substrate is hydrolyzed by the enzyme or enzymes at optimum pH around 7, their activity being almost completely inhibited by EDTA and o-phenanthroline. PMSF, the inhibitor of serine proteases, is slightly inhibitory. Gel filtration on a Sephadex column separated the protease activity to two or three fractions. The protease activity in cells with the repressed synthesis of protease corresponds to 5-20 mug of substrate degraded per hour by 1 mg of protein at 37 degrees C. It increases five to ten-fold during the derepression. When the intracellular protease activity increases the extracellular enzyme begins to be excreted into the medium. The intracellular protease activity rapidly decreases after the addition of chloramphenicol or of a mixture of amino acids to the derepressed culture. Half or even more of the protease activity is released from the cells during their conversion to protoplasts by means of lysozyme. This "periplasmic" activity remains mostly in the supernatant also after mesosomes have been centrifuged down from the periplasm. A portion of the activity bound in protoplasts sediments together with membrane fraction after their lysis.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies 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°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°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°C and stability from pH 4–12.5 at 60°C and below 75°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 Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium

Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C. The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48°C, pH optimum at pH 9 and a molecular weight of 87 500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48 degrees C, pH optimum at pH 9 and a molecular weight of 87500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

Degradation of an Fc‐fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease

A host‐cell‐related proteolytic activity was identified in a recombinant Fc‐fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post‐capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo‐protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. 2009; 104: 1132–1141. © 2009 Wiley Periodicals, Inc.     

Characterization of the dextranase purified from Streptococcus mutans Ingbritt.

We purified dextranase from the culture supernatant of Streptococcus mutans Ingbritt by procedures including ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the enzyme was estimated as 78 kDa by SDS-PAGE. The enzyme degraded dextran at the optimum pH of 5.5, but not other glucans and fructans at all. Paper chromatographic analysis revealed that the enzyme cleaved dextran by an endo-type mechanism. The enzyme was inhibited by Hg2+, Fe3+, Zn2+, and anionic detergents SDS and deoxycholic acid, but not inhibited by non-ionic detergents Triton X-100, Lubrol PX, Nonidet P-40, and Tween 80. SDS-blue dextran-PAGE analysis of the culture supernatant revealed that the enzyme activity detected in the 96 kDa band shifted gradually to the 78 kDa band during handling the supernatant. This shift was inhibited by phenylmethylsulfonyl fluoride, suggesting that the shift of the molecular size is due to proteolytic degradation of the enzyme by serine protease.     

Protease activity of CND41, a chloroplast nucleoid DNA-binding protein, isolated from cultured tobacco cells

CND41 is a 41 kDa DNA-binding protein isolated from chloroplast nucleoids of cultured tobacco cells. The presence of the active domain of aspartic protease in the deduced amino acid sequence of CND41 suggests that it has proteolytic activity. To confirm this, CND41 was highly purified from cultured tobacco cells and its proteolytic activity was characterized with fluorescein isothiocyanate-labeled hemoglobin as the substrate. The purified CND41 had strong proteolytic activity at an acidic pH (pH 2-4). This activity was inhibited by various chemicals, including the nucleoside triphosphates, NADPH, Fe(3+) and sodium dodecyl sulfate.     

Procollagen processing. Limited proteolysis of COOH-terminal extension peptides by a cathepsin-like protease secreted by tendon fibroblasts.

An enzymatic activity, capable of removing the COOH-terminal extensions of type I chick procollagen, has been demonstrated in embryonic chick tendons and in cultured tendon fibroblasts utilizing two new methods of analysis. The protease was purified by a combination of ultrafiltration concanavalin A affinity chromatography and gel filtration. The isolated protein has an apparent Mr of 43,000 by gel filtration and sodium dodecyl sulfate gel electrophoresis. The enzyme shows a major pH optimum at 4.2 and is susceptible to inhibitors such as pepstatin and leupeptin; it therefore seems related to the cathepsins. The possibility that this enzyme plays a role in the limited proteolytic processing of procollagen is discussed.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

球形芽孢杆菌C_3—41菌株胞外蛋白酶特性

球形芽孢杆菌能够合成具杀蚊活性的蛋白晶体,该晶体在蚊中肠碱性条件下降解产生毒性,尽管球形芽孢杆菌蛋白酶与杀蚊毒素的降解无关,但它在球形芽孢杆菌杀蚊制剂的产生中有重要意义。同时球形芽孢杆菌产生的碱性蛋白酶具有潜在的医疗价值。 我们以本实验室分离的高效杀蚊菌C_3—41菌株为材料,研究了球形芽孢杆菌蛋白酶的产生特性及其理化性质,在国内尚属首次报道。