Physiological Changes of Rhodotorula glutinis Induced by an Acid Protease Inhibitor (S–PI)

An acid protease inhibitor (S–PI) accelerated the growth of Rhodotorula glutinis. This acceleration of the growth was observed only when the pH of the culture fluid lowered during the cultivation, but was not observed when the pH of the culture fluid was adjusted to 2.8 with NaOH. The increase in number of cells was closely related to cell deaths. The cell deaths were observed in an active growing state but not in a resting state. S–PI increased the DNA, RNA and protein content in the cell and decreased the lipid, especially the phospholipid content. When the yeast was cultured in an acidic medium having an initial pH of 1.8 in the presence of S–PI, the decrease of the phospholipid in the cell preceded, followed by the leakage of potassium ion, UV absorbing materials and free pool amino acid from the cell which were accompanied with the cell death. On the basis of these results, physiological changes occuring in the yeast cell are discussed.     

Inhibition of Acid Proteases by a Pepsin Inhibitor (S-PI)

S–PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.

S–PI made an equimolar complex with these acid proteases. A part of the enzyme-S–PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S–PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S–PI free) enzyme, each of the enzyme-S–PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^?10M, Rh. glutinis acid protease; 1.8×10^?7M, Cladosporium acid protease; 3.0×10^?6M.

These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S–PI complex. S–PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.

Binding between these acid proteases and S–PI dissociated at around neutral pH. S–PI was separated from enzyme-S–PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S–PI complex by DEAE cellulose column chromatography as a native form.     

Acid Protease of Bovine Milk

Occurrence of milk acid protease in bovine casein in addition to alkaline protease was found and purification of this enzyme was achieved. The enzyme had a pH optimum at 4.0 and was most stable at pH 3.5. The molecular weight of the enzyme was 36,000 and no inhibition was observed by diisopropyl-fluorophosphate, EDTA etc. This enzyme is considered to be similar to cathepsin D.

Milk acid protease mainly hydrolyzed α_s-casein and similar change was observed in autolysis of casein at pH 5.5. It is suggested that milk acid protease may have some significance in cheese ripening.     

Purification and Some Enzymatic Properties of Acid Protease A and B of Scytalidium lignicolum ATCC 24568

Three kinds of acid proteases were purified from the culture filtrate of Scytalidium lignicolum ATCC 24568. About 3 mg of A–1, 6 mg of A–2 and 60 mg of B were obtained from one liter of culture broth. These purified enzymes were monodisperse by physicochemical criteria such as ultracentrifugal analysis and disc electrophoresis.

A–1 and A–2 were very similar to each other on their enzymatic properties except the small difference of isoelectric point. A–1 and A–2 were active between pH 3.0~3.5 toward casein, and stable between pH 2.5 and 5.5 for 20 hr at 37°C. Both enzymes were strongly inhibited by NBS, but not by EDTA, DFP and sulfhydryl reagents.

B was most active at pH 2.0, and stable at pH values between 1.5 and 5.0. This enzyme was also inhibited by NBS and KMnO₄, but not by EDTA, DFP and sulfhydryl reagents.

The molecular weights and isoelectric points of A–1, A–2 and B were 43,000, pH 3.6; 43,000, pH 3.8 and 22,000, pH 3.2, respectively.

A–1 and A–2 were not inhibited by S–PI and synthetic pepsin inhibitor such as diazoacetyl-dl-norleucine methylester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). B was inhibited by EPNP, but not by S–PI and DAN.     

Isolation and Identification of a Microorganism which Produces Non Streptomyces Pepsin Inhibitor and N-Diazoacetyl-dl-norleucine Methylester Sensitive Acid Proteases

Non pepsin inhibitor (S–PI) and diazoacetyl-dl-norleucine methylester (DAN) sensitive acid proteases producing microorganism was isolated from farm soil of Osaka Prefecture.

The isolated strain was identified as Scytalidium lignicolum M–133. When it was aerobically grown on a medium consisting of glucose 5%, meat extract 1.5%, yeast extract 0.1%, KH₂PO₄ 0.2%, MgSO₄·7H₂O 0.05% at pH 3.5 and 25°C, the strain produced two acid proteases, A and B, in the culture broth.

The acid proteases A and B were not at all inactivated by S–PI and DAN. These acid proteases were expected to be a new type of acid protease from the viewpoint of the active site.     

IgA Protease Produced by Streptococcus sanguis and Antibody Production against IgA Protease in Patients with Behçet's Disease

The production of IgA protease in twelve strains of Streptococcus sanguis isolated from patients with Behçet's disease (BD) was examined. Protease activity was detected in 10 out of 12 strains. The protease was purified from one representative strain, S. sanguis 113–20, by employing Rotofor and DEAE-Sephacel chromatography. The molecular mass of the purified protease was approximately 100 kDa, and it cleaved the proline-threonine site of the IgA. Both IgG and IgA titers against the cells (113–20) and the purified IgA protease in the sera of BD patients and healthy controls, 36 each, were assayed. The IgG titers against the cells and protease were not significant in the BD patients or controls, but the IgA titers against the cells and protease in the BD patients were significantly higher than those of the controls. These data indicate that the BD patients are infected with IgA protease-producing S. sanguis strains, which cause an increase of IgA titer against these organisms and IgA protease antigen. Since the organisms can proliferate in BD patients for a long period of time (years), it seems that IgA antibodies cannot effectively eliminate the organisms.     

Acid Protease Produced by Trametes sanguinea,a Wood-destroying Fungus

A wood-destroying fungus, Trametes sanguinea, produced a potent acid protease in a submerged culture. Maximum proteolytic activity of the culture was attained after 140-hours cultivation in a medium containing dextrin and corn steep liquor. The acid protease was obtained in crystalline form from the mycelium-free culture filtrate by the following successive treatments: acetone precipitation, ionexchange column chromatography, ammonium sulfate fractionation, dialysis, and crystallization by acetone. Throughout the over-all process, the acid protease was purified approximately 30-fold with about 8% recovery of the original activity.     

Thermostable Acid Protease Produced by Penicillium duponti K1014, a True Thermophilic Fungus Newly Isolated from Compost

A thermophilic fungus, K1014, newly derived from a compost was selected on the basis of protease productivity as the only one of 81 isolates to produce high levels of acid protease. The fungus was named Penicillium duponti K1014 based on taxonomical studies. It grew in the temperature range of 28 to 58 C, and the optimum was 45 to 50 C. These temperature characteristics showed that the fungus was the most strongly thermophilic of all the fungi next to Humicola lanuginosa. When P. duponti K1014 was grown on moistened wheat bran, maximal accumulation of acid protease occurred after 2 days at 45 to 50 C. The addition of ammonium salts, but not nitrate, was effective for the production of the acid protease. The acid protease of P. duponti K1014 was stable at 60 C for 1 hr and retained more than 65% of original activity after the treatment for 1 hr at 70 C at pH 4.7. This thermal property was different from those of the ordinary acid proteases, indicating that the enzyme is a thermostable protein.     

The State of Amino Acid Residues in Alkaline Protease Produced by Bacillus No. 221

The state of amino acid residues in alkaline protease of Bacillus No. 221 and that of subtiiisin BPN’ were compared by spectrophotometric tiiration of tyrosine residues and by several reagents: β-naphtoqumone-4,6-disulfonic acid and monochlorofluoroquinone for amino groups, H₂O₂-dioxane for tryptophan, glyoxal for arginine, and tetranitromethane for tyrosine.

The reactivity of both proteases was fairly similar to those reagents.

The helix content of alkaline protease of Bacillus No. 221 (37%) was higher than that of subtilisin BPN’ (20%).

The Km and V_max of alkaline protease of Bacillus No. 221 toward ATEE and BTEE were obtained from Lineweaver-Burk plot and compared with those of α-chymotrypsin and subtiiisin BPN’.     

Complete Amino Acid Sequence of Neutral Protease from Bacillus subtilis var. amylosacchariticus

The neutral protease of Bacillus subtilis var. amylosacchariticus was cleaved chemically or digested with proteolytic enzymes, and the resultant peptides were separated and purified by high performance liquid chromatography. The sequence analyses of these peptides by the manual Edman procedure established the complete amino acid sequence of the enzyme. The neutral protease consisted of 300 amino acid residues with Ala and Leu as its amino- and carboxyl-termini, respectively, and the molecular weight was calculated to be 32,633. The sequence was found to be identical to that of B. subtilis 1A72 neutral protease, which was deduced from nucleotide sequencing. Comparison of the sequence with those of other Bacillus proteases revealed that the putative active site amino acid residues, Zn-binding ligands, and two Ca-binding sites were well conserved among them, as compared with those of thermolysin.     

Isolation and Crystallization of Microbial Alkaline Protease Inhibitor,S-SI

S–SI, an microbial alkaline protease inhibitor, was produced in the culture broth of Streptomyces albogriseolus S–3253. S–SI was isolated from culture filtrate by salting out with ammonium sulfate, column chromatographies on DEAE-cellulose and Sephadex G–100. S–SI was also isolated by pH adjustment of the effluent from DEAE-cellulose column.

Crystallization of S–SI was carried out, and two distinct shaped crystals, needle shaped and rhombic crystal, were obtained. Homogeneity of these crystals were identified by polyacrylamide disc electrophoresis at various pHs.

S–SI retained its perfect inhibitory activity after treatment at 37°C for 25 hr in the pH range from 3 to 10, and 100°C for 10 min in the pH range from 4 to 6.

From the examination of molecular weight determination with Sephadex G–100, the molecular weight of S–SI was decided to be about 27,000.

S–SI was found to be highly specific towards microbial alkaline proteases.     

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.     

Protease inhibitors of Manduca sexta expressed in transgenic cotton

Summary To explore the effectiveness of insect derived protease inhibitors in protecting plants against insect feeding, anti-trypsin, anti-chymotrypsin and anti-elastase protease inhibitor (PI) genes from Manduca sexta L. were expressed in transgenic cotton (Gossypium hirsutum L.). From 198 independent transformants, 35 elite lines were further analyzed. Under the control of the 35S promoter of CaMV, PI accumulated to approximately 0.1% of total protein, depending on the tissue analyzed. Using cell-flow cytometry, DNA content/ nuclei of transgenic and non-transformed cotton were identical. On cotton plants expressing PIs, fecundity of Bemisia tabaci (Genn.), the sweetpotato whitefly, was reduced compared to controls. Expression of these protease inhibitors may reduce the developmental rate of B. tabaci and other insects, and provide a strategy for cotton protection.     

Expression and Secretion of Scytalidopepsin B,an Acid Protease from Scytalidium lignicolum,in Yeast

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.     

Enantioselectivity of Enzymatic Cleavage Reaction of 13-Hydroperoxylinolenic Acid to C6-Aldehyde and C12-Oxo Acid in Tea Chloroplasts

A protease with strict specificity to lysyl peptide bonds like that of Achromobacter protease I was purified from a crude enzyme powder obtained from a culture filtrate of Achromobacter lyticus M497-1 and characterized. The purified enzyme had the following differences from protease I. The enzyme had an isoelectric point of 5.3, lower than the value of 6.9 for protease I. The amino acid composition of the enzyme had higher proportions of His, Glu, and Gly and lower proportions of Arg and Thr than protease I. The enzyme was unstable (30% residual activity) in the presence of 7 m urea (pH 8.0, 30°C, 20 min); protease I was resistant to the same conditions (80% residual activity). The k_cat/Km values for the hydrolysis of Tos-Lys-OMe and Lys-pNA by the enzyme were lower than those of protease I.     

Purification and Characterization of a Cysteine Protease from Corms of Freesia,Freesia reflacta

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26,000 by SDS–PAGE. The optimum pH of the enzyme was 6.0–7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.     

Protease excretion during pineapple micropropagation in temporary immersion bioreactors

Summary Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine (BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. As happens in investigations focused on protease production, the specific protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion.     

The Bacteriophage-Inactivating Effect of Basic Amino Acids; Arginine,Histidine, and Lysine

Some physicochemical properties and substrate specificity of acid protease B isolated from Scytalidium lignicolum were investigated.

The molecular weight determined by the sedimentation equilibrium and sedimentation velocity method was 21,000 and 19,000~20,000, respectively. The isoelectric point was determined as 3.0 using the Tiselius electrophoresis apparatus, 3.2 by isoelectric focusing, respectively.

The enzyme did not contain histidine and was composed of 188 amino acid residues. Substrate specificity against various synthetic peptides was different from those of the acid proteases which were inactivated by S–PI and DAN.     

Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

BackgroundMajor protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs.MethodsSamples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain.ResultsWe cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC₅₀ to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC₅₀ to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure.ConclusionsHere we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally.     

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them ``SSI-like proteins' (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338–4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins. Received: 23 August 1996 / Accepted: 20 November 1996