Bacterial Neutral Protease Research Articles

Presence of Exopeptidase-Resistant and Susceptible Peptides in a Bacterial Protease Digest of Corn Gluten.

Corn gluten hydrolysate (CGH) was prepared by food-grade bacterial proteases, alcalase and neutral protease. Digestion of CGH with carboxypeptidase A and leucine aminopeptidase extensively changed the elution patterns of peptides as observed from reversed phase high performance liquid chromatography-mass spectrometry (LC-MS), whereas digestion with pepsin and trypsin hardly affected the elution patterns. Twenty-five major peptides in CGH were identified. After digestion with exopeptidases, only prolyl dipeptides and pyroglutamyl di- and tripeptides remained, whereas the other 17 peptides completely disappeared. On the other hand, all 25 peptides remained after digestion with pepsin and trypsin. These facts suggest that a majority of short-chain peptides in food protein hydrolysates are degraded by exopeptidases during digestion and absorption processes. Thus, susceptibility to exopeptidases should be considered for prediction of bioactive peptide upon ingestion, which has not been considered in most of previous studies on food-derived bioactive peptides.

Evolution of Enzyme Requirements for Human Islet Isolation

Islet transplantation is becoming an established treatment option for managing a subset of adult patients who have type 1 diabetes mellitus. The success of this procedure is dependent upon the recovery of a sufficient number of functional human islets from donor organs for subsequent transplant. Here, the use of optimized bacterial collagenase-neutral enzyme mixtures has been shown to affect the yield and quality (defined by viability and glucose-stimulated insulin secretion) of islets recovered from human pancreata. However, few reports provide a systematic approach to correlate the biochemical characteristics or dose of collagenases and proteases to the recovery of functional islets. The focus of this review is to close this gap. The review summarizes five key advances toward an understanding of how Clostridium histolyticum collagenase and bacterial neutral proteases support the release of islets from human pancreata and key collagenase biochemical characteristics that lead to higher human islet yields. This information provides a foundation to develop a model of enzymatic tissue dissociation that can serve as a guide in assessing the effectiveness of collagenase or neutral protease enzymes used for human islet isolation. One key conclusion is the importance of excess amounts of collagen degradation activity in the islet isolation procedure to ensure effective degradation of native collagen. If achieved, the selection and dose of neutral protease become the primary factors that determine the maximal release of islets from tissue. Maximal collagen degradation activity is achieved when an appropriate dose of C. histolyticum collagenase products containing primarily intact class I and class II collagenase are used in the isolation procedure. The review concludes by presenting future directions for research to develop new insights into enzymatic tissue dissociation with the goal of improving the robustness of the islet isolation process.

The effects of using a starter culture, lipase, and protease enzymes on ripening of Mihalic cheese

The purpose of this study was to determine the effects of fungal lipase from Mucor miehei and a bacterial neutral protease from Bacillus subtilis alone and combined with a starter culture on ripening properties of traditional Turkish Mihalic cheese. The use of protease with lipase (Cult + Prot + Lip) resulted in better flavour and texture with accelerated ripening. The obtained results pointed out that the gross compositions of the cheeses were changed by the type of enzymes and ripening time (P < 0.01). The acid degree value (ADV) of all cheeses showed a linear increase with ripening. The highest lipolysis rate was noted in lipase‐added cheese batch (as 5.56 ADV) with highest γ‐CN ratio and β‐CN degradation. At the end of ripening time, it was observed that αs‐CN ratios decreased in starter‐added (Cult), starter + protease–added (Cult + Prot), and protease‐added (Prot) cheese batches. The use of protease with lipase (Cult + Prot + Lip) resulted in better flavour and texture with accelerated ripening. Protease‐added cheeses, which were characterized by bitterness and crumbly textural properties owing to the intense breakdown of β‐casein, scored lower than lipase‐added cheeses. It was determined that the use of mesophilic aromatic starter culture with lipase and protease could be used to accelerate ripening of Mihalic cheese made from pasteurised milk.

Successful Isolation and Transplantation of Nonhuman Primate Islets Using a Novel Purified Enzyme Blend

Successful Isolation and Transplantation of Nonhuman Primate Islets Using a Novel Purified Enzyme Blend

Study of the effects of dispase in the chemotherapy of multicellular tumour spheroids of small-cell lung carcinoma in man.

Multicellular tumour spheroids (MTS), diameter 650 microm, from PC-6, SBC-1 and NCL-H60 small-cell lung carcinoma cell-lines in man were prepared by the liquid overlay culture method and used to study the influence of treatment with dispase (bacterial neutral protease from Bacillus polymyxa, 1000 units mL(-1)) on the effectiveness of carboplatin, as determined by colony-forming assay. When carboplatin alone was used on monolayers the curve of survival fraction against concentration was exponential in shape, indicating that the drug was active against the monolayer. When MTS were treated with medium concentrations (10(-5) and 10(-4) M) of carboplatin alone the survival fraction-concentration curve showed that the effectiveness of the treatment was less than that against the monolayer. On treatment of MTS with carboplatin and dispase the survival fraction-concentration curve was similar to that obtained for the monolayer and the survival fraction of the core of the MTS was also less than when carboplatin alone was used. These results imply that dispase dissolves the intercellular matrix of the MTS enabling enhanced infiltration of carboplatin into the core of the MTS. Dispase thus indirectly increases the effectiveness of carboplatin.

Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases

Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

Pilot Scale Production of a Lager Beer from a Grist Containing 50% Unmalted Sorghum

Pilot scale (1000 L) brews were carried out with a grist comprising of unmalted sorghum (50% of total wet weight of grain) (South African variety) and malted barley (50% of total wet weight of grain) grist using a mashing program with rests at 50°C, 95°C and 60°C. Mashes were supplemented with a high heat stable bacterial α-amylase, a bacterial neutral protease and a fungal α-amylase. A control brew containing 100% malted barley was also carried out. Saccharification difficulties were encountered during mashing, and extraction of the grist was lower for the sorghum mashes. The sorghum mashes showed comparable lautering behaviour to that of the control mash. At mashing off the sorghum worts were starch positive. Apparent degree of fermentation of the sorghum gyles were less than the control gyles. Green beer filtration proved unproblematic. The sorghum beers compared quite closely with the control beer with regard to colour, pH and colloidal stability. Foam stability deficiencies were apparent with the sorghum beer. However, the fermentability of the sorghum worts were lower. Hence the sorghum beers were lower in total alcohol. Sensory analysis indicated that no significant differences existed between the sorghum beer and both the control beer and a commercial malted barley beer with regard to aroma, mouth-feel, after-taste and clarity. However, the sorghum beer was found to be significantly different to both of the other beers with regard to colour, initial taste and foam stability.

Thermolysin in the absence of substrate has an open conformation.

The bacterial neutral proteases have been proposed to undergo hinge-bending during their catalytic cycle. However, in thermolysin, the prototypical member of the family, no significant conformational change has been observed. The structure of thermolysin has now been determined in a new crystal form that for the first time shows the enzyme in the absence of a ligand bound in the active site. This is shown to be an 'open' form of the enzyme. The relative orientation of the two domains that define the active-site cleft differ by a 5 degrees rotation relative to their positions in the previously studied ligand-bound 'closed' form. Based on structural comparisons, kinetic studies on mutants and molecular-dynamics simulations, Gly78 and Gly135-Gly136 have previously been suggested as two possible hinge regions. Comparison of the 'open' and 'closed' structures suggests that neither of the proposed hinge regions completely accounts for the observed displacement. The concerted movement of a group of side chains suggested to be associated with the hinge-bending motion is, however, confirmed.

The Use of Lipolytic and Proteolytic Enzymes in the Manufacture of Manchego Type Cheese from Ovine and Bovine Milk

The objective was to study the effect of two fungal lipases (Palatase 200 from Mucor miehei and Palatase 750L from Aspergillus niger), alone and combined with a fungal protease from Aspergillus oryzae (MKC fungal protease), and a bacterial neutral protease from Bacillus subtilis (Neutrase) on the maturation of Manchego type cheese to determine the practical application of these enzymes in cheese manufacture. The addition of both lipases, alone or in combination with the proteases, increased lipolysis. The M. miehei lipase affected mainly the longchain fatty acids of cheese, and the A. niger lipase affected the short- and long-chain fatty acids equally. The B. subtilis protease preparation increased lipolysis significantly, either by contamination of the enzymatic solution or by a synergetic effect with the added A. niger lipase. The protease from A. oryzae had the opposite effect on lipolysis, significantly decreasing the release of fatty acids by either partial proteolytic digestion or inhibition of the lipase. The combination of proteases and lipases increased proteolysis as measured by indexes of soluble N and HPLC of free amino acids. The PAGE of the casein fractions showed the intense breakdown of β-caseins but not of αs-caseins. The exclusive addition of lipases decreased proteolysis. Sensory analysis showed a slight but significant acceleration of ripening (16%) for the cheeses treated with A. niger lipase at the beginning of aging. The M. miehei lipase induced the appearance of soapy flavor in the cheeses because of the excessive release of long-chain fatty acids. The cheeses containing the B. subtilis protease developed bitterness and sticky and crumbly texture because of the intense breakdown of β-casein.

Inhibition of tumor-cell attachment to extracellular matrix as a method for preventing tumor recurrence in a surgical wound.

Studies with four different transplantable murine tumors demonstrated that surgical instruments contaminated by contact with a tumor mass could produce tumors in a surgical wound. Eighty-seven per cent of mice with wounds made by invisibly contaminated scissors developed tumors. Irrigation with water did not prevent tumor growth. Before spilled tumor cells can invade and grow into a recurrence in the wound site, they must first attach to underlying extracellular matrix. We have devised a simple in vitro assay to identify inhibitors of tumor-cell attachment to develop therapeutic compounds that can prevent tumor-cell reimplantation. Various test compounds, including proteases (trypsin and Dispase), known modulators of matrix metabolism (proline analogues, cycloheximide, heparin, cortisone, cortexolone, and heparin-steroid combinations), large molecular weight polymers (agarose, dextran, polyethylene oxide), and synthetic fibronectin peptides were tested for their ability to inhibit mouse melanoma (B16-F10) cell attachment to gelatinized dishes. Most of these compounds had little or no effect on tumor-cell adhesion when cells were plated in serum-containing medium. However we identified three compounds that inhibited tumor-cell attachment in a reversible fashion: (1) a specific inhibitor of collagen deposition (L-azetidine-2-carboxylic acid); (2) a bacterial neutral protease (Dispase); and (3) synthetic fibronectin peptides that contained the arginine-glycine-asparate (RGD) sequence that is responsible for cell binding. Dispase and the RGD-containing peptides also inhibited cell implantation and prevented tumor formation in a surgical wound. We propose that inhibitors of attachment might be used either alone or with other biologic modifiers to prohibit implantation of free tumor cells at the time of surgery and thus, to prevent local tumor recurrence.

Experimental study of treatment of high-velocity projectile wounds with bacterial neutral protease.

Experimental study of treatment of high-velocity projectile wounds with bacterial neutral protease.

Adenylate cyclase-cyclic AMP system in pure epidermis isolated by use of dispase.

AbstractEpidermal adenylate cyclase systems following dispase treatment were investigated. Dispase is a bacterial neutral protease obtained from Bacillus polymyxa. Following the treatment with dispase, the epidermal sheet is easily peeled off the dermis. Dispase‐treated pure epidermal sheets were shown to contain three major (beta‐adrenergic‐, adenosine‐, and histamine‐) receptor adenylate cyclase systems. Without phosphodiesterase inhibitors, the intracellular cyclic AMP (cAMP) level reached the maximal level at 3 min. This effect was markedly enhanced by the addition of cAMP phosphodiesterase inhibitor. Among these epidermal adenylate cyclase systems, the most marked cAMP accumulation was observed by histamine, followed by adenosine, and then by epinephrine. The separation of epidermis and dermis following dispase treatment revealed that epidermis contained most of the beta‐adrenergic response (87%), whereas the dermis retained a significant proportion of adenosine (26%) and histamine (40%) responses when 0.3 mm thickness skin was studied.Specific antagonists of epinephrine, adenosine, and histamine inhibited the effects of these agents completely. The simultaneous addition of two stimulators into the incubation medium resulted in an additive effect.Beta‐augmentations by hydrocortisone, colchicine, and retinoid all remained in the dispase‐treated pure epidermal sheets, but beta‐augmentations by these drugs were spoiled by trypsin treatment.These results indicate that dispase‐treated pure epidermis contains three major (beta‐adrenergic‐, adenosine‐, and histamine‐) specific and independent receptor adenylate cyclase systems. Dispase is a very useful tool for investigating the metabolism and regulatory system of keratinocytes without any significant damage to epidermal membrane receptor systems.

The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs--an in vitro study using EMT6 multicellular spheroids.

Multicellular spheroids of the EMT6/Ca/VJAC mouse mammary tumour cell line have been used in an investigation of the effect of tumour geometry on the response of tumour cells to 3 cytotoxic drugs, adriamycin (ADM), nitrogen mustard (HN2) and CCNU. In addition to the inherent cellular drug response, factors related to spheroid structure, namely cell-cycle distribution, intercellular contact, drug penetration and microenvironment (pH, oxygen, glucose, etc.) are believed to influence the response of cells within spheroids to cytotoxic drugs. Selective enzymatic dissociation (with bacterial neutral protease) has been used to separate the cells within large (approximately 800 micron in diameter) spheroids into 4 distinct subpopulations. The cells within the subpopulations have been characterized by their DNA content, RNA content, tritiated thymidine labelling index, cell size and clonogenic capacity. It was found that cells at the surface of spheroids are relatively larger and more proliferative than cells towards the centre while their clonogenic capacity is similar. Studies on the responses of EMT6/Ca/VJAC log and plateau-phase monolayer cells have been carried out in parallel and have shown that cycling cells are more sensitive to ADM and HN2 than are non-cycling cells but somewhat less sensitive for the response to CCNU. Since the response patterns of cells from different regions of spheroids to HN2, treated either before disaggregation (intact spheroid) or after disaggregation (isolated spheroid cells), are similar and the surviving fraction increases from the surface towards the centre of the spheroid, cell cycle distribution is thought to be the only factor involved in the cytotoxicity of HN2 towards cells within the spheroids. Although the patterns of response to ADM of cells within intact spheroids and isolated spheroid cells are similar to those for HN2, the initial slope of the curve for intact spheroids is much steeper than that of the isolated spheroid cells. Therefore, in addition to the factor of cell-cycle distribution, drug penetration also appears to be involved in the action of ADM on spheroids, while the factors of intercellular contact and microenvironment appear to be relatively less important. The reverse pattern was found for the response of cells within different regions of spheroids to CCNU, treated as intact spheroids or as isolated spheroid cells (i.e., greater killing of inner compared with outer cells).(ABSTRACT TRUNCATED AT 400 WORDS)

Role of calcium and calmodulin in hemidesmosome formation in vitro.

Intact epithelial sheets were removed from rabbit corneas using Dispase II, a bacterial neutral protease. The freed sheets were placed on denuded corneal basal laminae and incubated at 35 degrees C for 3, 6, 18, or 24 h. Epithelial-basal lamina preparations were incubated in culture medium that either contained (a) varying concentrations of Ca2+ ions, (b) calmodulin antagonists, (c) exogenous calmodulin following an initial 6-h incubation in the presence of antagonists, or that lacked (d) Mg2+ ions. Tissues were processed for electron microscopy, and micrographs were taken of basal cell membranes. At least four experiments were conducted for each treatment, and for each experiment the total number of hemidesmosomes were counted along the basal membrane-basal lamina surface of eight cells. The number of hemidesmosomes formed was directly proportional to the increasing concentration of Ca2+. The presence of absence of Mg2+ ions did not change the numbers of hemidesmosomes formed. Calmodulin antagonists inhibited hemidesmosome formation, and this inhibition was reversed by the addition of calmodulin. Thus, hemidesmosome formation is Ca2+ dependent and appears to be mediated by a calmodulin-regulated mechanism.

Separation of the epidermal sheet by dispase.

Dispase is a bacterial neutral protease which is obtained from the culture filtrate of Bacillus polymyxa. After 24-h treatment of the human skin with 500 and 1000 U/ml dispase, the epidermal sheet was easily peeled from the dermis, and its undersurface retained rete ridges. Electron microscopic observation showed that the basal surface was composed of cells with numerous slender villi and cytoplasmic projections. Although the intercellular spaces of the spinous as well as the basal layers were wide, all desmosomes were intact with their accompanying tonofilaments. An epidermal cell suspension was obtained by incubating the dispase-separated epidermal sheet in trypsin solution for 15 min and the viability of the cells was more than 95% as measured by the trypan blue dye exclusion test. A culture of keratinocytes was established by seeding the dissociated cells in the usual way.

Cultured rabbit gastric epithelial cells producing prostaglandin I2

A new method with Dispase, a bacterial neutral protease, was developed for purification of gastric mucosal cells from rabbit fetuses. These cells formed a typical polygonal monolayer after removal of fibroblastlike cells by Dispase treatment, and preserved the features of “normal” cells, exhibiting in vitro aging with a finite life span. The culture consisted mainly of surface mucous cells (50%–60% of the total cell population) and produced prostaglandins (predominantly I2). The addition of acetyl-salicylic acid to the culture medium caused marked inhibition of prostaglandin production by the cultured gastric epithelial cells. This epithelial cell strain will be useful in studies on cytoprotection of the stomach.

Solid KHT tumor dispersal for flow cytometric cell kinetic analysis.

We used a bacterial neutral protease to disperse KHT solid tumors into single cell suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral protease disaggregation under conditions which would be suitable for routine tumor dispersal was compared with a trypsin/DNase procedure. Cell yield, clonogenic cell survival, DNA distributions of untreated and drug-perturbed tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase-specific cell loss were investigated. Tumors dispersed with neutral protease yielded approximately four times more cells than those dispersed with trypsin/DNase and approximately a 1.5-fold higher plating efficiency in a semisolid agar system. Quantitative analysis of DNA distributions obtained from untreated and cytosine-arabinoside-perturbed tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated thymidine during the cell cycle were also similar with neutral protease and trypsin/DNase dispersal. Preferential phase-specific cell loss was not observed with either technique. We find that neutral protease provides good single cell suspensions of the KHT tumor for cell survival measurements and for cell kinetic analysis of drug-induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are often required.

Growth and surface properties of dispase dissociated human fibroblasts

The dissociation of human fibroblast cultures with the bacterial neutral protease (Dispase II) was monitored by viability and growth measurements and was compared to the effect of trypsin and EDTA. Cell suspensions with high viability and excellent growth were obtained after 10 min incubation at 37 °C in 4 U/ml dispase in 0.02% EDTA. A two to three-fold increase in mitotic index occurred in the cultures within 48 h after dispase dissociation. The initial rate of aggregation was comparable to that of trypsin or EDTA dissociated cells, but attachment to a substratum and agglutination by Wheat Germ Agglutinin were markedly enhanced. The results indicate that dispase-EDTA provides a valuable alternative to the enzymatic dissociation with trypsin. Moreover, it is an additional tool for the dissociation of cultured cells and for the study of the surface properties of single cells.

Aeromonas neutral protease: Specificity toward extended substrates

Kinetic measurements on the action of Aeromonas neutral protease toward blocked peptide substrates were made in order to determine the most favorable fit on the enzyme subsites that bind the residues flanking the scissile bond and to define the number of secondary sites involved in catalysis. Variations in the identity of P 1′, 3 3 The nomenclature of Schechter and Berger (1) is used for designating the amino acid residues (and such blocking groups as benzyloxycarbonyl, benzoyl, or acetyl) of substrates in relation to the scissile bond; i.e., residues toward the NH 2 terminus from the cleavage site are designated P 1, P 2, etc., and those toward the COOH terminus are designated P1 1′, P 2′, etc. S 1, S 2, etc., represent the corresponding subsites on the enzyme. the residue furnishing the amino group to the scissile bond, produced significant changes in k rmcat , whereas the identity of P 1′, the residue donating the carboxyl group, was of much less catalytic importance. Comparison of these results with those of previous investigators of other bacterial neutral proteases indicated distinct differences in specificity of the Aeromonas enzyme and revealed that phenylalanyl residues, rather than leucyl, were preferred in the P 1′ position. Additional binding sites on the carboxyl side of the scissile bond were shown to be important to catalytic efficiency and it is evident that at least three residues (P 1t , ́ P 2′, P 3′) are involved while only two residues (P 2, P 1) on the amino terminal side of the sensitive bond are implicated.

Use of a bacterial neutral protease for disaggregation of mouse tumours and multicellular tumour spheroids

A bacterial neutral protease (from Bacillus Polymixa) has been used to disaggregate both solid mouse tumours and multicellular tumour spheroids. The cell yields obtained were much higher than those obtained using trypsin whilst the plating efficiency of the resultant single cells were comparable. The use of this neutral protease is simple, inexpensive and highly efficient.