Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells

Certain monoclonal antibodies (mAbs) to human placental alkaline phosphatase (PLAP) block bromelain cleavage of a 2-kDa segment from each of the two polypeptide chains of PLAP. These mAbs also prevent the release of PLAP from cultured cancer cell surfaces by bromelain. Such proteolysis-blocking mAbs serve as tools to specifically modify the molecular topography of cell surfaces by protease treatment.     

Preparation and stability of N-terminal mono-PEGylated recombinant human endostatin

Endostatin can specifically inhibit endothelial proliferation and potently inhibit angiogenesis and tumor growth. N-Terminal site-specific mono-PEGylation of recombinant human endostatin (mPEG-rhES) was accomplished by using methoxy poly-ethylene glycol (mPEG) propionaldehyde with an average molecular weight of 5000 Da through a reactive terminal aldehyde group. The site-specific mPEG conjugation was conducted under optimal conditions, which were identified through a statistical L(9)(3(4)) orthogonal test. In this study, we have investigated the stability and antitumor activity of mPEG-rhES. SDS-PAGE, RP-HPLC, and UV spectrophotometric analysis were used to identify the purity and stability of mPEG-rhES. When incubated with protease or placed in an extreme environment, mPEG-rhES was more stable than rhES. The unmodified and PEGylated rhES were tested for their ability to inhibit the tumor growth of mouse H22 liver cancer in male mice. In a multiple versus single doses comparison study, daily administration of 0.25, 0.50, and 1.00 micromol/kg of unmodified rhES for 7 days resulted in 26.9%, 43.0%, and 64.9% reductions in tumor weight, respectively, while single doses of 0.13, 0.25, and 0.50 micromol/kg of the PEGylated protein per day resulted in 24.8%, 38.0%, and 64.5% reductions, respectively. Both treatments resulted in statistically significant reductions in mean tumor weight as compared to the physiological saline solution (control)-treated mice, with the dose of mPEG-rhES being a half of rhES, respectively, while the tumor inhibition rates were similar. Therefore, it is suggested that PEGylation enhances the stability of rhES and improves its antitumor activity.     

Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form

We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.     

Surface modification of cellulose with triazine derivative to improve printability with reactive dyes

Chemical modification of cellulose with triazine derivative, 2,4,6-tri-[(2-hydroxy-3-trimethyl-ammonium)propyl]-1,3,5-triazine chloride (Tri-HTAC), was investigated. Micro-FT-IR and nitrogen element analysis were applied to characterize molecular structure of the modified cellulose. The printing properties of the modified cellulose fabric with Tri-HTAC were discussed. Tri-HTAC was able to form covalent bonds with cellulose fibers. The apparent color strength of printed samples with three reactive dyes on the modified cellulose was higher than the corresponding color yields on the unmodified cellulose fabric. Compared with unmodified cellulose, the increases of the color yield were about 6–13%. The fixation rate was accelerated by the modification with Tri-HTAC. The wet rubbing and washing fastnesses of the printed cellulose fabrics modified with Tri-HTAC were better than those of the printed unmodified cellulose fabric. The modified cellulose with Tri-HTAC imparted good printing properties.     

Bromelain Decreases Neutrophil Interactions with P-Selectin,but Not E-Selectin,In Vitro by Proteolytic Cleavage of P-Selectin Glycoprotein Ligand-1

Stem bromelain, a cysteine protease isolated from pineapples, is a natural anti-inflammatory treatment, yet its mechanism of action remains unclear. Curious as to whether bromelain might affect selectin-mediated leukocyte rolling, we studied the ability of bromelain-treated human neutrophils to tether to substrates presenting immobilized P-selectin or E-selectin under shear stress. Bromelain treatment attenuated P-selectin-mediated tethering but had no effect on neutrophil recruitment on E-selectin substrates. Flow cytometric analysis of human neutrophils, using two antibodies against distinct epitopes within the P-selectin glycoprotein ligand-1 (PSGL-1) active site, revealed that bromelain cleaves PSGL-1 to remove one of two sites required for P-selectin binding, while leaving the region required for E-selectin binding intact. These findings suggest one molecular mechanism by which bromelain may exert its anti-inflammatory effects is via selective cleavage of PSGL-1 to reduce P-selectin-mediated neutrophil recruitment.     

Purification and characterization of a pineapple crown leaf thiol protease

A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.     

Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris

Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi‐component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54–65, 2017     

Low versus high molecular weight poly(ethylene glycol)-induced states of stem bromelain at low pH: stabilization of molten globule and unfolded states

The effect of low, medium, and high molecular weight poly(ethylene glycol) (e.g., PEG-400, -6000, and -20,000) on the structure of the acid unfolded state of unmodified stem bromelain (SB) obtained at pH 2.0 has been studied by various spectroscopic methods. The conformation of stem bromelain at pH 2.0 exhibits substantial loss of secondary structure and almost complete loss of native tertiary contacts and has been termed the acid unfolded state (A(U)). Addition of PEG-400 to A(U) led to an increase in the mean residue ellipticity (MRE) value at 222 nm, indicating formation of alpha-helical structure. On the other hand, PEG-6000 and 20,000 led to a decrease in the MRE value at 222 nm, indicating unfolding of the A(U) state. Interestingly, at 70% (w/v) PEG-400 and 40% (w/v) PEG-20,000, MRE values at 222 nm almost approach the native state at pH 7.0 and the unfolded state (6 M GnHCl) of stem bromelain, respectively. The probes for tertiary structure showed formation of nonnative tertiary contacts in the presence of 70% (w/v) PEG-400, while 40% (w/v) PEG-6000 and 20,000 were found to stabilize the unfolded state of SB. An increase in binding of 1-anilino 8-naphthalene sulfonic acid and a decrease in fractional accessibility of tryptophan residues (f(a)) compared to A(U) in the presence of 70% PEG-400 indicate that the PEG-400-induced state has a significant amount of exposed hydrophobic patches and is more compact than A(U). The results imply that the PEG-400-induced state has characteristics of molten globule, and higher molecular weight PEGs led to the unfolding of the A(U) state.     

Intestinal brush border hydrolase topography. Effects of vitamin D-3 and filipin

Intestinal brush borders were isolated from vitamin D-3-treated and vitamin D-deficient chicks, and protein topography in the paired preparations assessed by the enzymatic release of four marker hydrolases. Exposure of the brush borders to the protease bromelain resulted in soluble levels of alkaline phosphatase, leucine aminopeptidase, maltase, and sucrase activities from preparations of vitamin D-3-treated birds that were 42%, 75%, 64%, and 56%, respectively, of corresponding activities released in preparations from rachitic chicks. Analyses for recovery of enzyme activity revealed that bromelain treatment selectively inactivated 43% of the alkaline phosphatase activity of brush borders obtained from vitamin D-3-replete birds, and preferentially diminished recovered sucrase activity in preparations from vitamin D-deficient chicks. In additional experiments, brush borders isolated from rachitic birds were treated in vitro with the polyene antibiotic filipin or an equivalent volume of vehicle. Subsequent exposure of such preparations to bromelain resulted in little or no differences in levels of marker hydrolase specific activities released from filipin- or vehicle-treated brush borders. However, analyses of membrane-bound specific activities after treatment of brush border preparations with a range of filipin concentrations, revealed a biphasic inhibition of approx. 30% for both maltase and sucrase, relative to vehicle controls, and a smaller effect on alkaline phosphatase and leucine aminopeptidase.     

Induction of 'molten globule' like state in acid-denatured state of unmodified preparation of stem bromelain: implications of disulfides in protein folding

A denatured state of unmodified preparation of stem bromelain representing a structureless form has been characterized at pH 2.0 and the effect of increasing concentration of TFE on the acid-denatured state has been investigated by circular dichroism (CD), fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show considerable accumulation of secondary structure when the acid-denatured bromelain is subjected to 70% (v/v) TFE and exhibited close resemblance to spectral features of those of pH 7.0 preparation. Interestingly, the acid-denatured state also regained some tertiary structure/interactions, with increasing concentration of TFE and at 60% (v/v) TFE, these approached almost those of the native like state. However, further increase to 70% (v/v) TFE resulted in complete loss of tertiary structure/interactions. Tryptophan fluorescence emission studies also suggested the induction of significant compact structure at 60% (v/v) concentration of TFE. In addition the acid-denatured state showed enhanced binding of ANS in presence of 60% (v/v) TFE. Taken together these observations suggest the existence of a molten globule state in acid-denatured bromelain between 60 and 70% (v/v) TFE. A similar molten globule state under identical conditions has been identified in reduced and carboxymethylated preparation of stem bromelain as reported in our earlier communication [Arch. Biochem. Biophys. 413 (2003) 199]. Comparison suggests unfolding/folding behavior of the bromelain to be independent of the intactness of the disulfide bonds.     

Degradation of proteins with blocked amino groups by cytoplasmic proteases

The effect of blocking amino groups on the susceptibility of BSA and calmodulin to high molecular weight protease (HMP) and calpain, the two major cytosolic proteases, was studied. Both proteases hydrolyzed methylated vs. unmodified BSA more slowly. Methylation of BSA resulted in the accumulation of proteolytic intermediates, especially of larger sizes. However, similar fragments were generated from unmodified BSA indicating that rates of hydrolysis rather that sites of proteolytic cleavage were altered. Calmodulin from Dictyostelium discoideum was hydrolyzed rapidly by HMP whereas brain and muscle calmodulins which have a epsilon-N-trimethyl residue on the single surface lysine were relatively stable.     

Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu²⁺ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (K_m) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (V_max) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.     

Inhibitory effects of fruit juices on cytochrome P450 2C9 activity in vitro

There is limited information on the effect of fruits on human cytochrome P450 (CYP) 2C9 activity. The objective of this study was to determine the effect of fruit juice on CYP2C9-mediated drug metabolism. Nine citrus fruits and eight tropical fruits were chosen. We investigated effects of the fruits on diclofenac 4'-hydroxylation and tolbutamide hydroxylation by human liver microsomes. Among the fruits, pineapple juice showed potent inhibition of CYP2C9 activity. The addition of 25 microl (5.0% v/v) of pineapple juice resulted in almost complete inhibition. Next we examined the inhibitory effect of bromelain, a cysteine protease in pineapple. Bromelain also strongly inhibited CYP2C9 activity. In addition, E-64, a cysteine protease inhibitor, almost entirely blocked inhibition by pineapple juice and bromelain. Thus we found that pineapple juice was a potent inhibitor of CYP2C9, and that the inhibitory effect might be due to the bromelain contained in pineapple.     

Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens

Aim: This study aimed to evaluate the effect of bromelain, a cysteine protease isolated from pineapple (Ananas comosus), on growth of several agronomically important fungal pathogens. Methods and Results: Purification of bromelain from pineapple stems was carried out by chromatography techniques, and its antimicrobial activity was tested against the fungal pathogens Fusarium verticillioides, Fusarium oxysporum and Fusarium proliferatum by broth microdilution assay. A concentration of 0·3 μmol l^?1 of bromelain was sufficient for 90% growth inhibition of F. verticillioides. The capability of bromelain to inhibit fungal growth is related to its proteolytic activity. Conclusions: The study demonstrates that stem bromelain exhibits a potent antifungal activity against phytopathogens and suggests its potential use as an effective agent for crop protection. Significance and Impact of the Study: The results support the use of a natural protease that accumulates at high levels in pineapple stems as alternative to the use of chemical fungicides for crop protection.     

Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle

An endogenous inhibitor of calcium-activated neutral protease was purified to homogeneity from rabbit skeletal muscle using ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex A-50 columns, chromatofocusing, and hydrophobic interaction chromatography on a phenyl-Sepharose CL-4B column. The purified inhibitor was shown to be a dimer of identical subunits and each subunit has a molecular weight of about 34,000. This inhibitor was remarkably thermo- and acid-stable. It was specific for calcium-activated neutral protease and had no effect on any other protease examined (trypsin, papain, alpha-chymotrypsin, bromelain, etc.). It is demonstrated that the inhibition is due to the formation of stoichiometric complex between two enzyme molecules and one inhibitor molecule.     

Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain

A new engineering strain, Bacillus pumilus c172-14 (pBX 96), was obtained by introducing the pBX 96 plasmid, which carries the alpha-amylase amy gene, into the host strain of alkalophilic Bacillus pumilus c172 via transformation. The newly constructed strain was found to express the amy gene and could use starch instead of glucose or starch hydrolysate as carbon source for its fermentation of alkaline protease. The pBX 96 plasmid in the new host was found to be segregationally and structurally stable. The expression of amy gene did not affect the host strain's resistance to bacteriophages. Moreover, the level of alkaline protease was improved significantly compared with the parent strain. The constructed strain gave a maximum alkaline protease activity of 14,014 U/ml in shaking flask after 48 h cultivation when growing in a medium containing 6% corn meal, 4% soybean flour, 0.4% Na2HPO4, 0.03% KH2PO4, 0.02% MgCl2, 0.3% CaCl2, 0.25% Na2CO3, 0.1% glucose, and 20 microg/ml kanamycin (pH 7.0). The optimal pH value and temperature of the alkaline protease were 11.0 and 40 degrees C, respectively. This enzyme was stable over a pH range of 8-11. Its residual activity remained at 100% when treated under a temperature of less than 45 degrees C for 30 min. The corresponding residual activity reduced to 65% of its optimal value at 60 degrees C for 30 min. The alkaline protease was a kind of serine protease, which was demonstrated by the complete inactivation by PMSF (1 mM). This newly constructed strain will be useful in the alkaline protease industry.     

Stability,purification, and applications of bromelain: A review

Bromelain is a cysteine protease found in pineapple tissue. Because of its anti‐inflammatory and anti‐cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid–liquid extractions by aqueous two‐phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up‐to‐date compilation of the data reported on this enzyme. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:5–13, 2016     

Purification and Characterization of Soymilk-clotting Enzymes from Bacillus sp. K-295G-7

Enzymes I and II, which have a high soymilk-clotting activity, produced from K-295G-7 were purified by chromatographies on Sephadex G-100, CM-cellulose, hydroxylapatite, and 2nd Sephadex G-100.

The two purified enzymes were found to be homogeneous by polyacrylamide gel elec-trophoresis (PAGE) at pH 4.3. The molecular weights of enzymes I and II were 28,000 and 29,500 by SDS-PAGE, and their isoelectric points were 9.22 and 9.45, respectively. Enzymes I and II coagulated soymilk optimally at 65°C and were stable up to 45°C. Both enzymes were most active at pH 5.8, for soymilk coagulation between pH 5.8 to 6.7, and were stable with about 50 ~ 100% of the original activity from pH 5 to 10.

Each of the purified enzymes was a serine protease with an optimum pH of 9.0 for soy protein isolate (SPI) and casein digestions, because these enzymes were inhibited completely by diisopropylfluoro-phosphate (DFP).

The soymilk-clotting activity to proteolytic activity ratio of the enzyme II was 3 times higher than that of enzyme I. Enzymes I and II were more sensitive to the calcium ion concentration in soymilk than bromelain is.     

Functional Characterization of Recombinant Bromelain of Ananas comosus Expressed in a Prokaryotic System

Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.     

Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.