Low Proteolytic Activity Research Articles

Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products

A total of 38 lactic acid bacteria, belonging to Lactobacillus, isolated from 24 samples of traditional Egyptian dairy products, were screened for antimicrobial activity against different Gram-positive and Gram-negative bacteria. A strain of Lactobacillus brevis showed the best inhibitory activity when tested by well diffusion assay. The antibacterial activity was pronounced between early logarithmic and early stationary phases. The strain produced a heat-stable antimicrobial compound showing no reduction in activity after heat treatment from 60 to 100°C for 15 and 30 min. Since it was inactivated by proteolytic enzymes, it is considered to be proteinaceous in nature and, therefore, referred to as a bacteriocin-like substance. This compound was also active over a wide pH range (pH 2–6). The antimicrobial compound was partially purified by 40% ammonium sulfate precipitation. Lactobacillus brevis was tested for its in vitro antibiotics susceptibility, tolerance to bile salts, resistance to low pH values, acidifying activity, proteolytic activity, and haemolytic activity. The results showed the potential of L. brevis strain as a probiotic culture, and hence it can be utilized in the manufacturing of pharmaceuticals and dietary supplements.

An efficient transformation method for Bacillus subtilis DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10(3) transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is "easy" in handling and allows for the first time to generate large libraries (1.5 × 10(5) transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.

Production and characterization of milk-clotting enzyme from Bacillus amyloliquefaciens JNU002 by submerged fermentation

Milk-clotting enzymes produced by microorganisms have been developed to replace calf rennet, yet the enzymatic level and the ratio of milk-clotting activity to proteolytic activity still need further improvement. This work described a strain Bacillus amyloliquefaciens JNU002 that screened from wheat bran that has a promising characterization. After optimization, B. amyloliquefaciens JNU002 showed a high milk-clotting activity (4969 SU/mL) and low proteolytic activity (4.02 U/mL) at 48 h with an inoculum size of 0.2% (v/v) at initial pH 6.0 in 15-L bioreactor. After purification, the purified enzyme gave a single protein band on SDS–PAGE, corresponding to 28 kDa. The purified enzyme showed a high ratio (2,575) of milk-clotting activity to proteolytic activity at 35 °C, and the ratio would be even higher (22,992) at 70 °C. The milk-clotting reaction and proteolytic reaction were prevented at 75 °C. This enzyme was stable at pH 4–6 and below 40 °C, and this was convenient for storage and transportation.

A Fermented Infant Milk Formula Reduces Ileal Proteolytic Activity

Background and aims: Gastrointestinal (GI) proteases are best known for their function in protein digestion. However, GI luminal serine protease activity is also associated with inflammation, pain and visceral hypersensitivity via activation of protease-activated receptor-2 (PAR-2). An infant milk formula comprising product fermented by Streptococcus thermophilus 065 and Bifidobacterium breve C50 has previously been reported to improve gut comfort in infants with digestive problems. Here, the effect of this fermented formula (Lactofidus) on proteolytic activity was investigated at the terminal ileum. Methods: The fermented formula and a non-fermented standard formula were tested in three-week-old piglets (N=6) fitted with a T-cannula. Each formula was provided in a Latin square design. Ileal digesta were collected during eight hours and subsequently analyzed for total proteolytic activity and protein digestibility. Proteolytic activity was further characterized either by specific substrates or inhibitors as serine protease, trypsin, or chymotrypsin activity. Results: Fermented formula yielded a more than threefold lower total proteolytic activity at the terminal ileum compared to standard formula (477 ± 84 ·103 and 1599 ± 343 ·103 units/8h, respectively). This proteolytic activity was found to be mainly constituted by the pancreatic serine proteases trypsin and chymotrypsin. Despite lower proteolytic activity in the fermented formula, protein was fully digested. Conclusions: The fermented formula causes lower exposure of the small intestine to pancreatic serine proteases. This could contribute to improved gut comfort via diminished activation of PAR-2, which could be particularly beneficial in infants with digestive problems.

Characterization of the Structure and Function of Escherichia coli DegQ as a Representative of the DegQ-like Proteases of Bacterial HtrA Family Proteins

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists invivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.

English

Three strains, Bacillus subtilis B1, B. subtilis B18 and Bacillus thuringiensis B12,were screened from wheat bran to produce milk-clotting enzyme. Among them, B. subtilis B1 exhibited considerable milk-clotting activity with low proteolytic activity. After response surface methodology optimization, milk-clotting activity wasimproved from 782 SU ml-1 to 1129.05 ± 74.55 SU mL-1 with a small inoculum size (0.130%, v/v). The optimized medium contained glucose (16.2 gL-1), wheat bran (30 gL-1), NaCl (5 gL-1), MgSO4·7H2O (5 gL-1), KH2PO4 (2 gL-1) and CaCO3 (3 gL-1). The milk-clotting enzyme from B. subtilis B1 has the optimum pH 5.5 and CaCl2concentration 50 mmol L-1. It is completely inactivated after 5 min at 70°C. The result showed that B. subtilis B1 was a promising strain for industrial milk-clotting enzyme production.   Key words: Milk-clotting enzyme, Bacillus subtilis, submerged fermentation, wheat bran.

Phenotypic and molecular characterization of rhizobacterium Burkholderia sp. strain R456 antagonistic to Rhizoctonia solani, sheath blight of rice

The effect of rhizobacterium Burkholderia sp. strain R456 on the inhibition of Rhizoctonia solani, sheath blight of rice was examined. Results from this study indicated that strain R456 not only suppressed the in vitro mycelial growth of R. solani, but also reduced the incidence and severity of rice sheath blight under greenhouse conditions. However, similar to plant pathogenic strain LMG 1222T of Burkholderia cepacia, the type species of the genus, infiltration of tobacco leaves with cell suspension of strain R456 resulted in typical hypersensitivity reactions while the two bacterial strains were unable to cause disease symptoms on rice seedlings. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that the antagonistic rhizobacterium R456 is a member of the genus Burkholderia. Furthermore, strain R456 was differentiated from B. cepacia LMG 1222T and was identified as Burkholderia seminalis based on recA gene sequence analysis and multilocus sequence typing. In addition, this rhizobacterium had a lower proteolytic activity compared with that of the pathogenic B. cepacia LMG 1222T while no cblA and esmR marker genes were detected for the two bacterial strains. Overall, this is the first characterization of rhizobacterium B. seminalis that protected rice seedlings from infection by R. solani.

Factor VII activating protease.

Factor VII activating protease (FSAP) is a circulating serine protease with high homology to fibrinolytic enzymes. A role in the regulation of coagulation and fibrinolysis is suspected based on in vitro studies demonstrating activation of FVII or pro-urokinase plasminogen activator (uPA). However, considering the paucity of any studies in animal models or any correlative studies in humans the role of FSAP in haemostasis remains unclear. In relation to vascular remodeling processes or inflammation it has been convincingly shown that FSAP interacts with growth factors as well as protease activated receptors (PAR). Against this sparse background there are a plethora of studies which have investigated the linkage of single nucleotide polymorphisms (SNP) in the FSAP gene (HABP2) to various diseases. The G534E SNP of FSAP is associated with a low proteolytic activity due to an amino acid exchange in the protease domain. This and other SNPs have been linked to carotid stenosis, stroke as well as thrombosis in the elderly and plaque calcification. These SNP analyses indicate an important role for FSAP in the regulation of the haemostasis system as well as fibroproliferative inflammatory processes.

Comparative analysis of newborn and adult Bothrops jararaca snake venoms

Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercial antivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 5′-nucleotidase activities. However, newborn snake venom showed higher l-amino oxidase, hyaluronidase, platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities of B. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciously neutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.

Apparent Tradeoff of Higher Activity in MMP-12 for Enhanced Stability and Flexibility in MMP-3

The greater activity of MMP-12 than MMP-3 toward substrates from protein fibrils has been quantified. Why is MMP-12 the more active protease? We looked for behaviors associated with the higher activity of MMP-12 than MMP-3, using nuclear magnetic resonance to monitor backbone dynamics and residue-specific stabilities of their catalytic domain. The proteolytic activities are likely to play important roles in inflammatory diseases of arteries, lungs, joints, and intestines. Nuclear magnetic resonance line broadening indicates that regions surrounding the active sites of both proteases sample conformational substates within milliseconds. The more extensive line broadening in MMP-3 suggests greater sampling of conformational substates, affecting the full length of helix B and β-strand IV forming the active site, and more remote sites. This could suggest more excursions to functionally incompetent substates. MMP-3 also has enhanced subnanosecond fluctuations in helix A, in the β-hairpin of strands IV and V, and before and including helix C. Hydrogen exchange protection in the EX2 regime suggests that MMP-3 possesses 2.8 kcal/mol higher folding stability than MMP-12(E219A). The β-sheet of MMP-3 appears to be stabilized still more. The higher stability of MMP-3 relative to MMP-12 coincides with the former's considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.

Identification of Pep4p as the Protease Responsible for Formation of the SAGA-related SLIK Protein Complex

The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex is a coactivator for transcription by RNA polymerase II and has various activities, including acetylation and deubuiqitination of histones and recruitment of TATA-binding protein to promoters. The Spt7p subunit is subject to proteolytic cleavage at its C terminus resulting in removal of the Spt8p-binding domain and generation of the SAGA-related SALSA/SAGA-like (SLIK) protein complex. Here, we report identification of the protease responsible for this cleavage. Screening of a protease knock-out collection revealed PEP4 to be required for cleavage of Spt7p within SAGA in vitro. Endogenous formation of truncated Spt7p was abolished in cells lacking PEP4. Purified Pep4p but not catalytic dead mutant Pep4p or unrelated Prc1p protease specifically cleaved Spt7p within SAGA into SLIK-related Spt7p. Interestingly, SAGA lacking Spt8p was more sensitive to Pep4p-mediated truncation of Spt7p, suggesting that Spt8p counteracted its own release from SAGA. Strains mimicking constitutive SLIK formation showed increased resistance to rapamycin treatment, suggesting a role for SLIK in regulating cellular responses to nutrient stress.

Milk-clotting activity of berries extracts from nine Solanum plants

Nine Solanum species (Solanum aculeastrum, Solanum aethiopicum, Solanum anomalum, Solanum cerasiferum, Solanum dasyphyllum, Solanum indicum, Solanum nigrum, Solanum nodiflorum and Solanum terminale) berries were studied in order to find a new source of milk-clotting enzymes for artisanal use by Cameroonian farmers. The results showed that milk-clotting activity was obtained by soaking fresh and dried berries in either distilled water or 5% NaCl solution. Maximum milk-clotting activity was obtained by soaking dried berries in 5% NaCl solution at optimum time of extraction for each species. Dried berries released active enzymes more easily than fresh ones, and addition of salt facilitated the extraction process. Extract from S. aethiopicum berries had the highest milk-clotting activity. All extracts had proteolytic activity. High milk-clotting and proteolytic activities were found in extract from S. cerasiferum obtained after 24 h of soaking dried berries in 5% NaCl solution. High milkclotting and low proteolytic activities were found in extract from S. aethiopicum obtained under same conditions

Proteolytic activity among psychrotrophic bacteria isolated from refrigerated raw milk

Psychrotrophic bacteria were isolated from refrigerated raw milk from a processing plant in Southern Brazil. Psychrotrophic counts were between 4.9 and 7.8 log cfu/mL, and 5.3 to 7.2 log cfu/mL, for samples collected at the truck and the milk storage silo, respectively. Among the bacterial isolates, 90% were Gram‐negative. Most strains presented low proteolytic activity, but strains of Burkholderia cepacia, Klebsiella oxytoca and Aeromonas sp. showed higher than 20 U/mL on azocasein as substrate. Crude proteases from selected strains were resistant to conventional heat treatments and caused coagulation of UHT milk after 5 days storage at room temperature.

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.

History of the Study of Thrombolytic and Fibrinolytic Enzymes of Higher Basidiomycetes Mushrooms at the V.L. Komarov Botanical Institute in Saint Petersburg, Russia

The thrombolytic, fibrinolytic, milk-clotting, and general proteolytic activities of fruiting bodies of 400 species of Basidiomycetes belonging to various taxonomical and ecological groups were studied. A sufficient distinction between saprotrophic and symbiotrophic groups of Basidiomycetes in proteinase biosynthesis was established. It was found that the most pronounced proteolytic activity in combination with wide-range substrate specificity was characteristic for a group of saprotrophic mushrooms, whereas the symbiotrophic fungi were characterized by a low level or even complete absence of the aforementioned types of activity. The most pronounced fibrinolytic and thrombolytic activities were noted for lignotrophic saprotrophs and litter saprotrophs associated with lignin-cellulose complexes (rotten wood, compost, substrata, etc.). The list of species of Basidiomycetes that synthesize enzymes possessing high thrombolytic activity and low general proteolytic activity is presented.

Identification and characterization of hydrolytic enzymes from the midgut of Sunn Pest of wheat (Eurygaster integriceps)

To help in the development of Sunn Pest-resistant transgenic plants employing protease or α-amylase inhibitors, midgut hydrolytic enzymes of Sunn Pest (Eurygaster integriceps, Put.) (Heteroptera: Scutelleridae) were identified and characterized biochemically. We observed levels of very low proteolytic activity of trypsin (3 nmoles/min/mg), elastase (0.66 nmoles/min/mg) and leucine aminopeptidase-like (14.4 nmoles/min/mg) proteases, but no chymotrypsin and papain-like activity. Proteolytic activities were insensitive to inhibition by soybean trypsin inhibitor (SBTI) and aprotinin, but were inhibited to varying degrees (40–100%) by the synthetic protease inhibitors PMSF, TPCK, CdCl2 and CuCl2. Compared to proteolytic activity, significantly higher amylolytic activity (4.45 mmoles/min/mg) was observed. Αlpha-amylase activity was found to be resistant to inhibition by bean and chickpea α-amylase inhibitors, and only slightly inhibited by wheat (36.9 ± 1.8%) and maize (40.2 ± 2.8) α-amylase inhibitors. Here, we report on the biochemical properties of digestive enzymes from Sunn Pest midgut, and their inhibition patterns by several synthetic and natural hydrolytic enzyme inhibitors.

Proteolytic activity and cysteine protease expression in wheat leaves under severe soil drought and recovery

The involvement of acidic proteases in soil drought response of winter wheat (Triticum aestivum L.) at seedling stage in three cultivars differing in water stress tolerance was studied. Withholding irrigation for seven days resulted in severe drought stress corresponding to 60% leaf water deficit. Stressed plants were recovered by providing optimal water supply for 3 days. Reversible changes in leaf pigment and protein content were registered, being least expressed in the drought-resistant cultivar Katya. Protein loss was inversely related to the increase in total proteolytic activity at pH 5 and in aminopeptidase activity at pH 7. Quantitative differences among the cultivars were established only for azocaseinolytic activity (pH 5). The drought-resistant cultivar (Katya) showed relatively little increase in acid protease activity whereas the highest values of this activity were detected in cultivar Pobeda. In-gel staining for cysteine-activated proteases revealed four to five separate activity bands. The upper band, specifically inhibited by E-64, was raised at severe drought. Transcript abundance of two wheat cysteine proteases -Ta.61026 putative thiol protease, and WCP2 peptidase of papain type was analyzed by RT-PCR. Gene expression of the cysteine proteases under study was suppressed in the drought-tolerant cultivar, while in the less resistant ones it remained unchanged or augmented. The results suggest that lower proteolytic activity and decreased expression of certain cysteine protease genes under water deficit during early developmental stage could be regarded as an indicator for drought resistance of winter wheat cultivars.

Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31 : (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

Microbial rennet-like milk-clotting enzymes are aspartic proteinases that catalyze milk coagulation, substituting calf rennet. Crude enzymatic extract produced by the thermophilic fungus, Thermomucor indicae-seudaticae N31, on solid state fermentation (SSF) using wheat bran, exhibited high milk-clotting activity and low proteolytic activity after 24 h of fermentation. Highest milk-clotting activity (MCA) was at pH 5.7, at 70 °C and in 0.04 M CaCl 2; it was stable in the pH range 3.5–4.5 for 24 h and up to 45 °C for 1 h. MCA was highly inhibited by pepstatin A. Hydrolytic activity profile of the crude enzymatic extract on whole bovine casein, analyzed by gel electrophoresis (Urea–PAGE) and RP-HPLC revealed low proteolytic action towards casein fractions and a peptide profile similar to the one obtained with commercial Rhizomucor miehei protease (Hannilase).

Differential HIV epitope processing in monocytes and CD4 T cells affects cytotoxic T lymphocyte recognition.

The ability of cytotoxic T lymphocytes (CTLs) to clear virus-infected cells is dependent on the presentation of viral peptides processed intracellularly and displayed by major histocompatibility complex class I. Most CTL functional assays use exogenously added peptides, a practice that does not account for the kinetics and quantity of antigenic peptides produced by infectable cells. Here, we examined the relative ability of 2 major human immunodeficiency virus-infectable cell subsets-CD4 T lymphocytes and monocytes-to produce antigenic peptides, using cytosol as a source of peptidases and mass spectrometry to define the degradation products. We show clear subset-specific differences in the kinetics of peptide production and the ability of the peptides produced to sensitize cells for lysis by CTLs, with primary CD4 T lymphocytes having significantly lower proteolytic activity than monocytes. These differences in epitope processing by cell subsets may affect the efficiency of CTL-mediated clearance of infected subsets and contribute to the establishment of chronic infection.