Non-proteolytic Enzymes Research Articles

Probiotic Characteristics and Antimicrobial Potential of a Native Bacillus subtilis Strain Fa17.2 Rescued from Wild Bromelia sp. Flowers.

In the present study, we identified the Bacillus subtilis strain annotated Fa17.2 isolated from Bromelia flower inflorescences collected from the subtropical humid mesothermal region, Santo Domingo de Los Tsachilas Province, Ecuador. The probiotic capacity and antimicrobial potential against four foodborne pathogens were assessed. The cell culture of Fa17.2 is highly resistant to synthetic gastric acid (pH 2.5, 3.0, and 3.5), bile salts (0.3%), tolerating different sodium chloride concentrations (1, 3, and 5%), and growth conditions (15 °C and 45 °C), suggesting its potential probiotic features. The isolate showed no antibiotic resistance and was considered safe as no hemolysis was detected on sheep blood agar. The optimum medium for bacterial growth and the release of antimicrobial compounds was MRS with 10% glucose. The active components released in the neutralized crude extract (NCE) were insensitive to organic solvents, surfactants, and nonproteolytic enzymes and sensitive to proteolytic enzymes suggesting their proteinaceous nature. The antimicrobial activity was enhanced by heat and maintained active over a wide range of pH (2.0–8.0). Moreover, the crude extract (CE) showed inhibitory activity against several Gram-negative and Gram-positive bacteria. The molecular weight of partially purified precipitated bacteriocin-like substances (BLISs) was about 14 kDa in 20% Tricine-SDS-PAGE. The CE obtained from Fa17.2 inhibits the growth of four foodborne pathogens, Staphylococcus aureus, Escherichia coli, Kosaconia cowanii, and Shigella dysenteriae, which implies its potential as an antimicrobial producer strain.

Novel in vitro inhibitory functions of potato tuber proteinaceous inhibitors.

Plant protease inhibitors are a structurally highly diverse and ubiquitous class of small proteins, which play various roles in plant development and defense against pests and pathogens. Particular isoforms inhibit in vitro proteases and other enzymes that are not their natural substrates, for example proteases that have roles in human diseases. Mature potato tubers are a rich source of several protease inhibitor families. Different cultivars have different inhibitor profiles. With the objective to explore the functional diversity of the natural diversity of potato protease inhibitors, we randomly selected and sequenced 9,600 cDNA clones originated from mature tubers of ten potato cultivars. Among these, 120 unique inhibitor cDNA clones were identified by homology searches. Eighty-eight inhibitors represented novel sequence variants of known plant protease inhibitor families. Most frequent were Kunitz-type inhibitors (KTI), potato protease inhibitors I and II (PIN), pectin methylesterase inhibitors, metallocarboxypeptidase inhibitors and defensins. Twenty-three inhibitors were functionally characterized after heterologous expression in the yeast Pichia pastoris. The purified recombinant proteins were tested for inhibitory activity on trypsin, eleven pharmacological relevant proteases and the non-proteolytic enzyme 5-lipoxygenase. Members of the KTI and PIN families inhibited pig pancreas elastase, β-Secretase, Cathepsin K, HIV-1 protease and potato 5-lipoxygenase. Our results demonstrate in vitro inhibitory diversity of small potato tuber proteins commonly known as protease inhibitors, which might have biotechnological or medical applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-014-0906-5) contains supplementary material, which is available to authorized users.

Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer

BackgroundSince volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis.ResultsTo develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel’s industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an α-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer.ConclusionsIn this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the basis for a next generation production host, since the strain has still a large potential for further genetic engineering. The final amylase titer of 65% in reference to B. licheniformis protease titer suggests that the developed B. pumilus expression platform is also suitable for an efficient production of non-proteolytic enzymes reaching a final titer of several grams per liter without complex process modifications.

A broad-spectrum antimicrobial activity of Bacillus subtilis RLID 12.1.

In the present study, an attempt was made to biochemically characterize the antimicrobial substance from the soil isolate designated as RLID 12.1 and explore its potential applications in biocontrol of drug-resistant pathogens. The antimicrobial potential of the wild-type isolate belonging to the genus Bacillus was determined by the cut-well agar assay. The production of antimicrobial compound was recorded maximum at late exponential growth phase. The ultrafiltered concentrate was insensitive to organic solvents, metal salts, surfactants, and proteolytic and nonproteolytic enzymes. The concentrate was highly heat stable and active over a wide range of pH values. Partial purification, zymogram analysis, and TLC were performed to determine the preliminary biochemical nature. The molecular weight of the antimicrobial peptide was determined to be less than 2.5 kDa in 15% SDS-PAGE and in zymogram analysis against Streptococcus pyogenes. The N-terminal amino acid sequence by Edman degradation was partially determined to be T-P-P-Q-S-X-L-X-X-G, which shows very insignificant identity to other antimicrobial peptides from bacteria. The minimum inhibitory concentrations of dialysed and partially purified ion exchange fractions were determined against some selected gram-positive and gram-negative bacteria and some pathogenic yeasts. The presence of three important antimicrobial peptide biosynthesis genes ituc, fend, and bmyb was determined by PCR.

Physical chemical and biological characterization of a new bacteriocin produced by Bacillus cereus NS02

ObjectiveTo screen the bacteriocinogenic isolate from buffalo milk and to characterize it on physical, chemical and biological aspects for the application in biopreservation. MethodsBacillus cereus (B. cereus) was isolated and assessed for its baceteriocinogenic activity. Bacteriocin was produced and purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography. Purified bacteriocin was used to check its antimicrobial activity against food borne bacteria. Effect and stability of bacteriocin was determined with the respect to temperature, pH, enzymes, organic solvents and chemicals. Bacteriocin was also subjected to SDS PAGE analysis to determine its molecular weight. In addition, functional groups exist in the bacteriocin was determined by FTIR analysis. ResultsB. cereus was identified by 16S rRNA sequence analysis. Bacteriocin showed increased activity against all the bacteria used and its activity unit was found to be 51, 200 AU/mL. It was stable to high temperature (100 °C) and wide range of pH (3-10), sensitive to proteolytic enzymes and resistant to nonproteolytic enzymes. It was low molecular weight (3.5 – 6 KDa) protein and FTIR study revealed the presence of amide group and NH stretching. ConclusionsBacteriocin produced in this study possesses the highest antimicrobial activity against both gram positive and gram negative bacteria thereby it has immense application as biopreservative agent. FTIR proved its peptide nature.

Wrapping tissues with a pre-established cage-like layer composed of living cells

We report a method for wrapping tissues with a pre-established cage-like layer composed of living cells. We encapsulated multicellular aggregates of human hepatoma HepG2 cells as a model of tissues such as pancreatic islets and hepatocyte spheroids in alginate-based hydrogel microcapsules and subsequently coated the microcapsule surface with a gelatin derivative through a horseradish peroxidase-catalyzed reaction. Human aorta endothelial (HAE) cells grew on the surface and formed a cell layer within 24 h of incubating the microcapsules in a medium containing the cells. Subsequent degradation of the hydrogel microcapsule using a non-proteolytic enzyme, alginate lyase, resulted in a cage-like structure of HAE cells formed around the microcapsule. The HAE cell layer shrank without fragmenting and wrapped the inner spherical tissue. This method was also effective for wrapping multiple cellular aggregates within a single cage of HAE cells. In addition, it was possible to wrap tissue grown from individual cells in spherical cavities within the microcapsules.

New opportunities in drug design of metalloproteinase inhibitors: combination between structure–function experimental approaches and systems biology

Introduction: MMPs (matrix metalloproteinases) and ADAMs (a disintegrin and metalloproteinases) are endopeptidases central to the degradation and remodeling of the extracellular matrix. These proteases also exhibit regulatory activity in cell signaling pathways and thus tissue homeostasis under normal conditions and in many diseases. Consequently, individual members of the MMP and ADAM protein families were identified as important therapeutic targets. However, designing effective inhibitors in vivo for this class of enzymes appears to be extremely challenging. This is attributed to the broad structural similarity of their active sites and to the dynamic functional interconnectivity of MMPs with other proteases, their inhibitors, and substrates (the so-called degradome) in healthy and disease tissues.Areas covered: The article covers the progress in designing metalloproteinase inhibitors, based on recent advancements in our understanding of enzyme structures and their function as master regulators. It also discusses the potential of utilizing structure-based drug design strategies in conjunction with systems biology experimental approaches for designing potent and therapeutically effective metalloproteinase inhibitors.Expert opinion: We highlight the use of protein-based drug design strategies, for example, antibodies and protein scaffolds, targeting extracatalytic domains, which are central to proteolytic and non-proteolytic enzyme functions. Such rationally designed function-blocking inhibitors may create new opportunities in disease management and in emerging therapies that require control of dysregulated MMP activity without causing severe side effects. Importantly, the lessons learned from studying these protein-based inhibitors can be implemented to design new and effective small or medium sized synthetic antagonists.

Detection, partial purification and characterization of bacteriocin produced by Lactobacillus brevis FPTLB3 isolated from freshwater fish

Lactobacillus brevis FPTLB3 was isolated from freshwater fish, capable of producing bacteriocin that had broad spectrum of inhibition (3200 AU/ml) against Escherichia coli MTCC 1563, Enterococcus faecalis MTCC 2729, Lactobacillus casei MTCC 1423, Lactobacillus sakei ATCC 15521 and Staphylococcus aureus ATCC 25923. The antimicrobial activity of crude supernatant fluid was stable after heating at 121 °C for 60 min and declined thereafter. Stability of antimicrobial activity was observed at pH range of 2.0 to 8.0. Its active principle was proteinaceous in nature since the bacteriocin was inactivated by proteolytic enzymes, but not by other non-proteolytic enzymes. Mitomycin C and UV light did not affect the activity of the bacteriocin, while chloroform extraction completely destroyed their activity. Exposure to surfactant resulted in an increase in titre, except Nonidet P-40, which led to total loss of activity. No bacteriocin adsorption was detected at pH 1 to 2, whereas 100% bacteriocin adsorption was found at pH 6.5. Based on Tricine SDS-PAGE the estimated molecular mass of bacteriocin was 54 kDa. No plasmid was found to present in the isolate.

ChemInform Abstract: Comprehensive Survey of Chemical Libraries for Drug Discovery and Chemical Biology: 2009

AbstractReview: 383 refs.

Comprehensive survey of chemical libraries for drug discovery and chemical biology: 2009.

This is the thirteenth installment of the comprehensive survey series in high throughput chemistry.1 Biologically active libraries reported in 2009 are captured in Tables 1–5 under the headings of proteases, nonproteolytic enzymes, GPCRs, nonGPCRs, and oncolytics/antiinfectives. Table 6 lists molecular probes. Compound collections without disclosed biological activity are delineated in Tables 7–10 under the headings of scaffold derivatization/acyclic synthesis, monocyclic-, bicyclic/spirocyclic-, and polycyclic/macrocyclic synthesis. Polymer-supported reagents/scavengers/linkers are presented in Tables 11 (nonfluorous) and Table 12 (fluorous). There are 370 libraries and 24 molecular probes extracted from 355 literature citations.2–491 Approximately 90% of the citations originated from academic laboratories, with the majority of these from European and Asian laboratories. Solution-phase methodology accounted for ca. 85% of chemical library synthesis.

ANGIOTENSIN A AN ANG-II LIKE PEPTIDE STIMULATING THE AT2 RECEPTOR: PP.24.461

The angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. The other angiotensin peptides nevertheless may importantly contribute to vascular regulation, showing different affinities to the angiotensin receptors and hence different vascular effects. The present experiments revealed that after incubation of human mononuclear lymphocytes a vasocontrictive substance appeared time dependently. After chromatographic purification structural analysis by matrix-assisted laser desorption/ionisation and electrospray ionization mass spectrometry revealed an angiotensin-octapeptide with the sequence Asp - Arg - Val - Tyr - Ile - His - Pro – Phe, which differs from Ang II in Ala1 instead of Asp1. In the presence of lymphocytes Ang II is converted to des[Asp1]-[Ala1]-Ang II by decarboxylation of Asp1 group of Ang II. des[Asp1]-[Ala1]-Ang II had a same affinity to the AT1 receptor as Ang II, but was a weaker vasoconstrictor, suggesting partial AT1 receptor agonism. Being a partial AT1 receptor agonist with the same affinity as Ang II, and showing a higher AT2 receptor affinity, des[Asp1]-[Ala1]-Ang II has a specific profile of actions. Therefore the generation of des[Asp1]-[Ala1]-Ang II from Ang II may modulate the Ang II effects on vasculature. des[Asp1]-[Ala1]-Ang II showed a higher affinity to the AT2 receptor than Ang II. In summary des[Asp1]-[Ala1]-Ang II is a further human active angiotensin peptide which is transformed by a lymphocyte-derived aspartat-decarboxylase activity from angiotensin II and which is a further player in the angiotensin system. This is the first time that a non-proteolytic enzyme activity is demonstrated, which transforms an active peptide hormone into an also active derivative.

In Situ Simultaneous Protein−Polysaccharide Bioconjugation and Hydrogelation Using Horseradish Peroxidase

We propose the peroxidase-catalyzed simultaneous conjugation and hydrogelation of polysaccharide and protein derivatives, each possessing phenolic hydroxyl (Ph) moieties, as a novel route for obtaining protein-polysaccharide conjugate hydrogels. We used alginate, gelatin, and albumin derivatives bearing Ph moieties (Alg-Ph, Gela-Ph, and Alb-Ph) to demonstrate the feasibility. The gelation time of conjugate gels decreased with decreasing H(2)O(2) concentration and with increasing horseradish peroxidase concentration. Gelation time was controllable from a few seconds to 6 min. The repulsion force detected at 40% compression of a conjugate gel obtained from a mixture of Alg-Ph and Gela-Ph at 1.0% (w/v), respectively, was more than 2.8 times larger than that detected for gels produced from 3.0% (w/v) Gela-Ph or 2.0% (w/v) Alg-Ph alone. Cell adhesiveness of gels was tunable by changing the type of protein derivative. A gel from Gela-Ph and Alg-Ph showed higher cell adhesiveness than Alg-Ph gel, but a gel produced from Alb-Ph and Alg-Ph showed a lower cell adhesiveness than Alg-Ph gel. The conjugate gel was degradable by degrading alginate molecules using the nonproteolytic enzyme alginate lyase. The tunable gelation, mechanical properties, and cell adhesiveness of polysaccharide-protein conjugate hydrogels obtained through peroxidase-catalyzed gelation indicates great potential for a wide range of applications, such as scaffolds for tissue engineering and carriers for drug delivery system.

Quantification and kinetics of the decline in grass grub endopeptidase activity during initiation of amber disease

Amber disease in the grass grub ( Costelytra zealandica White) (Coleoptera: Scarabaeidae), caused by strains of the bacteria Serratia entomophila or S. proteamaculans, is characterised by cessation of feeding and clearance of the midgut. Analysis of the midgut enzyme activity in diseased grass grub larvae showed that proteolytic activity was reduced to low levels. The endopeptidases, trypsin, elastase, and chymotrypsin, were all markedly reduced in activity whereas the exopeptidases (leucine-aminopeptidase and carboxypeptidase A and B) were much less affected. There was no effect on the non-proteolytic enzymes, esterase and α-amylase. Sequential analysis of enzyme levels in the gut during onset of disease showed that proteolytic activity dropped after cessation of feeding and preceded gut clearance. In starved, uninfected larvae enzyme activity levels remained high, indicating that decline in enzyme activity is not associated with absence of food and cessation of feeding, but with the onset of disease.

Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905 ready-to-eat salad.

Lactobacillus plantarum BFE 905 isolated from 'Waldorf' salad produced a bacteriocin termed plantaricin D which was active against Lact. sake and Listeria monocytogenes strains. Plantaricin D was heat stable, retaining activity after heating at 121 degrees C. The bacteriocin was inactivated by alpha-chymotrypsin, trypsin, pepsin and proteinase K, but not by papain and other non-proteolytic enzymes tested. Plantaricin D was stable at pH values ranging from 2.0 to 10.0. The bacteriocin inhibited growth of L. monocytogenes in automated turbidity assays. Although Lact. Plantarum BFE 905 harboured plasmids ranging in size from 3 to 55 kilobase pairs, loss of bacteriocin production could not be correlated with plasmid loss. A role for bacteriocin-producing Lact. plantarum of vegetable origin in assuring the safety of vegetable foods is suggested.

Comprehensive survey of chemical libraries yielding enzyme inhibitors, receptor agonists and antagonists, and other biologically active agents: 1992 through 1997.

This review is a historical accounting of chemical libraries from which biologically active agents have been obtained. The comprehensive tabulation includes citations as early as 1992, when the first descriptions of biologically active libraries were disclosed, and continues through 1997. Four tables are provided listing libraries screened against (1) proteolytic enzymes, (2) non-proteolytic enzymes, (3) G-protein coupled receptors (GPCRs), and (4) other targets not classified in the first three tables (e.g. non-GPCRs, integrins, antiinfectives). A name, generic structure, and size is provided for each library citation, accompanied by the molecular screen and the structure and potency of the most active library member. In total, 86 libraries are presented with 60% of the contributions reported from pharmaceutical and biotechnology companies. Approximately 70% of the libraries have used alpha-amino acid synthons in their construction and 85% of the libraries include one or more amide bonds.

Discovery of enzyme inhibitors through combinatorial chemistry.

This review serves to highlight the recent examples of combinatoric methodology as applied to the discovery and optimization of enzyme inhibitors. Early research efforts focused on the identification of polypeptides from libraries as inhibitors of proteases. As solution- and solid-phase chemistries gain in sophistication, libraries containing less peptidic structural motifs have been created. A recurring design stratagem relies on the synthesis of libraries incorporating pharmacophores with known affinity for the target enzyme. Screening of these structure-based libraries has led to the discovery of small-molecule inhibitors of both proteolytic and non-proteolytic enzymes alike. Two tables are provided listing the enzyme targeted libraries through 1996. A name, generic structure and size is given for each library citation, accompanied by the enzyme screen and the structure and potency of the most active library member.

Production and characterization of enterocin 900, a bacteriocin produced by Enterococcus faecium BFE 900 from black olives

Enterococcus faecium BFE 900 isolated from black olives produced a bacteriocin termed enterocin 900, which was antagonistic towards Lactobacillus sake, Clostridium butyricum, enterococci as well as Listeria spp. including Listeria monocytogenes. Enterocin 900 was inactivated by pepsin, α-chymotrypsin, proteinase K and trypsin but not by catalase, α-amylase, or other non-proteolytic enzymes tested. The bacteriocin was heat stable, retaining activity after heating at 121 °C for 15 min. Enterocin 900 was active at pH values ranging from 2.0–10.0, with highest activity at pH 6.O. Bacteriocin production occurred in the late logarithmic growth phase when culture density was ca. log 8.0 CPU ml −1. Enterocin 900 was produced in media with initial pH ranging from 6.0–10.0, but not in media with a pH lower than 6.0. Medium composition, especially the concentrations of peptone and yeast extract influenced bacteriocin production, with no bacteriocin being produced in the absence of either of these compounds. No plasmids could be isolated from Enterococcus faecium BFE 900, indicating that the gene for bacteriocin activity is located on the chromosome.

5281356 Heavy duty liquid detergent compositions containing non-proteolytic enzymes comprising capsules comprising proteolytic enzyme and composite polymer: Liang Tsaur, Michael Aronson, Leslie J Morgan, John Hessel, Jack McCown, John L Gormley assigned to Lever Brothers Company

5281356 Heavy duty liquid detergent compositions containing non-proteolytic enzymes comprising capsules comprising proteolytic enzyme and composite polymer: Liang Tsaur, Michael Aronson, Leslie J Morgan, John Hessel, Jack McCown, John L Gormley assigned to Lever Brothers Company

5281357 Protease containing heavy duty liquid detergent compositions comprising capsules comprising non-proteolytic enzyme and composite polymer: Leslie J Morgan, Michael Aronson, Liang Tsaur, John Hessel, Jack McCown assigned to Lever Brothers Company Division of Conopco Inc

5281357 Protease containing heavy duty liquid detergent compositions comprising capsules comprising non-proteolytic enzyme and composite polymer: Leslie J Morgan, Michael Aronson, Liang Tsaur, John Hessel, Jack McCown assigned to Lever Brothers Company Division of Conopco Inc

Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus.

A bacteriocin-producing Bacillus cereus strain was isolated. The bacteriocin, here called cerein, was shown to be active specifically against other B. cereus strains and inactive against all other bacterial species tested. Cerein was detected in the culture supernatants of stationary-phase cells, and its appearance was inhibited by induction of sporulation. The bacterial activity of cerein was insensitive to organic solvents and nonproteolytic enzymes, partially stable to heat, and active over a wide range of pH values. Direct detection of antimicrobial activity on sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of about 9 kDa.