Proteases from an Environmental Isolate of Pseudomonas Aeruginosa MCCB 123 and Their Applications

In the present study,heterotrophic protease producing bacterial isolates were screened for protease activity and a potent protease producing bacterial isolate was selected,identified and coded as Pseudomonas aeruginosa MCCB 123.The organism was capable of producing three different types of enzymes each having potential industrial applications.The non-toxic nature of the bacterial strain and the relatively non-toxic nature of three enzymes suggested their poetential application in various industries.Application of LasA protease and beta-1,3 glucanase in DNA extraction is a promising area for commercial utilization. LasB protease can find its potential application in detergent and tanning industries.As on today Bacillus sp.has been the source of commercial proteases,and the ones produced form P.aeruginosa 123 can pave way for making the industrial and biomedical processes more cost effective and refined.

Alkaline Proteases From Bacteria Isolated From Cochin Estuary

Microorganisms distributed in the marine and brackish environments play an important role in the decomposition of organic matter and mineralisation in the system (Seshadri and lgnacimuthu, 2002). Estuary is one of the most productive ecosystems, at the same time one among the least explored ecosystems on earth, which has immense potential as a source of potent microorganisms that produce valuable compounds particularly, enzymes such as proteases. In this scenario, it is very appropriate to embark on finding novel alkaline protease producers from the estuarine system. The area where the present investigation was carried out is a part of the extensive estuarine system of South India viz. Cochin Estuary. There is meagre knowledge regarding the microbial composition, particularly the protease producers of Cochin Estuary. Hence, the present study has been undertaken with the objective of finding novel alkaline protease producing bacteria from Cochin Estuary

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin

Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043×10−10 M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4–10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases

Evolutionary analysis of novel serine proteases in the venom gland transcriptome of Bitis gabonica rhinoceros

Background: Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. Methodology and Principal Findings: Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. Conclusions and Significance: Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites.

Sequence and phylogenetic analysis of viper venom serine proteases

Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues.

Protease-activated receptor 2, dipeptidyl peptidase I, and proteases mediate Clostridium difficile toxin A enteritis

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Temperature regulation of extracellular proteases in ectomycorrhizal fungi (Hebeloma spp.) grown in axenic culture

Strains of Hebeloma representative of different climatic zones were grown in axenic culture at either 2 °C and 22° or 6° and 22°. Culture filtrates were assayed for proteolytic activity using FITC labelled BSA as a substrate. Assays were run between 0–37°. Growth at low temperature induced greater proteolytic activity (g−1 D.W. mycelium). Many of the strains produced protease(s) which retained significant activity at temperatures as low as 0°, and a thermal optimum between 0–6° with a second optimum at higher temperature. The results are discussed in relation the nutrient acquisition potential of ectomycorrhizal fungi at low temperature and the contribution such cold active proteases might make to the soil enzyme pool.

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis, genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of similar to 1.7 kb located in 2 chromosomal bands of 600-720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Trypanosoma cruzi: Isolation and characterization of aspartyl proteases

Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO(2))-Phe-Val-Leu-O(4)MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (>= 68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (>= 80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation. (c) 2009 Elsevier Inc. All rights reserved.

Immobilized analogues of sunflower trypsin inhibitor-1 constitute a versatile group of affinity sorbents for selective isolation of serine proteases

Sunflower trypsin inhibitor-1 (SFI-1), a natural 14-residue cyclic peptide, and some of its synthetic acyclic variants are potent protease inhibitors displaying peculiar inhibitory profiles. Here we describe the synthesis and use of affinity sorbents prepared by coupling SFTI-1 analogues to agarose resin. Chymotrypsinand trypsin-like proteases could then be selectively isolated from pancreatin; similarly, other proteases were obtained from distinct biological sources. The binding capacity of [Lys5]-SFTI-1-agarose for trypsin was estimated at over 10 mg/mL of packed gel. SFTI-1-based resins could find application either to improve the performance of current purification protocols or as novel protease-discovery tools in different areas of biological investigation. (C) 2009 Elsevier B.V. All rights reserved.

Atividade de proteases e disponibilidade de nitrogênio para laranjeira cultivada em Latossolo Vermelho distrófico

O conhecimento do potencial da mineralização dos compostos orgânicos nitrogenados do solo é fundamental para o uso eficiente do N em sistemas de produção agrícola. Com o objetivo de avaliar a atividade de proteases e suas relações com os teores de diferentes formas de N em Latossolo Vermelho distrófico cultivado com laranjeira, foram coletadas amostras de solo em duas profundidades (0-10 e 10-20 cm), na linha de projeção da copa e nas entrelinhas, nos meses de novembro e dezembro/98, fevereiro e março/99. em março/99, época recomendada para a diagnose foliar, também foram coletadas amostras de folhas. As amostras de solo foram avaliadas com relação à atividade de proteases e aos teores de N-amoniacal, N-nitrato, N-total, N-amoniacal obtido após incubação anaeróbia (N-pot) e N-amoniacal extraído por solução de CaCl2 sob autoclavagem (N-autoclave). Nas amostras de folha, determinou-se o teor de N-total. A atividade de proteases foi influenciada pelo local e pela profundidade de amostragem, tendo sido mais elevada na linha de projeção da copa e profundidade de 0-10 cm. A aplicação de fertilizante orgânico causou aumento na atividade de proteases, principalmente na camada de 0-10 cm. O coeficiente de correlação entre atividade de proteases e outros métodos para avaliação da disponibilidade de N dependeu da época, do local e da profundidade de amostragem. A correlação entre o N-foliar, obtido na época indicada para diagnose foliar, a atividade de proteases e as demais formas para avaliar disponibilidade de N (N-amoniacal, N-nitrato, N-autoclave e N-pot) variou com a época de amostragem. A atividade de proteases foi positiva e altamente correlacionada com o N-foliar, obtido na época indicada para fins de diagnose foliar, para amostras de terra retiradas nas entrelinhas (0-10 cm), no mês de dezembro/1998.