Key active sites of proteases and protease inhibitors: a review / 生物工程学报

Proteases are widely found in organisms participating in the decomposition of proteins to maintain the organisms' normal life activities. Protease inhibitors regulate the activities of target proteases by binding to their active sites, thereby affecting protein metabolism. The key amino acid mutations in proteases and protease inhibitors can affect their physiological functions, stability, catalytic activity, and inhibition specificity. More active, stable, specific, environmentally friendly and cheap proteases and protease inhibitors might be obtained by excavating various natural mutants of proteases and protease inhibitors, analyzing their key active sites by using protein engineering methods. Here, we review the studies on proteases' key active sites and protease inhibitors to deepen the understanding of the active mechanism of proteases and their inhibitors.

Obtención de hidrolizados proteicos de leguminosas usando una proteasa recombinante de Pseudomonas aeruginosa M211 / Obtaining protein hydrolysates from leguminous using a recombinant protease from Pseudomonas aeruginosa M211

El género Pseudomonas es una fuente importante de proteasas; sin embargo, su uso está restringido en la industria alimentaria. El clonaje permite aprovechar la capacidad catalítica de estas enzimas mediante su producción en microorganismos inocuos. Por otro lado, las leguminosas son fuentes ricas en proteínas, a partir de las cuales se pueden obtener compuestos con valor agregado mediante procesos de hidrólisis enzimática. En este estudio, se produjo y caracterizó una proteasa recombinante (PT4) alcalina y termoestable de Pseudomonas aeruginosa M211, para la obtención de hidrolizados proteicos de leguminosas. Para ello, el gen de la proteasa se clonó en el vector pJET1.2/blunt utilizando E. coli DHalfa como hospedero. El análisis de la secuencia nucleotídica parcial de la proteasa indicó un 99 % de similitud con Peptidasas de la Familia M4 de Pseudomonas aeruginosa. La enzima recombinante presentó un peso molecular de 80 kDa, demostró ser activa y estable en condiciones alcalinas y termófilas con un pH y temperatura óptimos de 8 y 60 °C, respectivamente, y fue inhibida por EDTA. Además, hidrolizó proteínas de semillas de Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis, obteniéndose fracciones peptídicas menores a 40 kDa. Esta proteasa recombinante se podría utilizar en la elaboración de hidrolizados proteicos funcionales a partir proteínas de distintas fuentes y residuos agroalimentarios.

The genus Pseudomonas is an important source of proteases; however, in the food industry the use of this bacterium is restricted. Cloning allows for the use of the proteolytic activity of Pseudomonas proteases through their production in innocuous microorganisms. Leguminous are protein-rich sources from which value-added compounds can be obtained through enzymatic hydrolysis. In this study, an alkaline and thermostable recombinant protease (PT4) from Pseudomonas aeruginosa M211 was cloned and characterized in order to obtain protein hydrolysates from leguminous. Therefore, protease gene was cloned into the pJET1.2 / blunt vector using E. coli DHalpha as a host. Analysis of protease partial nucleotide sequence showed 99% homology with Peptidases M4 Family from Pseudomonas aeruginosa. The molecular weight of the recombinant enzyme was 80 kDa, it was active and stable under alkaline and thermophilic conditions, presented an optimum pH and temperature of 8 and 60 °C, respectively, and was inhibited by EDTA. In addition, it hydrolysed Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis proteins, obtaining peptide fractions less than 40 kDa. This recombinant protease could be used in the elaboration of functional hydrolysates using protein from different sources and agricultural waste.

Genome-wide identification of the Sec-dependent secretory protease genes in Erwinia amylovora and analysis of their expression during infection of immature pear fruit / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The general secretory (Sec) pathway represents a common mechanism by which bacteria secrete proteins, including virulence factors, into the extracytoplasmic milieu. However, there is little information about this system, as well as its associated secretory proteins, in relation to the fire blight pathogen Erwinia amylovora. In this study, data mining revealed that E. amylovora harbors all of the essential components of the Sec system. Based on this information, we identified putative Sec-dependent secretory proteases in E. amylovora on a genome-wide scale. Using the programs SignalP, LipoP, and Phobius, a total of 15 putative proteases were predicted to contain the N-terminal signal peptides (SPs) that might link them to the Sec-dependent pathway. The activities of the predicted SPs were further validated using an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system that confirmed their extracytoplasmic property. Transcriptional analyses showed that the expression of 11 of the 15 extracytoplasmic protease genes increased significantly when E. amylovora was used to inoculate immature pears, suggesting their potential roles in plant infection. The results of this study support the suggestion that E. amylovora might employ the Sec system to secrete a suite of proteases to enable successful infection of plants, and shed new light on the interaction of E. amylovora with host plants.

Construction and application of a novel genetically engineered Aspergillus oryzae for expressing proteases

Background: We aimed to test the possibility of improving polypeptide production from soybean meal fermentation by engineered Aspergillus oryzae strains. Four different protease genes were cloned and transformed into wild-type A. oryzae, and the engineered A. oryzae strains were then used for soybean meal fermentation. Results: The results showed different degrees of improvement in the protease activity of the four transformants when compared with wild-type A. oryzae. A major improvement in the polypeptide yield was achieved when these strains were used in soybean meal fermentation. The polypeptide conversion rate of one of the four transformants, A. oryzae pep, reached 35.9%, which was approximately twofold higher than that exhibited by wild-type A. oryzae. Amino acid content analysis showed that the essential amino acid content and amino acid composition of the fermentation product significantly improved when engineered A. oryzae strains were used for soybean meal fermentation. Conclusions: These findings suggest that cloning of microbial protease genes with good physicochemical properties and expressing them in an ideal host such as A. oryzae is a novel strategy to enhance the value of soybean meal.

Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk

The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli. The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca+2. The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca+2, on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.

Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

Biochemical and molecular characterization of Bacillus pumilus isolated from coastal environment in Cochin, India / Caracterização bioquímica e molecular de Bacillus pumilus isolado do ambiente costeiro de Cochin, Índia

Bacillus species constitute a diverse group of bacteria widely distributed in soil and the aquatic environment. In this study, Bacillus strains isolated from the coastal environment of Cochin, India were identified by detailed conventional biochemical methods, fatty acid methyl ester (FAME) analysis and partial 16S rDNA sequencing. Analysis of the data revealed that Bacillus pumilus was the most predominant species in the region under study followed by B. cereus and B. sphaericus. The B. pumilus isolates were further characterized by arbitrarily primed PCR (AP-PCR), antibiotic sensitivity profiling and PCR screening for known toxin genes associated with Bacillus spp. All B. pumilus isolates were biochemically identical, exhibited high protease and lipase activity and uniformly sensitive to antibiotics tested in this study. One strain of B. pumilus harboured cereulide synthetase gene cesB of B. cereus which was indistinguishable from rest of the isolates biochemically and by AP-PCR. This study reports, for the first time, the presence of the emetic toxin gene cesB in B. pumilus.

As espécies de Bacillus constituem um grupo diversificado de bactérias amplamente distribuídas no solo e no ambiente aquático. Neste estudo, cepas de Bacillus isoladas do ambiente costeiro de Cochin, Índia, foram identificadas através de métodos bioquímicos convencionais, análise de ésteres metílicos de ácidos graxos (FAME) e sequenciamento de 16S rDNA. A análise dos dados revelou que Bacillus pumilus foi a espécie predominante na região estudada, seguido de B. cereus e B. sphaericus. Os isolados de B. pumilus foram caracterizados através da reação em cadeia da polimerase com primers arbitrários (AP-PCR), perfil de sensibilidade a antibióticos e triagem por PCR de genes de toxinas associadas com Bacillus spp. Todos os isolados de B. pumilus foram bioquimicamente idênticos, apresentaram elevada atividade de protease e lipase e foram uniformemente sensíveis aos antibióticos estudados. Um dos isolados de B. pumilus apresentou o gene cesB de B. cereus, que não foinão distinguível dos demais isolados por testes bioquímicos nem por AP-PCR. Este é o primeiro relato da presença do gene cesB da toxina eméticaem B. pumilus.

The role of autolysis loop in determining the specificity of coagulation proteases

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

Influence of jasmonic acid as potential activator of induced resistance against Karnal bunt in developing spikes of wheat.

Induction of defense response against Karnal bunt (KB)by suppressing the pathogenesis was observed upon exogenous application of jasmonic acid (JA)as evident from decrease in the coefficient of infection and overall response value in both susceptible and resistant varieties of wheat. The ultra-structural changes during disease progression showed the signs of programmed cell death (PCD). However, JA strengthened the defense barrier by enhancing the lignifications of cell walls as observed in spikes of both varieties by histochemical analysis. Compared to the plants inoculated with pathogen alone, plants of resistant line (RJP) first treated with JA followed by inoculation with pathogen showed more lignifications and extracellular deposition of other metabolites on cells, which is supposed to prevent mycelial invasions. Contrary to this, susceptible (SJP)lines also showed lignifications but the invasion was more compared to resistant line.Induction of protease activity was higher in resistant variety than its corresponding susceptible variety. The protease activity induced during the colonization of the pathogen and its proliferation inside the host system gets inhibited by JA treatment as demonstrated by the quantitative and in-gel protease assay. The results indicate the role of JA signalling in inhibiting the proteases due to expression of certain protease inhibitor genes. SDS-PAGE analysis shows differential gene expression through induction and/or suppression of different proteins in wheat spikes of resistant and susceptible varieties under the influence of JA. Thus, exogenously applied JA provides the conditioning effect prior to the challenge of infection and induces defense against KB probably by maintaining a critical balance between proteases and protease inhibitors and/or coordinating induction of different families of new proteins.

Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.

Transcriptome overview of Paracoccidioides brasiliensis proteases

Proteases perform a wide variety of functions inside and outside cells, regulating many biological processes. Infectious microorganisms use proteases, either secreted or attached to their cell surface to weaken and invade their hosts. Therefore, proteases are targets for drugs against a diverse set of diseases. Paracoccidioides brasiliensis is the most prevalent fungal pathogen causing systemic mycosis in Latin America. The development of paracoccidioidomycosis depends on interactions between fungal and host components and proteases have been described as important factors implicated in the mechanism of host colonization by fungi. The primary goal for this study is to present an overview of the transcriptome sequences--identified cDNAs that encode proteases. We obtained a total of 53 cDNAs encoding proteases; 15 were classified as ATP-independent, 12 as ATP-dependent, 22 as proteasome subunits, and 4 as deubiquitinating proteases. The mechanisms and biological activity of these proteases differ in substrate specificity and in catalytic mechanisms.