Effectiveness of Sal Deoiled Seed Cake as an Inducer for Protease Production from Aeromonas sp. S1 for its Application in Kitchen Wastewater Treatment

The present study is an attempt to demonstrate the feasibility of sal (Shorea robusta) deoiled cake—a forest-based industrial by-product—as a cheaper media supplement for augmented protease production from Aeromonas sp. S1 and application of protease in the treatment of kitchen wastewater. Under optimized conditions, protease production could successfully be enhanced to 5.13-fold (527.5 U mL^?1) on using sal deoiled seed cake extract (SDOCE), as medium additive, compared to an initial production of 102.7 U mL^?1 in its absence. The culture parameters for optimum production of protease were determined to be incubation time (48 h), pH (7.0), SDOCE concentration (3 % (v/v)), inoculum size (0.3–0.6 % (v/v)), and agitation rate (100 rpm). The enzyme was found to have an optimum pH and temperature of 8.0 and 60 °C, respectively. The protease preparation was tested for treatment of organic-laden kitchen wastewater. After 96 h of wastewater treatment under static condition, enzyme preparation was able to reduce 74 % biological oxygen demand, 37 % total suspended solids, and 41 % oil and grease. The higher and improved level of protease obtained using sal deoiled seed cake-based media hence offers a new approach for value addition to this underutilized biomass through industrial enzyme production. The protease produced using this biomass could also be used as pretreatment tool for remediation of organic-rich food wastewater.     

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Kinetic Analysis, Expression Pattern, and Production of a Recombinant Fungal Protease Inhibitor of Tasar Silkworm Antheraea mylitta

Antheraea mylitta, a tasar silk-producing insect of Saturniidae family, expresses a fungal protease inhibitor named as A. mylitta fungal protease inhibitor-1 (AmFPI-1). AmFPI-1 inhibits alkaline protease of Aspergillus oryzae but its mechanism of action is not known. To understand the mode of inhibition of AmFPI-1 against the fungal protease, it was purified from the hemolymph of A. mylitta larvae and inhibitory activity against A. oryzae protease was studied. Kinetic analysis of purified AmFPI-1 on alkaline protease of A. oryzae showed that AmFPI-1 acts as a canonical-type competitive inhibitor with equilibrium dissociation constant (K _i ) of 60?nM. Expression of AmFPI-1 in different body tissues of fifth instar A. mylitta larvae was determined by real-time PCR, and the highest expression was observed in fat body followed by integument, silk gland, and gut, indicating that AmFPI-1 has pleiotropic functions including protection from invading fungi. The cDNA of AmFPI-1 was expressed in Escherichia coli, and recombinant His-tagged fusion protein was purified by Ni-NTA chromatography. Recombinant AmFPI-1 showed inhibitory activity against A. oryzae protease and suggested its use in various biological applications to prevent proteolysis.     

Expression of the Acid Protease Gene from Saccharomycopsis fibuligera in the Marine-Derived Yarrowia lipolytica for Both Milk Clotting and Single Cell Protein Production

In this study, the native acid protease gene in Yarrowia lipolytica 22a-2 with high content of protein was disrupted, and the disruptant 3-13-10 obtained had very low acid protease activity. Then, the acid protease gene (AP1 gene) from Saccharomycopsis fibuligera A11 was actively expressed in the disruptant 3-13-10, and the transformant 43 carrying the AP1 gene had high specific acid protease activity (46.7 U/mg). The recombinant acid protease produced by the transformant 43 was found to be able to actively clot milk, and the transformant 43 still kept high content of protein. The hydrolysis products of κ-casein under catalysis of the recombinant acid protease and the commercial calf rennet had the same molecular mass, suggesting that the recombinant acid protease and its producer can be used both in cheese manufacturing and as protein source in food industry.     

A fluorescence turn-on method for real-time monitoring of protease activity based on the electron transfer between a fluorophore labeled oligonucleotide and cytochrome c

A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1 nM trypsin and the IC₅₀ values are 3.23 μg mL⁻¹ and 0.303 μg mL⁻¹ for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.     

Synthèse racémique et énantiosélective d’énol-lactones et leur évaluation comme inhibiteurs de la protéase du VIH-1

Racemic and enantioselective synthesis of enol-lactone and their biological activity as potent HIV-1 protease inhibitors. A series of enol-lactones was prepared and evaluated for its activity against HIV-1 protease. The preparation of (R,S)-5,6-dihydro-4-hydroxy-6-phenyl-2H-pyran-2-one (3) is the key intermediate for the synthesis. The synthesized enol-lactones (5 and 8–11) were kinetically evaluated against a HIV-1 protease substrate and were found to exhibit moderate inhibitory activity. The enantioselective synthesis of enol-lactone is achieved to give the two enantiomers. To cite this article: S. Ibrahimi et al., C. R. Chimie 8 (2005).     

Purification and Biochemical Characterization of a Novel Alkaline Protease Produced by Penicillium nalgiovense

Penicillium nalgiovense PNA9 produces an extracellular protease during fermentation with characteristics of growth-associated product. Enzyme purification involved ammonium sulfate precipitation, dialysis, and ultrafiltration, resulting in 12.1-fold increase of specific activity (19.5 U/mg). The protein was isolated through a series of BN-PAGE and native PAGE runs. ESI-MS analysis confirmed the molecular mass of 45.2 kDa. N-Terminal sequencing (MGFLKLLKGSLATLAVVNAGKLLTANDGDE) revealed 93 % similarity to a Penicillium chrysogenum protease, identified as major allergen. The protease exhibits simple Michaelis-Menten kinetics and K _m (1.152 mg/ml), V _max (0.827 mg/ml/min), and k _cat (3.2?×?10²) (1/s) values against azocasein show that it possesses high substrate affinity and catalytic efficiency. The protease is active within 10–45 °C, pH 4.0–10.0, and 0–3 M NaCl, while maximum activity was observed at 35 °C, pH 8.0, and 0.25 M NaCl. It is active against the muscle proteins actin and myosin and inactive against myoglobin. It is highly stable in the presence of non-ionic surfactants, hydrogen peroxide, BTNB, and EDTA. Activity was inhibited by SDS, Mn²⁺ and Zn²⁺, and by the serine protease inhibitor PMSF, indicating the serine protease nature of the enzyme. These properties make the novel protease a suitable candidate enzyme in meat ripening and other biotechnological applications.     

Complementary regioselective esterification of non-reducing oligosaccharides catalyzed by different hydrolases

The enzymatic transesterification of several tri- and tetrasaccharides with vinyl laurate is described. The lipases from Candida antarctica B (Novozym 435) and Thermomyces lanuginosus (Lipozyme TL IM) and the alkaline protease from Bacillus licheniformis (subtilisin Carlsberg) have been used with each carbohydrate to obtain different regioisomers. By using the sugars in their amorphous form, complete solubility is achieved in the reaction media (tert-butanol/pyridine mixtures for the lipases and pyridine for the protease) and high isolated yields of the corresponding monoesters are obtained. Good to excellent regioselectivity is observed for all the enzymes, showing a final complementary picture respect to the primary hydroxyls of the oligosaccharides studied.     

An efficient enzymatic preparation of rhinovirus protease inhibitor intermediates

The development of an efficient route for the preparation of (2S)-2-[3-{[(5-methylisoxazol-3-yl)carbonyl]amino}-2-oxopyridin-1(2H)-yl]pent-4-ynoic acid (4), a key intermediate in the synthesis of a human rhinovirus (HRV) protease inhibitor, is presented. In the presence of 40% acetonitrile, the alkaline protease from Bacillus lentus can catalyze the kinetic resolution of racemic ester 7 to afford (S)-acid 4 in 49% chemical yield/per cycle with 98% ee and >98% HPLC purity. The (R)-ester can then be readily recycled via a DBU catalyzed epimerization. The enzymatic preparation described here is superior to the existing chemical resolution route, exhibiting lower costs as well as higher yields, enantioselectivity, and substrate loads. In addition, this protease displays broad substrate specificity toward this class of compounds and can be easily extended to the preparation of other tripeptide mimetics of rhinovirus protease inhibitors.     

Isolation,Purification and Characterisation of an Organic Solvent-Tolerant Ca2+-Dependent Protease from Bacillus megaterium AU02

A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl₂, and it retained 90 % activity at 50 °C for 3 h. The K _m and V _max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH₂) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.     

MS Analysis and Molecular Characterization of Botrytis cinerea Protease Prot-2. Use in Bioactive Peptides Production

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8 %) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74 %) and with chymotrypsin from Metarhizium anisopliae (71 %). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.     

Enantioselective Synthesis of Cyclopentyltetrahydrofuran (Cp-THF), an Important High-Affinity P2-Ligand for HIV-1 Protease Inhibitors

A convenient optically active synthesis of (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan, a high-affinity nonpeptidyl ligand for HIV-1 protease inhibitor 2, is described. The synthesis utilizes commercially available (1R,5S)-(+)-2-oxabicyclo[3.3.0]oct-6-en-3-one as the starting material and oxymercuration or bromohydrin reaction as the key step. Enantiopure ligand was converted to protease inhibitor 2.     

Protease determination using an optimized alcohol enzyme electrode

A new method for the determination of protease activities is described. In this large family, trypsin is used as a protease model that cleaves the enthyl or methyl ester of artificial substrates producing ethanol or methanol. Alcohol is detected using an alcohol oxidase enzyme electrode. The H₂O₂ production that occurs is measured amperometrically. At 30°C, in a 0.1M phosphate buffer, pH 7.5, the enzyme electrode response for ethanol was calibrated at 3.10^?6–3.10^?3 M and for methanol from 3.10^?7 to 4.10^?4 M in the cell measurement. Trypsin levels as determined by the proposed method and by a conventional spectrophotometric method are in good agreement when using the same measurement conditions. A detection limit of 10 U·L^?1 and a linear calibration curve of 10–100,000 U·L^?1 in the sample were obtained. Measuring time for the required trypsin solution concentration was from 4 min (for the most dilute samples) to 1 min (for the most concentrate samples). In a typical experiment, protease measurements did not inactivate the alcohol oxidase on the probe, nor did a more classical use for alcohol detection. The procedure developed could permit any protease estimation on the condition that they hydrolyze ester bonds from synthetic substrate.     

Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease

Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.     

Salt-tolerant and thermostable alkaline protease fromBacillus subtilis NCIM No. 64

The proteolytic activity produced by aBacillus subtilis isolated from a hot spring was investigated. Maximum protease production was obtained after 38 h of fermentation. Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production. Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5–11. The partially purified enzyme was stable at 60°C for 30 min. Calcium ions were effective in stabilizing the enzyme, especially at higher temperature. The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h. The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000. The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.     

Protease Inhibitor from Insect Silk-Activities of Derivatives Expressed In Vitro and in Transgenic Potato

Several recombinant derivatives of serine protease inhibitor called silk protease inhibitor 2 (SPI2), which is a silk component in Galleria mellonella (Lepidoptera, Insecta), were prepared in the expression vector Pichia pastoris. Both the native and the recombinant protease inhibitors were highly active against subtilisin and proteinase K. The synthetic SPI2 gene with Ala codon in the P1 position was fused with mGFP-5 to facilitate detection of the transgene and its protein product. A construct of the fusion gene with plant regulatory elements (promoter 35S and terminator OCS) was inserted into the binary vector pRD400. The final construct was introduced into Agrobacterium tumefaciens that was then used for genetic transformation of the potato variety Velox. The transgene expression was monitored with the aid of ELISA employing polyclonal antibody against natural SPI2. In vitro tests showed increased resistance to the late blight Phytophthora infestans in several transformed lines. No effect was seen on the growth, mortality, life span or reproduction of Spodoptera littoralis (Lepidoptera, Insecta) caterpillars, while feeding on transformed potato plants expressing the fusion protein, indicating that the transformed potatoes may be harmless to non-target organisms.     

Application of Co-Culture Technology to Enhance Protease Production by Two Halophilic Bacteria,Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus

Although axenic microbial cultures form the basis of many large successful industrial biotechnologies, the production of single commercial microbial strains for use in large environmental biotechnologies such as wastewater treatment has proved less successful. This study aimed to evaluate the potential of the co-culture of two halophilic bacteria, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus for enhanced protease activity. The co-culture was significantly more productive than monoculture (1.6–2.0 times more growth), with Marinobacter hydrocarbonoclasticus being predominant (64%). In terms of protease activity, enhanced total activity (1.8–2.4 times) was observed in the co-culture. Importantly, protease activity in the co-culture was found to remain active over a much broader range of environmental conditions (temperature 25 °C to 60 °C, pH 4–12, and 10–30% salinity, respectively). This study confirms that the co-culturing of halophilic bacteria represents an economical approach as it resulted in both increased biomass and protease production, the latter which showed activity over arange of environmental conditions.     

The enzymatic asymmetric aldol reaction using acidic protease from Aspergillus usamii

AUAP (acidic protease from Aspergillus usamii) could catalyze the direct aldol reactions between aromatic aldehydes and cyclic ketones in acetonitrile (MeCN) in the presence of water. The enantioselectivities of up to 88% ee and diastereoselectivities of up to 97:3 (anti/syn) were achieved. This new activity of protease expands the application of the biocatalyst and provides a novel example of enzymatic promiscuity.     

Purification and Characterization of a New Metallo-Neutral Protease for Beer Brewing from Bacillus amyloliquefaciens SYB-001

The increased additive amount of adjuncts in the raw materials of Chinese beer requires the usage of protease to release more water-soluble proteins. Here, a metallo-neutral protease suited for brewing industry was purified from Bacillus amyloliquefaciens SYB-001. A 5.6-fold purification of the neutral protease was achieved with a 4-step procedure including ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel-filtration chromatography. The molecular mass of the enzyme was estimated to be 36.8 kDa. The protease was active and stable at a wide range of pH from 6.0–10.0 with an optimum at pH 7.0. The highest activity of the purified enzyme was found at 50 °C. The existence of manganese ion would specifically enhance the protease activity. Comparing with other commercial neutral proteases in China, adding the new neutral protease during mashing process would release more amino acids from wort such as aspartic acid, arginine, methione, and histidine, resulting in a better amino acid profile in wort. Moreover, the wort processed with the new neutral protease had a higher α-amino nitrogen concentration, which would ensure a vigorous yeast growth and better flavor. The study of the enzyme could lay a foundation for its industrial application and further research.     

Cloning,Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus

An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.