Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA-10

Aspergillus nidulans is a highly potent fungus used in the production of alkaline protease. Extracellular alkaline protease was purified from A. nidulans in a two-step procedure involving ammonium sulphate precipitation and Sephadex G-100 column chromatography. The molecular mass of the enzyme was determined to be 42 kDa by SDS-PAGE. The enzyme activity was also analyzed by zymogram with gelatin. The enzyme was more stable over a wide range of pH (6-10) and the temperatures up to 50 degrees C. It showed optimum enzyme activity at pH 8.0 and a temperature of 35 degrees C. The protease enzyme was completely inhibited by the serine protease inhibitor of phenylmethylsulfonyl fluoride (PMSF). The crystallization of the purified enzyme was performed by hanging drop vapour diffusion method using PEG 6000 as the precipitant. The micro crystals occurred in 40% of PEG 6,000.     

Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries

An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component.     

Purification, characterization and thermodynamics of antifungal protease from Streptomyces sp. A6

A 20 kDa antifungal serine protease from Streptomyces sp. A6 was purified to 34.56 folds by gel permeation chromatography. The enzyme exhibited highest activity at neutral to near alka- line pH 7-9 and 55 °C. Neutral surfactant triton X-100 enhanced the activity by 4.12 fold. The protease activity also increased (109.9-119%) with increasing concentration of urea (2-8 mole/l). The enzyme was identified as serine protease with 67% similarity to SFase 2 of Streptomyces fradiae by MALDI-LC-MS/MS analysis. Determination of kinetic constants k(m) , V(max) , k(cat) and k(cat) /k(m) suggested higher affinity of enzyme for N-Suc-Ala-Ala-Val-Ala-p NA (synthetic substrate for chymotrypsin activity). The enzyme was highly stable at temperature prevailing under field conditions (40 °C) as apparent from K(d) and t(1/2) values, 0.0065 and 106.75 min, respectively and high ΔG* and negative ΔS * values, 87.17 KJ/mole and -126.95 J/mole, respectively. Thermal stability and increased activity of protease in presence of commonly used chemical fertilizer, urea, suggested its feasibility for agricultural applications. The present study is the first report on thermodynamic and kinetic properties of an antifungal protease from Streptomyces sp. A6. The study reflects potential of this enzyme for biocontrol of fungal plant pathogens.     

Ambient alkaline pH prevents maturation but not synthesis of ASPA, the aspartyl protease from Penicillium roqueforti

Penicillium roqueforti secretes an aspartyl protease, ASPA, which represents the main extracellular proteolytic activity. Alkaline pH of the medium plays a major role by inhibiting the enzymatic activity and stopping aspA expression in the presence of casein, an inducing protein. However, casein degradation by the mature enzyme produces peptides which can induce aspA expression at acidic and alkaline pH. ASPA synthesized as a proenzyme is processed at an acidic pH but not at an alkaline pH. The data indicate that, in P. roqueforti, alkaline pH has an indirect repressive effect by inhibiting ASPA maturation and the release of inducers. At an acidic pH, the mature enzyme degrades extracellular proteins and peptides are released to induce aspA. In contrast, at an alkaline pH, the proenzyme remains inactive, the inducing substances are consequently not produced and aspA is no longer expressed. Received: 10 April 2000 / Accepted: 18 July 2000     

Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6

A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249‐amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p‐nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p‐nitrophenyl butyrate. When p‐nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H‐257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X‐100, sodium deoxycholate, urea, and guanidine hydrochloride.

     

Serine protease inhibitors and cytotoxic T lymphocytes

Summary: Serine proteases control a wide variety of physiological and pathological processes in multi-cellular organisms, including blood clotting, cancer, cell death, osmoregulation, tissue remodeling, and immunity to infection. Cytotoxic T lymphocytes (CTLs) are required for adaptive cell-mediated immunity to intracellular pathogens by killing infected cells and through the development of memory T cells. Serine proteases not only allow a CTL to kill but also impose homeostatic control on CTL number. Serine protease inhibitors (serpins) are the physiological regulators of serine proteases’ activity. In this review, I discuss the role of serpins in controlling the recognition of antigen, effector function, and homeostatic control of CTLs through the inhibition of physiological serine protease targets. An emerging view of serpins is that they are important promoters of cellular viability through their inhibition of executioner proteases. This view is discussed in the context of the T-lymphocyte survival during effector responses and the development and persistence of long-lived memory T cells. Given the important role serpins play in CTL immunity, I discuss the potential for developing new immunotherapeutic approaches based directly on serpins or knowledge gained from identifying their physiologically relevant protease targets.     

Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients

NS3 protease is essential for hepatitis C Virus (HCV) replication, and is one of the most promising targets for specific anti-HCV therapy. Its natural polymorphism has not been studied at the quasispecies level. In the present work, the genetic heterogeneity of the NS3 protease gene was analyzed in 17 HCV genotype 1 (5 subtypes 1a and 12 subtypes 1b) samples collected from infected patients before anti-viral therapy. A total of 294 clones were sequenced. Although the protease NS3 is considered to be one of the less variable genes in the HCV genome, variability of both nucleotide and amino acid sequences was found. In variants belonging to 1a and 1b subtypes, 224 and 267 of 543 positions showed one or more nucleotide substitutions, respectively. Forty and 74 of the 181 NS3 amino acid positions showed at least one mutation in HCV-1a and HCV-1b isolates, respectively. Most substitutions were conservative. This substantial polymorphism of the NS3 protease produced by HCV-1a and HCV-1b suggests that, despite the numerous functional and structural constraints, the enzyme is sufficiently flexible to tolerate substitutions.     

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

A novel extracellular alkaline serine protease secreted by Vibrio metschnikovii (V. metschnikovii) ATCC700040 cells was purified by three chromatographic steps and characterized in terms of enzymatic kinetics and substrate specificity. The purified enzyme (named AKP-Vm) was composed of a single polypeptide with an apparent molecular weight of 50 kDa on 12% SDS-polyacrylamide gel in the presence of CuCl?. The optimal temperature and the pH for the enzyme were found to be 37?C and 9.5, respectively. However, the enzyme activity was inhibited by inhibitors such as PMSF and aprotinin. AKP-Vm could hydrolyze a peptide bond at the carboxyl side of the arginine residue, as revealed by its amidolytic activity toward a chromogenic substrate, Boc-Val-Pro-Arg-pNA. The kinetic parameters of the enzyme were as follows: KM=0.91 mM, kcat=0.8 sec?1 and kcat/KM=0.88 mM?1sec?1. AKP-Vm protease could cleave various blood coagulation-associated proteins, including fibrinogen, prothrombin and thrombin. In particular, the enzyme showed powerful fibrinogenolytic and fibrinolytic activities, as it could cleave all major chains of fibrinogen and also digest cross-linked fibrin. The results obtained suggest that AKP-Vm is a novel alkaline serine protease that can actively cleave fibrinogen and cross-linked fibrin.     

Purification and characterization of the ncgl2923 ‐encoded 3‐hydroxybenzoate 6‐hydroxylase from Corynebacterium glutamicum

Corynebacterium glutamicum ATCC 13032 metabolizes 3‐hydroxybenzoate via gentisate. We have now characterized the ncgl2923 ‐encoded 3‐hydroxybenzoate 6‐hydroxylase involved in the initial step of 3‐hydroxybenzoate catabolism by this strain, a first 3‐hydroxybenzoate 6‐hydroxylase molecularly and biochemically characterized from a Gram‐positive strain. The ncg12923 gene from Corynebacterium glutamicum ATCC 13032 was shown to encode 3‐hydroxybenzoate 6‐hydroxylase, the enzyme that catalyzes the NADH‐dependent conversion of 3‐hydroxybenzoate to gentisate. Ncgl2923 was expressed with an N‐terminal six‐His tag and purified to apparent homogeneity by Ni²⁺‐nitrilotriacetic acid affinity chromatography. The purified H₆‐Ncgl2923 showed a single band at apparent molecular mass of 49 kDa on a sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be most likely a trimer as determined by gel filtration chromatography. It had a specific activity of 6.92 ± 0.39 U mg^–1 against 3‐hydroxybenzoate and with a K_m value of 53.4 ± 4.7 μM using NADH as a cofactor. The product formed from the 3‐hydroxybenzoate hydroxylation catalyzed by H₆‐Ncgl2923 was identified by high‐performance liquid chromatography as gentisate, a ring‐cleavage substrate in the microbial aromatic degradation. The enzyme exhibited a maximum activity at pH 7.5 in phosphate buffer, and adding flavin adenine dinucleotide to a final concentration of 15 μM would enhance the activity by three‐fold. Although this enzyme shares no more than 33% identity with any of reported 3‐hydroxybenzoate 6‐hydroxylases from Gram‐negative bacterial strains, there is little difference in subunit sizes and biochemical characteristics between them. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Production and characterization of clinical grade exosomes derived from dendritic cells

We describe methods for the production, purification, and characterization of clinical grade (cGMP) exosomes derived from antigen presenting cells (APCs). Exosomes have been shown to have immunotherapeutic properties through their presentation of biologically relevant antigens [Nat. Med. 4 (1998) 594] and are being developed as an alternative to cellular therapies. Exosomes are 50–90-nm-diameter vesicles secreted from multivesicular bodies (MVBs) found in a variety of both hematopoietic and tumor cells. These particles contain antigen presenting molecules (MHC class I, MHC class II, and CD1), tetraspan molecules (CD9, CD63, CD81), adhesion molecules (CD11b and CD54), and costimulatory molecules (CD86); hence, providing them the necessary machinery required for generating a potent immune response [J. Biol. Chem. 273 (1998) 20121; J. Cell. Sci. 113 (2000) 3365; J. Immunol. Methods 247 (2001) 163; J. Immunol. 166 (2001) 7309]. Exosomes from monocyte-derived dendritic cells (MDDCs) were rapidly purified (e.g. 4–6 h of a 2–3 l culture) based on their unique size and density. Ultrafiltration of the clarified supernatant through a 500-kDa membrane and ultracentrifugation into a 30% sucrose/deuterium oxide (D₂O) (98%) cushion (density 1.210 g/cm³) reduced the volume and protein concentration approximately 200- and 1000-fold, respectively. The percentage recovery of exosomes ranged from 40% to 50% based on the exosome MHC class II concentration of the starting clarified supernatant. This methodology was extended to a miniscale process with comparable results. Conversely, the classical differential centrifugation technique is a more lengthy and variable process resulting in exosomes being contaminated with media proteins and containing only 5–25% of the starting exosome MHC class II concentration; hence, making it difficult for their use in clinical development. Lastly, we developed the following quality control assays to standardize the exosome vaccine: quantity (concentration of MHC class II) and protein characterization (FACS). The combination of a rapid and reproducible purification method and quality control assays for exosomes has allowed for its evaluation as a cancer vaccine in clinical trials [Proc. Am. Soc. Oncol. 21 (2002) 11a].