Gene cloning and expression of a detergent stable alkaline protease from Aspergillus clavatus ES1

The genes, cDNA alpES1 and alpES1, encoding Aspergillus clavatus ES1 alkaline protease were amplified from complementary DNA (cDNA) and genomic DNA, respectively, cloned in pCR^®II-TOPO plasmid and then sequenced. Sequence analysis of the cDNA alpES1 gene revealed an open reading frame (ORF) of 1212 bp encoding a pre–pro-protein of 403 amino acid residues consisting of a 21-aa signal peptide, a 100-aa pro-peptide and a 282-aa mature protein with a calculated molecular weight of 28.5 kDa. Compared to the cDNA alpES1 gene, the alpES1 gene contained three introns, which had 53, 57 and 54 bp, respectively. The cDNA alpES1 gene was then sub-cloned in pET-30b(+) and expressed in Escherichia coli BL21 (λDE3). The purified recombinant protease had a molecular weight of about 32 kDa estimated by SDS-PAGE. Kinetic parameters, K_m and k_cat values of the recombinant AlpES1 for casein, were 0.23 mM and 12.38 min⁻¹, respectively. The catalytic efficiency (k_cat/K_m) was 53.82 min⁻¹ mM⁻¹.     

A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21. Purification and characterization

An extracellular bleach stable protease producing strain was isolated from marine water sample and identified as Bacillus mojavensis A21 on the basis of the 16S rRNA gene sequencing and biochemical properties. The A21 alkaline protease was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange chromatography, with a 6.43-fold increase in specific activity and 16.56% recovery. The molecular weight of the purified enzyme was estimated to be 20 kDa by SDS-PAGE and gel filtration. The enzyme was highly active over a wide range of pH from 7.0 to 13.0, with an optimum at pH 8.5. The relative activities at pH 11.0 and 12.0 were about 80 and 71.7% of that obtained at pH 8.5. The enzyme was extremely stable in the pH range of 7.0–12.0. It exhibited maximal activity at 60 °C. The thermostability of the enzyme was significantly increased by the addition of CaCl₂. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease.The N-terminal amino acid sequence of the first 20 amino acids of the purified protease was DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with bacterial peptidases, suggesting that the enzyme is a new protease.The alkaline protease showed high stability towards anionic (0.1% SDS) and non-ionic (1 and 5% Tween 80 and 1% Triton X-100) surfactants. In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 79 and 70% of its initial activity after 1 h incubation in the presence of 1% H₂O₂ and 0.1% sodium perborate, respectively. The enzyme showed excellent stability with a wide range of commercial solid and liquid detergents at 30 and 40 °C. Considering its promising properties, B. mojavensis A21 may find potential application in laundry detergents.     

Purification and characterization of an invertase produced by Aspergillus ochraceus TS.

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.     

Patulin produced by an Aspergillus clavatus isolated from feed containing malting residues associated with a lethal neurotoxicosis in cattle

A severe neurotoxicosis, comprising tremors, ataxia, paresis, recumbency and death, occurred simultaneously among several herds of beef cattle in the region of Flanders (Belgium). After a first multi-toxin screening of some suspected diet elements, verruculogen was detected in a sample of a common feed ingredient. However, when the first animal necropsies revealed serious nervous lesions, including neuronal degeneration of the central nervous system and axonal degeneration in the peripheral nervous system, further investigations focused on fungal isolation. As expected from the pathological lesions, Aspergillus clavatus was found to be the dominant fungal species in a sample of compacted fodder, containing malting residues, consumed by all the affected herds. The isolated fungus appeared to produce patulin in culture medium. Traces of patulin were also detected in the fodder. These findings and their possible role in the intoxication are discussed.     

Purification and characterization of an alkaline lipase from a newly isolated Acinetobacter radioresistens CMC-1

A novel alkaline lipase showing a broad range of specificity towards long chain triacylglycerols or p-nitrophenyl esters was purified to homogeneity from Acinetobacter radioresistens CMC-1. Its molecular mass was 45 kDa (by SDS-PAGE), pI of approx. 5.2, and optimally activity at 10.5 and 40°C. Using triolein as substrate, the lipase showed 1,3-positional specificity for hydrolyzing ester bonds. The enzyme was activated in 40% (v/v) dimethylsulfoxide and 20% (v/v) dimethylformamide. © Rapid Science Ltd. 1998     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two β-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50°C and 65°C for exo-glucanases and endo-glucanases but 35°C and 65°C for β-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40–50°C. Exo-II and Exo-III ( K _m, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I ( K _m, 50.00 mg/ml). The K _m values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities ( V _max, 12.0 and 10.0 IU/mg protein) were comparable. β-Glucosidases exhibited K _m values of 0.24 and 0.12 mmol and V _max values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29000; Exo-II, 72500; Exo-III, 138000; Endo-I, 25000; Endo-II, 32500; β-Gluco-I, 14000 and β-Gluco-II, 26000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas β-glucosidases did not. Hg²⁺ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two beta-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50 degrees C and 65 degrees C for exo-glucanases and endo-glucanases but 35 degrees C and 65 degrees C for beta-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40-50 degrees C. Exo-II and Exo-III (Km, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I (Km, 50.00 mg/ml). The Km values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities (Vmax, 12.0 and 10.0 IU/mg protein) were comparable. beta-Glucosidases exhibited Km values of 0.24 and 0.12 mmol and Vmax values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29,000; Exo-II, 72,500; Exo-III, 138,000; Endo-I, 25,000; Endo-II, 32,500; beta-Gluco-I, 14,000 and beta-Gluco-II, 26,000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas beta-glucosidases did not. Hg2+ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21,000 and 36,000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10-12 (protease I) and pH 10.5 (protease II). The optimum temperture for the activity of protease I was 70 degrees C and that for protease II was 60 degrees C. Protease I was stable in the range of pH 4.0-8.0 up to 60 degrees C and protease II was stable in the range of pH 6.0-12.0 up to 50 degrees C.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

T sujibo , H., M iyamoto , K., H asegawa , T. & I namori , Y. 1990. Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete. Journal of Applied Bacteriology 69 , 520–529.
Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21000 and 36000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10–12 (protease I) and pH 10.5 (protease II). The optimum temperature for the activity of protease I was 70°C and that for protease II was 60°C. Protease I was stable in the range of pH 4.0–8.0 up to 60°C and protease II was stable in the range of pH 6.0–12.0 up to 50°C.     

Brefeldin A,a cytotoxin produced by Paecilomyces sp. and Aspergillus clavatus isolated from Taxus mairei and Torreya grandis

Paecilomyces sp. and Aspergillus clavatus, which were isolated from Taxus mairei and Torreya grandis from southeast China, produced toxic metabolites when grown in liquid culture. Nuclear magnetic resonance techniques, infrared spectrometry, electrospray ionization mass spectroscopy and X-ray analysis identified brefeldin A, a bioactive metabolite produced by a number of fungal species belonging to the genera Alternaria, Ascochyta, Penicillium, Curvularia, Cercospora and Phyllosticta. This is the first report of the isolation of the cytotoxin from Paecilomyces sp. and A. clavatus. The relevance of brefeldin A to the association between these fungi and their host plants is discussed.     

Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.     

Purification and characterization of a prolyl endopeptidase isolated from Aspergillus oryzae

A new fungal strain that was isolated from our library was identified as an Aspergillus oryzae and noted to produce a novel proly endopeptidase. The enzyme was isolated, purified, and characterized. The molecular mass of the prolyl endopeptidase was estimated to be 60 kDa by using SDS-PAGE. Further biochemical characterization assays revealed that the enzyme attained optimal activity at pH 4.0 with acid pH stability from 3.0 to 5.0. Its optimum temperature was 30 °C and residual activity after 30 min incubation at 55 °C was higher than 80 %. The enzyme was activated and stabilized by Ca²⁺ but inhibited by EDTA (10 mM) and Cu²⁺. The K _m and k _cat values of the purified enzyme for different length substrates were also evaluated, and the results imply that the enzyme from A. oryzae possesses higher affinity for the larger substrates. Furthermore, this paper demonstrates for the first time that a prolyl endopeptidase purified from A. oryzae is able to hydrolyze intact casein.     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48 degrees C, pH optimum at pH 9 and a molecular weight of 87500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48°C, pH optimum at pH 9 and a molecular weight of 87 500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

Purification and characterization of acid-stable protopectinase produced by Aspergillus awamori in solid-state fermentation

Aspergillus awamori IFO 4033 produced an acid-stable protopectinase in solid-state fermentation using wheat bran as the medium. The enzyme was purified to a homogeneous preparation with anion-exchange, hydrophobic, and size-exclusion chromatography. The enzyme was a monomeric protein of 52 kDa, by SDS-PAGE analysis, with an isoelectric point of pH 3.7. The optimum pH for enzyme activity was 2.0, and it was most active at 50 degrees C (at pH 2.0) and was stable up to 50 degrees C (at pH 2.0). The enzyme showed pectin-releasing activity toward protopectins from various origins, especially on lemon protopectin. An outstanding characteristic of the enzyme was its extreme stability in acidic conditions: the enzyme activity was not lost after incubating at pH 2.0 and 37 degrees C for 24 h.     

Purification and characterization of glucoamylase produced by Aspergillus niger in solid state fermentation

Glucoamylases produced by Aspergillus niger grown on wheat bran in solid cultures were purified. Four different forms, GA I, GA I', GA II and GA III, were found having apparent molecular weights of 112 000, 104 000, 74 000 and 61 000 Da respectively. The enzymes are glycoproteins with a carbohydrate content of 16%, and optimal activity at 60C and pH 4.4. Activity was strongly inhibited by Hg²⁺ while Mn²⁺ and Fe²⁺ were stimulatory. The K_m values for the degradation of starch and maltose were 3.5 and 7.8 mg ml^-1, respectively.     

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 degrees C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine.HCl and 1% SDS) at 23 degrees C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.     

Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures

Polygalacturonases are pectinolytic enzymes that catalyze the hydrolysis of the plant cell-wall pectin backbone. They are widely used in the food industry for juice extraction and clarification. Aspergillus giganteus produces one polygalacturonase (PG) on liquid Vogel medium with citrus pectin as the only carbon source. In specific applications, such as those used in the food and medicine industries, the PG must be free of substances that could affect the characteristics of the product and the process, such as color, flavor, toxicity, and inhibitors. We present here an efficient, simple, and inexpensive method for purifying the A. giganteus PG and describe the characteristics of the purified enzyme. Purified PG was obtained after two simple steps: (1) protein precipitation with 70% ammonium sulfate saturation and (2) anion-exchange chromatography on a DEAE-Sephadex A-50 column. The final enzyme solution retained 86.4% of its initial PG activity. The purified PG had a molecular weight of 69.7 kDa, exhibited maximal activity at pH 6.0 and 55–60°C, and was stable in neutral and alkaline media. It had a half-life of 115, 18, and 6 min at 40, 50 and 55°C, respectively. Purified PG showed its highest hydrolytic activity with low-esterified and nonesterified substrates, releasing monogalacturonic acid from substrate, indicating that it is an exopolygalacturonase. PG activity was enhanced in the presence of β-mercaptoethanol, dithiothreitol, Co²⁺, Mn²⁺, Mg²⁺, NH₄ ⁺, and Na⁺ and was resistant to inhibition by Pb²⁺.     

Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.