Purification,characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.     

Purification and characterization of a high molecular weight endoxylanase from the solid-state culture of an alkali-tolerant Aspergillus fumigatus MKU1

Summary A high molecular weight endoxylanase (XylF2) from the solid state culture of Aspergillus fumigatus MKU1 was purified to homogeneity by a combination of tube gel electrophoresis and electroelution methods. The purity was demonstrated by SDS-PAGE and the molecular mass of the XylF2 was found to be 66 kDa. The optimal pH and temperature for activity were 5.0 and 90 °C, respectively. The apparent K _m and V _max values of XylF2 with oat spelt xylan as substrate were 1.6 mg/ml and 3.25 mmol/min/mg protein respectively. The enzyme showed high activity towards oat spelt xylan while negligible activity was observed on carboxymethylcellulose. The activity of XylF2 was strongly inhibited by Hg²⁺, Ni²⁺, Zn²⁺, SDS and N-bromosuccinimide and stimulated by l-cysteine and iodoacetamide. The hydrolysis of oat spelt xylan by XylF2 released only xylo-oligosaccharides.     

Purification and properties of glutaminase from Aspergillus oryzae

Glutaminase activity was found in a water extract of a wheat bran koji (extracellular fraction) of Aspergillus oryzae strains Lee-1, H-16 and MA-27-IM isolated from a commercial koji ssed for soy sauce fermentation, as well as in thier mycelia (intracellular fraction). Both the intracellular and the extracellular glutaminases were purified from strain MA-27-IM. Polyacrylamide gel electrophoresis of each purified preparation gave a single band with identical electrophoretic mobility. The molecular weight of the intracellular and the extracellular glutaminases were estimated to be approximately 113, 000. Both preparations hydrolyzed various γ-glutamyl compounds besides l-glutamine but did not exhibit asparaginase activity. Further investigation of these preparations inidicated that these glutaminases possessed almost the same properties, suggesting their similarity.     

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C₄-C₆ and C₃-C₈ chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.This revised version was published online in February 2005 with corrections to Table 1.     

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 rat liver glutaminase

Phosphate-dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400-fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein. For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o-phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion-exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half-life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h. The relative molecular mass of the protein (Mr 170,500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57,000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney.     

Purification and Characterization of the Antioxidative Substance Produced by Aspergillus sojae K

An antioxidative substance produced by Aspergillus sojae K is absolutely soluble in water and strongly inhibits autoxidation of Na-ascorbate. The substance, produced extracellularly in the culture ftuid by the mold, was purified by ion exchange chromatography, gel filtration, and HPLC. The substance was purified 34-fold with an activity recovery of 38% from culture fluid. Purity of the substance was confirmed with a single peak through HPLC using an analytical column as well as a single spot on TLC. The purified substance consists of equimolar aspartic acid and glycine, indicating that the substance is a peptide. From mass spectral analysis the molecular weight was 710, but the precise sequence of the amino acids is not clear. The substance is stable at 70°C, but about 80% of the activity was lost at 80°C after 60 min. Besides, the substance is completely stable at pH 3–14. This substance efficiently suppressed the oxidation of fish oil.     

Purification and characterization of a low molecular weight endoxylanase from solid-state cultures of alkali-tolerant Aspergillus fischeri

A low molecular weight, alkaline-stable endoxylanase (XylB) was purified to homogeneity from solid-state culture of Aspergillus fischeri Fxn1. XylB had a molecular mass of 13 kDa which is the lowest of reported xylanases. Optimal activity was at pH 6 and 55 degrees C. XylB was stable from pH 4.5 to 10 and up to 60 degrees C. It was non-glycosylated. The apparent K(m) and V(max) values of XylB on birch wood xylan were 0.53 mg ml(-1) and 0.2 mmol min-1 mg-1, respectively. The activity of XylB was not inhibited by Cd2+, Zn2+, Co2+, EDTA, iodoacetamide, beta-mercaptoethanol and acetic anhydride but strongly inhibited by 10 mm of N-bromosuccinimide, Hg2+, Pb2+ and p-hydroxymercuric benzoate. XylB is an endoxylanase since it hydrolysed xylan resulting the formation of xylo-oligomers but not of xylose residues.     

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae

 A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of l-glutamine but also of d-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137–143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5′-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae. Received: 23 August 1999 / Received last revision: 7 January 2000 / Accepted: 14 January 2000     

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

Purification and characterization of a ketimine-reducing enzyme

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.     

Purification and characterization of a dimethoate-degrading enzyme of Aspergillus niger ZHY256, isolated from sewage

A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50 degrees C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu(2+). The Michaelis constant (K(m)) and V(max) for dimethoate were 1.25 mM and 292 micromol min(-1) mg of protein(-1), respectively.     

Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

 A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates. Received: 24 October 1995/Received last revision: 27 March 1996/Accepted: 30 March 1996     

Gene cloning, purification, and characterization of a novel peptidoglutaminase-asparaginase from Aspergillus sojae

Glutaminase is an enzyme that catalyzes the hydrolysis of l-glutamine to l-glutamate, and it plays an important role in the production of fermented foods by enhancing the umami taste. By using the genome sequence and expressed sequence tag data available for Aspergillus oryzae RIB40, we cloned a novel glutaminase gene (AsgahA) from Aspergillus sojae, which was similar to a previously described gene encoding a salt-tolerant, thermostable glutaminase of Cryptococcus nodaensis (CnGahA). The structural gene was 1,929 bp in length without introns and encoded a glutaminase, AsGahA, which shared 36% identity with CnGahA. The introduction of multiple copies of AsgahA into A. oryzae RIB40 resulted in the overexpression of glutaminase activity. AsGahA was subsequently purified from the overexpressing transformant and characterized. While AsGahA was located at the cell surface in submerged culture, it was secreted extracellularly in solid-state culture. The molecular mass of AsGahA was estimated to be 67 kDa and 135 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahA was a dimer. The optimal pH of the enzyme was 9.5, and its optimal temperature was 50°C in sodium phosphate buffer (pH 7.0). Analysis of substrate specificity revealed that AsGahA deamidated not only free l-glutamine and l-asparagine but also C-terminal glutaminyl or asparaginyl residues in peptides. Collectively, our results indicate that AsGahA is a novel peptidoglutaminase-asparaginase. Moreover, this is the first report to describe the gene cloning and purification of a peptidoglutaminase-asparaginase.     

Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.     

Purification and characterization of shikimate kinase enzyme activity in Bacillus subtilis.

In Bacillus subtilis shikimate kinase enzyme activity can be demonstrated when a small polypeptide forms a trifunctional complex with the bifunctional enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase-chorismate mutase. The shikimate kinase polypeptide whoch carries the catalytic site has been purified to homogeneity by a five-step procedure. The skikimate kinase was determined to have a molecular weight of 10,000 by superfine Sephadex G-75 thin layer chromatography and by calculation of the minimum chemical molecular weight from its amino acid composition. This number corresponds closely to the molecular weight determined by the mobility of the protein following electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The enzyme aggregates with itself forming larger molecular weight proteins. Thes aggregational pattersn depend on protein concentration and sulfhydryl bridges. The enzyme activity is completely inhibited by EDTA and the requirement for Mg2+ can be partially replaced by Mn2+, Ca2+, and Co2+. The inhibition of shikimate kinase activity by p-hydroxymercuribenzoate is reversed completely when the enzyme complex is treated with dithiothreitol, suggesting the sulfhydryl groups may be involved with the active site. The trifunctional complex is relatively unstable, and the nonidentical subunits dissociate readily. This dissociation results in a 99% loss in shikimate kinase activity and a 30% decrease in the chorismate mutase-DAHP synthetase activities. Shikimate kinase activity is subject to a variety of controls. It is inhibited by the allosteric effectors chorismate and prephenate, the products of the reaction, ADP, and shikimate 5-phosphate. The activity responds to changes in the energy charge of the cell. Because of the variety of controls exerted on this enzyme, this member of the regulatory complex may represent the key enzyme in the allosteric control of the synthesis of the common precursors of aromatic acid synthesis.