Arthromyces ramosus peroxidase produces two chlorinating species

We previously reported that the hemes of horseradish peroxidase (HRP) and Arthromyces ramosus peroxidase (ARP) undergo vinyl and meso-carbon modifications when the enzymes oxidize chloride ion. Here we demonstrate for ARP that, although both modifications exhibit the same pH profile with an optimum at approximately pH 4.0, monochlorodimedone suppresses the vinyl but not meso-carbon modifications. Furthermore, meso-chlorination occurs when ARP reacts with exogenous HOCl, implicating an Fe(III)-O-Cl intermediate in the reaction. These results establish that (a) the chloro species involved in meso-modification differs from that which reacts with the vinyl groups, (b) equilibration of the vinyl modifying species (HOCl) into the medium occurs more rapidly than vinyl group modification, and (c) the oxidation of chloride by ARP produces two reactive species: HOCl, which adds to the heme vinyl but not meso-positions, and a distinct second species that adds to the meso-carbon.     

Enhanced production of peroxidase in continuous culture of Arthromyces ramosus by selective bleeding of mycelium

A new method of continuous culture with selective bleeding of mycelia using 9-mesh screen was developed to improve the production rate of peroxidase (POD) by Arthromyces ramosus. At the dilution rate of 0.05 h⁻¹ with the mycelium leakage rate of 60%, a high production rate (average value was 1.67 U·ml⁻¹·h⁻¹) was maintained for over 100 h: the rate was 3.2 times that in a glucose-fed batch culture. At the same dilution rate, the volumetric and specific production rates of POD in the continuous culture without the screen were lower than those in the first continuous culture and decreased gradually in the later phase of the culture. In the continuous culture with low mycelium leakage rate of 1.6%, the POD production rate was not improved further, although the mycelial concentration (43 g·l⁻¹) increased 2.9 times. It is suggested that the high agitation rate required to meet the oxygen demand is unfavorable for the POD production.     

Study and immunoanalytical applications for peroxidase from Arthromyces ramosus

Some biochemical and catalytic properties of peroxidase from Arthromyces ramosus (EC 1.11.7.1) in chemiluminescent reaction of luminol oxidation by hydrogen peroxide were investigated. The second order rate constants were determined by the stopped-flow technique. Optimal conditions to quantity the enzyme were found, the detection limit being 5.10(-13) M. The peroxidase was used as a marker in the human IgG immunoassay.     

Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP)

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment V_HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mg l⁻¹ fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V_HH R9 (R9-ARP) is the preferred orientation.     

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus

Peroxidase (donor: H₂O₂ oxi-doreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4₂2₁2, with unit cell dimensions of a = b = 74.5 Å, c = 117.6 Å. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 Å resolution and are stable to X-rays. © 1993 Wiley-Liss, Inc.     

Peroxidase production by carbon and nitrogen sources fed-batch culture of Arthromyces ramosus

Summary Carbon and nitrogen sources were investigated for improving peroxidase production by Arthromyces ramosus, a hyperproducer of peroxidase. Glucose as carbon source and a mixture of yeast extract and polypeptone at the ratio of 3 to 5 as nitrogen source in a production medium were shown to give the highest peroxidase activity. During the culture amino acids such as alanine, arginine, methionine, leucine, tyrosine and tryptophan were depleted. Therefore, glucose supplemented nitrogen source fed-batch culture was carried out and a peroxidase activity of 73 U/ml was obtained. This activity was 1.7 times higher than that of glucose fed-batch culture. This indicates that an adequate nitrogen source supply during the culture is effective for improving the peroxidase production by A. ramosus.     

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.     

Enhancement of acid amylase production by an isolated Aspergillus awamori

AIMS: Evaluation of the influence of fermentation components on extracellular acid amylase production by an isolated fungal strain Aspergillus awamori. METHODS AND RESULTS: Eight fungal metabolic influential factors, viz. soluble starch, corn steep liquor (CSL), casein, potassium dihydrogen phosphate (KH(2)PO(4)) and magnesium sulfate (MgSO(4) x 7H(2)O), pH, temperature and inoculum level were selected to optimize amylase production by A. awamori using fractional factorial design of Taguchi methodology. Significant improvement in acid amylase enzyme production (48%) was achieved. The optimized medium composition consisted of soluble starch--3%; CSL--0.5%; KH(2)PO(4)--0.125%; MgSO(4) x 7H(2)O--0.125%; casein--1.5% at pH 4.0 and temperature at 31 degrees C. CONCLUSION: Optimization of the components of the fermentation medium was carried out using fractional factorial design of Taguchi's L-18 orthogonal array. Based on the influence of interaction components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. Least significant factors of individual level have higher interaction severity index and vice versa at enzyme production in this fungal strain. The pH of the medium and substrate (soluble starch) showed maximum production impact (60%) at optimized environment. Temperature and CSL were the least influential factors for acid amylase production. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid amylase production by isolated A. awamori is influenced by the interaction of fermentation factors with fungal metabolism at individual and interaction levels. The pH of the fermentation medium and substrate concentration regulates maximum enzyme production process in this fungal strain.     

Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation

The production of exo-polygalacturonase (exo-PG) and endo-PG by Aspergillus awamori grown on wheat in solid-state fermentation was studied. Endo- and exo-PG activities were detected after 24 h of inoculation. Glucose released from starch hydrolysis acted as a catabolite repressor for the exo-PG enzyme. In contrast, endo-PG production was not affected by glucose repression. When milled grains were used, the particle-size distribution and the chemical composition of the medium influenced the rate of micro-organism growth and therefore the trend followed by endo- and exo-PG production. However, these two parameters did not affect the maximum production of exo-PG and endo-PG. For one of the milled samples, three different moisture contents were used (50, 55, 60%). Moisture contents of 60% provide a higher yield of pectinases by A. awamori.     

Redox properties of the Fe3+/Fe2+ couple in Arthromyces ramosus class II peroxidase and its cyanide adduct

The thermodynamics of the one-electron reduction of the ferric heme in free and cyanide-bound Arthromyces ramosus peroxidase (ARP), a class II plant peroxidase, were determined through spectro-electrochemical experiments. The data were compared with those for class III horseradish peroxidase C (HRP) and its cyanide adduct, and were interpreted in terms of ligand binding features, electrostatic effects and solvent accessible surface area of the heme group and of catalytically relevant residues in the heme distal site. The values for free and cyanide-bound ARP (−0.183 and −0.390 V, respectively, at 25 °C and pH 7) are higher than those for HRP and HRP-CN. ARP features an enthalpic stabilization of the ferrous state and a remarkably negative reduction entropy, which are both unprecedented for heme peroxidases. Once the compensatory contributions of solvent reorganization are partitioned from the measured reduction enthalpy, the resulting protein-based value for ARP turns out to be less positive than that for HRP by +10 kJ mol⁻¹. The smaller stabilization of the oxidized heme in ARP most probably results from the less pronounced anionic character of the proximal histidine, and the decreased polarity in the heme distal site as compared with HRP, as indicated by the X-ray structures. The surprisingly negative value for ARP is the result of peculiar reduction-induced solvent reorganization effects.     

Rheological changes of Aspergillus awamori broth during amyloglucosidase production

Two kinds of mathematical correlations are proposed, one between the biomass concentration and the rheological parameter consistency index (K) from the 'Power law' and another between either the specific growth rate or the specific glucoamylase production rate and K. Experimental data from Aspergillus awamori batch cultivations, with initial polysaccharide concentrations in the range of 20 to 180 g.L, were used to define these correlations.     

Direct binding of hydroxylamine to the heme iron of Arthromyces ramosus peroxidase. Substrate analogue that inhibits compound I formation in a competetive manner

The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H(2)O(2) in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mm at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56).     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation

The feasibility of using grape pomace for the production of xylanase and exo-polygalacturonase by Aspergillus awamori in solid state fermentation has been evaluated. Solid state fermentation experiments indicated that the particle size did not influence the enzyme production. The addition of extra carbon sources and the initial moisture content of the grape pomace were found to have a marked influence on the enzymes yields. Xylanase and exo-PG activities were high at 65% (w/w) initial moisture content and glucose supplementation.     

Ethanol production from starch by immobilized Aspergillus awamori and Saccharomyces pastorianus using cellulose carriers

A simultaneous saccharification and fermentation (SSF) process was investigated to produce ethanol using two kinds of cellulose carriers that were respectively suitable for immobilization of Aspergillus awamori and Saccharomyces pastorianus. The maximum ethanol concentration attained by the batch operation was 25.5 g l⁻¹. Under suitable conditions, both cellulose carriers with immobilized cells could be reused efficiently for three cycles. The total amount of ethanol production was 66.0 g (per 1 l working volume) after the repeated operation. Ethanol productivity mainly depends on a saccharification process. There is a limit in durability in the repeated batch operation, and it is important to maintain high activity of the fungus in order to produce ethanol efficiently. Journal of Industrial Microbiology & Biotechnology (2001) 27, 52–57. Received 11 December 2000/ Accepted in revised form 02 June 2001     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

We report the expression and production of llama variable heavy-chain antibody fragments (V_HHs) by Aspergillus awamori. Fragments encoding V_HHs were cloned in a suitable Aspergillus expression vector and transformants secreting V_HH fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V_HHs were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V_HHs were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.An erratum to this article can be found at     

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.