Inducive effect of cresoquinone on microbiological transformation of L-tyrosine to 3,4 dihydroxy phenyl L-alanine by Aspergillus oryzae NG-11(P1)

The present work describes the inducive effect of cresoquinone on microbiological transformation of L-tyrosine to 3,4 dihydroxy phenyl L-alanine ( L-DOPA) by Aspergillus oryzae NG-11(P1). Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinases, beta-carboxylases and tyrosine hydroxylases are intracellular enzymes. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved after 60 min of biochemical reaction. To enhance the production of L-DOPA, cresoquinone was added to the reaction mixture. Best L-DOPA biosynthesis results were observed when the concentration of cresoquinone was 3.5 x 10(-6) M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). Cresoquinone not only increased enzyme activity but also enhanced cell membrane permeability to facilitate secretion of enzymes into the reaction broth. Comparison of kinetic parameters revealed the ability of the mutant to yield L-DOPA [Y(p/x) [i.e., mg L-DOPA formed (mg cells formed)(-1)] =7.360+/-0.04]. When the culture grown on various cresoquinone levels was monitored for Q(p), Q(s) and q(p) [ Q(p): mg L-DOPA produced ml(-1) x h(-1); Q(s): mg substrate consumed ml(-1) x h(-1); q(p): mg L-DOPA formed (mg cells)(-1) h(-1)], there was significant enhancement ( P<0.025) of these variables.     

Fermentation Kinetics of Media Optimization for the Production of Alpha Amylase by a New Isolate of Aspergillus Oryzae

The starch-degrading enzyme‘α-amylase’is widelydistributedin nature .This extracellular enzyme randomlyhydrolyzesα1~4 glucosidic linkage throughout the starchmolecule in an endo-fashion producing oligosaccharidesand monosaccharides including maltose ,…     

Stimulatory effect of alcohols (methanol and ethanol) on citric acid productivity by a 2-deoxy D-glucose resistant culture of aspergillus niger GCB-47

The present study describes citric acid fermentation by Aspergillus niger GCB-47 in a 15-1 stainless steel stirred fermentor. Among the alcohols tested as stimulating agents, 1.0% (v/v) methanol was found to give maximum amount of anhydrous citric acid (90.02 +/- 2.2 g/l), 24 h after inoculation. This yield of citric acid was 1.96 fold higher than the control. Methanol has a direct effect on mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from the mycelial cells. The sugar consumed and % citric acid was 108 +/- 3.8 g/l and 80.39 +/- 4.5%, respectively. The desirable mycelial morphology was in the form of small round pellets having dry cell mass 14.5 +/- 0.8 g/l. Addition of ethanol, however, did not found to enhance citric acid production, significantly. The maximum value of Yp/x (i.e., 5.825 +/- 0.25 g/g) was observed when methanol was used as a stimulating agent. The best results of anhydrous citric acid were observed, 6 days after inoculation when the initial pH of fermentation medium was kept at 6.0.     

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 +/- 5 g/l).     

Innovative Effect of Illite on Improved Microbiological Conversion of L-Tyrosine to 3,4 Dihydroxy Phenyl L-Alanine (L-DOPA) by Aspergillus oryzae ME2 Under Acidic Reaction Conditions

In the present investigation, the previous ultraviolet irradiated mutant strain of Aspergillus oryzae UV-7 was further improved in terms of 3,4 dihydroxy phenyl L-alanine (L-DOPA) activity after chemical mutagenesis through 1-methyl 3-nitro 1-nitroso guanidine (MNNG = 250–1500 μg/ml) treatment (0–30 min). Among several mutant variants, the one that produced a larger amount of L-DOPA from L-tyrosine was designated to as ME₂ and it was made 2-deoxy-D-glucose-resistant by growing it at various concentrations of 2 dg (0.01–0.025 %, w/v) in Vogel’s agar medium. Relatively better production of L-DOPA (> 0.60 mg/ml) was obtained when 2.0% (w/v) glucose was used as a carbon source in the mycelium production medium and the tyrosinase activity increased constitutively (1.08 mg/ml), which resulted in a greater production of L-DOPA. At optimum pH₀ (pH 6.0) and reaction time (60 min), more than 65% sugar was utilized for cell mass formation. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved 60 min after the biochemical reaction. Mould mycelium was used for microbiological conversion of L-tyrosine to L-DOPA because tyrosinases, β-carboxylases, and tyrosine hydroxylases are intracellular enzymes. The effect of illite (1.0 × 10⁶–6.0 × 10⁶ M) on biochemical conversion of L-tyrosine to L-DOPA by Aspergillus oryzae ME₂ was also carried out. Best results of L-DOPA biosynthesis were observed when the concentration of illite was 3.5 × 10⁻⁶ M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). It was noted that the addition of illite not only increased enzyme activity but also enhanced the permeability of cell membrane to facilitate the secretion of enzymes into the reaction broth. The comparison of kinetic parameters showed the ability of mutant to yield L-DOPA (i.e., Y_p/x 7.360 ± 0.04 mg/mg). When the culture grown on various illite concentrations was monitored for Q_p, Q_s, and q_p, there was significant enhancement (p < 0.025) in these variables over the control, which indicate that the study can be commercially applicable on stirred and magnetic rotary drums. Overall, there was up to 3-fold (Q_p = 0.290 mg/L-DOPA produced/ml/h) enhancement in the product formation rate, which is highly encouraging (HS, LSD 0.456).     

莱氏野村菌紫外线诱变抗敌敌畏菌株的生理特性

[背景]夜蛾科害虫易对化学杀虫剂产生高抗性,但一些化学农药可以对部分虫生真菌的毒力作用效果起增幅作用,目前缺乏对莱氏野村菌(Nomuraea rileyi)的该方面研究.[目的]探究对常用有机磷杀虫剂敌敌畏具有较强耐药性的紫外线诱变莱氏野村菌突变菌株的生理特性,包括菌丝生长、产孢情况和产几丁质酶活性.[方法]在紫外线诱...     

Enhancement of L+-lactic acid production using mycelial flocs of Rhizopus oryzae

L(+)-Lactic acid production was enhanced in the culture of Rhizopus oryzae using mycelial flocs formed by addition of 3 g/L mineral support and 5 ppm polyethylene oxide. By addition of the mineral support, an electrostatic repulsion between mycelia increased by 3.5-fold compared to that of mycelia, which allowed a dispersed growth of R. oryzae in the early growth phase. In conventional culture the morphology of R. oryzae is that of a pellet-like cake, however, when support and polyethylene oxide are added to the culture, the morphology of R. oryzae takes on a cotton-like appearance. The formation of these cotton-like mycelial flocs was induced by the addition of 5 ppm polyethylene oxide into a 14 h culture containing the mineral support before the formation of the conventional pellet morphology. The cotton-like flocs were also formed in cultures grown in a fermentor. This morphology allowed effective mass transfer inside the flocs and effective fluidity of culture broth in the reactor. L(+)-Lactic acid concentration produced by mycelial flocs in fermentor, with the support and polyethylene oxide, was 103.6 g/L with the yield of 0.86 using 120 g/L of glucose as the substrate for this cultures without both, the concentration was 65.2 g/L. It demonstrates that cotton-like mycelial flocs are the optimal morphology in the culture of R. oryzae. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 461-470, 1997.     

Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis of Streptomyces fradiae NRRL-2702

Tylosin is a macrolide antibiotic used as veterinary drug and growth promoter. Attempts were made for hyper production of tylosin by a strain of Streptomyces fradiae NRRL-2702 through irradiation mutagenesis. Ultraviolet (UV) irradiation of wild-type strain caused development of six morphologically altered colony types on agar plates. After screening using Bacillus subtilis bioassay only morphological mutants indicated the production of tylosin. An increase of 2.7±0.22-fold in tylosin production (1500 mg/l) in case of mutant UV-2 in complex medium was achieved as compared to wild-type strain (550 mg/l). Gamma irradiation of mutant UV-2 using ⁶⁰Co gave one morphologically altered colony type γ-1, which gave 2500 mg/l tylosin yield in complex medium. Chemically defined media promoted tylosin production upto 3800 mg/l. Maximum value of q_p (3.34 mg/gh) was observed by mutant γ-1 as compared to wild strain (0.81 mg/gh). Moreover, UV irradiation associated changes were unstable with loss of tylosin activity whereas mutant γ-1 displayed high stability on subsequent culturing.     

Dimorphism inBenjaminiella poitrasii: Involvement of intracellular endochitinase andN-acetylglucosaminidase activities in the yeast-mycelium transition

The chitinase and N-acetylglucosaminidase activities in cell-wall-bound and free fractions in the dimorphic fungus Benjaminiella poitrasii were studied as a function of morphological (unicellular yeast-mycelium) transition. The specific activities of chitinases of cell-wall-free, particularly in the membrane fraction, were significantly different in the yeast and mycelial forms. During the yeast-mycelium transition, the N-acetylglucosaminidase activity isolated in a membrane preparation increased steadily. The activity of the yeast cells (0.83 +/- 0.17 nkat/mg protein) increased 17-fold to 14.2 +/- 1.7 nkat/mg protein in 1-d-old mycelial cells. The endochitinase activity increased 12-fold between 6 and 12 h and thereafter practically remained unchanged up to 24 h. A reverse trend in the chitinolytic activities was observed during the mycelium-yeast transition. Isoelectrofocussing (pH range 3.5-10) of mixed membrane fraction free of particulate fraction of parent and morphological (Y-5, yeast-form) mutant cells separated endochitinase and N-acetylglucosaminidase activity into two pH ranges, viz. 4.3-5.7 and 6.1-7.7, respectively. The predominant N-acetylglucosaminidase activity observed at pH 6.9 and 7.1 for the parent strain membrane fraction was undetected in the mutant preparation. The results suggested that the membrane-bound (either tightly or loosely) chitinolytic enzymes, particularly, N-acetylglucosaminidase, significantly contributed to the morphological changes in B. poitrasii.     

Dilution rate as a determinant of mycelial morphology in continuous culture

The morphology of mycelial fungi in liquid culture effects culture rheology and this in turn may affect product yield. It is therefore important to understand how environmental factors influence mycelial morphology and this paper describes the effect of dilution rate on two strains of Fusarium graminearum, the relatively sparsely branched parental strain (A3/5) and a relatively highly branched "colonial" variant (C106). At any given dilution rate, the concentration of mycelial fragments present at steady state of both strains remained approximately constant with time, suggesting that mycelial fragmentation occurred in a regular manner. However, for both strains fragment concentration decreased with increasing dilution rate. The strains had a similar morphology at a dilution rate of 0.07 h(-1). The length of the hyphal growth unit of A3/5 increased with increase in dilution rate, while that of C106 decreased with increase in dilution rate. At all dilution rates, C106 produced up to ten times more macroconidia than A3/5.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.     

Treatment of germinated wheat to increase levels of GABA and IP6 catalyzed by endogenous enzymes

We found that the levels of bioactive products from wheat can be increased dramatically by manipulating germination conditions and taking advantage of the activity of endogenous enzymes. The yield of phytic acid (IP(6)) from wheat germinated in the presence of high, controlled levels of dissolved oxygen (188 +/- 28 mg/100 g wheat) was almost three times greater than that from wheat germinated with no supplemental oxygen (74 +/- 10 mg/100 g wheat). The yield of gamma-aminobutyric acid (GABA) from wheat germinated in the presence of uncontrolled levels of dissolved oxygen was 18 +/- 3 times greater than that from nonsupplemented wheat (1 mg/100 g wheat). The concentration of GABA was much greater in wheat germ than in whole wheat, and the yield of GABA from wheat germ processed with supplemental water (163 +/- 7 mg/100 g wheat germ) was notably greater than that from wheat germ processed with no supplemental water (100 +/- 2 mg/100 g wheat germ). In contrast, IP(6) was more concentrated in wheat bran, and the yield of IP(6) from wheat bran processed with supplemental water (3100 +/- 12 mg/100 g wheat bran) was notably higher than that from wheat bran processed with no supplemental water (2420 +/- 13 mg/100 g wheat bran). We conclude that the large amount of GABA extracted from wheat germ is likely due to high glutamate decarboxylase activity and low aminotransferase activity and that the large amount of IP(6) extracted from wheat bran is likely due to high levels of tyrosinase activity. Our findings indicate that bioactive molecules such as GABA and IP(6) can be successfully mass-produced by taking advantage of endogenous enzymatic activities.     

Hydroxy- or methoxy-substituted benzaldoximes and benzaldehyde-O-alkyloximes as tyrosinase inhibitors.

Several benzaldoximes, benzaldehyde-O-ethyloximes, and acetophenonoximes were synthesized and evaluated as tyrosinase inhibitors by an assay based on tyrosinase catalyzed L-DOPA oxidation. Whereas benzaldoxime itself is only a weak inhibitor, its derivatives with one or two hydroxy or methoxy moieties in para and meta positions depress tyrosinase activity. Acetophenonoximes and trisubstituted benzaldoximes show no inhibitory activity. The IC(50) of 3,4-dihydroxybenzaldehyde-O-ethyloxime (0.3 +/- 0.1 micromol L(-1)) is of the same magnitude as tropolone (0.13 +/- 0.08 micromol L(-1)), one of the best tyrosinase inhibitors known so far.     

Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.     

Magnaporthe oryzae endopolygalacturonase homolog correlates with density-dependent conidial germination.

Endopolygalacturonases (endoPGs) of some phytopathogens are virulent factors for dicots. To investigate the function of the endoPG of Magnaporthe oryzae, a disruption mutant of MGG_08938, the homolog of endoPG found in the genome database of this fungus, was generated. The pathogenicity, mycelial growth, and appressorium formation of this mutant were comparable with those of the wild-type strain; however, the germination of conidia in a highly concentrated suspension of conidia was affected by the mutation. Whereas the germination of the wild-type strain was inhibited at high concentrations, this effect was canceled out by disruption by the endoPG homolog gene. The authors named the gene MDG1 (M. oryzae density-dependent germination), which delineates this new function in the fungus.     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

Fed-batch culture of a metabolically engineered Escherichia coli strain designed for high-level succinate production and yield under aerobic conditions

An aerobic succinate production system developed by Lin et al. (Metab Eng, in press) is capable of achieving the maximum theoretical succinate yield of 1.0 mol/mol glucose for aerobic conditions. It also exhibits high succinate productivity. This succinate production system is a mutant E. coli strain with five pathways inactivated: DeltasdhAB, Delta(ackA-pta), DeltapoxB, DeltaiclR, and DeltaptsG. The mutant strain also overexpresses Sorghum vulgare pepc. This mutant strain is designated HL27659k(pKK313). Fed-batch reactor experiments were performed for the strain HL27659k(pKK313) under aerobic conditions to determine and demonstrate its capacity for high-level succinate production. Results showed that it could produce 58.3 g/l of succinate in 59 h under complete aerobic conditions. Throughout the entire fermentation the average succinate yield was 0.94+/-0.07 mol/mol glucose, the average productivity was 1.08+/-0.06 g/l-h, and the average specific productivity was 89.77+/-3.40 mg/g-h. Strain HL27659k (pKK313) is, thus, capable of large-scale succinate production under aerobic conditions. The results also showed that the aerobic succinate production system using the designed strain HL27659k(pKK313) is more practical than conventional anaerobic succinate production systems. It has remarkable potential for industrial-scale succinate production and process optimization.     

Inactivation of the Escherichia coli K-12 twin-arginine translocation system promotes increased hydrogen production

The effect of deleting the genes encoding the twin-arginine translocation (Tat) system on H2 production by Escherichia coli strain MC4100 and its formate hydrogenlyase upregulated mutant (DeltahycA) was investigated. H2 evolution tests using two mutant strains defective in Tat transport (DeltatatC and DeltatatA-E) showed that the rate doubled from 0.88+/-0.28 mL H2 mg dry weight-1 L culture-1 in the parental strain, to 1.70+/-0.15 and 1.75+/-0.18 mL H2 mg dry weight-1 L culture-1, respectively, in the DeltatatC and DeltatatA-E strains. This increase was comparable to that of a previously characterized hydrogen over-producing E. coli strain carrying a DeltahycA allele. Construction of a tatC, DeltahycA double deletion strain did not increase hydrogen production further. Inactivation of the Tat system prevents correct assembly of the uptake hydrogenases and formate dehydrogenases in the cytoplasmic membrane and it is postulated that the subsequent loss of basal levels of respiratory-linked hydrogen and formate oxidation accounts for the observed increases in formate-dependent hydrogen evolution.