Phytase fromAspergillus niger

132 microorganisms, isolates from soil and decayed fruits, were tested for phytase production. All isolates intensively producing active extracellular phytase were of fungal origin. The most active fungal isolates with phytase activity were identified asAspergillus niger. At the end of the growth phase, the extracellular phytase activity produced byA. niger strain 92 was 132 nkat/mL, with strain 89 it was 53 nkat/mL. In both strains the extracellular enzyme activity exhibited two marked activity optima at pH 1.8 and 5.0 and a temperature optimum at 55°C.     

Production and properties of hemicellulases by a Thermomyces lanuginosus strain

Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications. Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source. Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch. Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs. No cellulase activity was observed. The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5. 5 and 9.5 were optimal for beta-xylanase activity. The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase. The pH optima of the other hemicellulases were between 5.0 and 6.5. Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable. These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.     

Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg-1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS-PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated Km is 6.3 microM and Vmax 9.8 nKat.     

Improved mannan-degrading enzymes' production by Aspergillus niger through medium optimization

The effect of different carbon and nitrogen sources on the production of mannan-degrading enzymes, focussing on β-mannanase, by Aspergillus niger was investigated using shake flask culture. The β-mannanase activity obtained during growth of A. niger on guar gum (GG, 1495 nkat mL(-1)) was much higher than those observed on other carbon substrates, locust bean gum (1148 nkat mL(-1)), α-cellulose (10.7 nkat mL(-1)), glucose (8.8 nkat mL(-1)) and carboxymethylcellulose (4.6 nkat mL(-1)). For fermentation using GG as a carbon source, bacteriological peptone gave the highest β-mannanase activity (1744 nkat mL(-1)) followed by peptone from meat (1168 nkat mL(-1)), yeast extract (817 nkat mL(-1)), ammonium sulphate (241 nkat mL(-1)), ammonium nitrate (113 nkat mL(-1)) and ammonium chloride (99 nkat mL(-1)) when used as a nitrogen source. The composition of bacteriological peptone and initial pH of the medium were further optimized using response surface methodology (RSM). Medium consisted of 21.3 g L(-1) GG and 57 g L(-1) peptone with initial culture pH of 5.5 was optimum for β-mannanase production (2063 nkat mL(-1)) by A. niger. The β-mannanase production obtained in this study using A. niger was significantly higher than those reported in the literature.     

Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity

The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K(M)) towards β-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer β-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K(M) for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K(M) for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-β-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.     

Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes.

The beta-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor alpha1 signal peptide (MFalpha1(s)) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant beta-xylosidase showed optimum activity at 60 degrees C and pH 3.2 and optimum stability at 50 degrees C. The K(i(app)) value for D-xylose and xylobiose for the recombinant beta-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The XLO2 fusion gene and the XYN2 beta-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of beta-xylanase activity when expressing only the beta-xylanase and 860 nkat/ml when coexpressing the beta-xylanase with the beta-xylosidase. The maximum beta-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the beta-xylanase. Coproduction of the beta-xylanase and beta-xylosidase enabled S. cerevisiae to degrade birchwood xylan to D-xylose.     

黑曲霉菊糖酶的纯化及性质

黑曲霉(Aspergillus niger)319发酵液经硫酸铵分级沉淀、DEAE-纤维素52离子交换层析和Sephadex G-100分子筛层析,得到了电泳纯的菊糖酶组分。提纯倍数为67,收率为25.5％。菊糖酶的最适pH为5.0,最适温度为60℃。此酶为单亚基蛋白,凝胶过滤法测得分子量为28000,含糖13.9％,用等电聚焦法测得等电点为5.4,该酶对温度有较高的稳定性,对pH的稳定范围较窄。Hg²⁺、Pb²⁺和Cu²⁺对该酶有强烈的抑制作用。此酶对菊糖有较强的底物专一性,产物为果糖,但它也可作用于蔗糖,I/S值为0.348。当以菊糖为底物时,K_m为6.25mmol/L,V_m为67.11 μmol·mg~(-1)·min~(-1)。     

Production of phytase (myo-inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter

The growth and production pattern of phytase by a filamentous fungus, Aspergillus niger van Teighem, were studied in submerged culture at varying agitation rates and controlled and uncontrolled pH conditions. Allowing the initial culture to grow under neutral condition with subsequent decline in pH resulted in increased phytase productivity. A maximum of 141 nkat/mL phytase was obtained when the broth pH was maintained at pH 2.5 as compared to 17 nkat/mL units at controlled pH 5.5. The culture morphology and rheological properties of the fermentation broth significantly varied with the agitation rate. The volumetric oxygen transfer coefficient was determined at different phases of fungal growth during batch fermentation using static gassing out and dynamic gassing out methods. The oxygen transfer coefficient (k(L)a) of the fermenter was found to be 125 h(-)(1) at 500 rpm as compared to 38 h(-)(1) at 200 rpm. The oxygen transfer rates at different phases of growth were significantly affected by cell mass concentration and fungal morphology. During the course of fermentation there was a gradual decline of k(L)a from 97 h(-)(1) on day 2 to 63 h(-)(1) on day 6 of fermentation, after which no significant change was observed. The degree of agitation considerably influenced the culture morphology where shear thinning of filamentous fungus was observed with the increase in agitation.     

Heterologous Expression and Optimized Production of an <Emphasis Type="Italic">Aspergillus aculeatus</Emphasis> Endo-1,4-β-mannanase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The Aspergillus aculeatus MRC11624 man1 gene, encoding an endo-β-1,4-mannanase, was cloned and expressed in the promising heterologous enzyme producer, the ascomycetous yeast Yarrowia lipolytica. Both single- and multi-copy transformants were constructed, and the secretion of the enzyme was evaluated as an in-frame fusion with the LIP2 secretion signal, as well as with its natural secretion signal. In shake-flask analysis, the highest volumetric enzyme activity (13,073 nkat/ml) and specific enzyme activity (1,020 nkat/(mg dcw)) were obtained with a multi-copy integrant utilizing β-mannanase’s own secretion signal. The best β-mannanase-producing strain was subsequently evaluated in batch fermentation and resulted in a maximum volumetric enzyme activity of 6,719 nkat/ml. Fed batch fermentations resulted in a 3.9-fold increase in volumetric enzyme activity compared with batch fermentation, and a maximum titre of 26,139 nkat/ml was obtained. The results reported in this study indicate that Y. lipolytica is a promising producer of A. aculeatus β-mannanase, producing higher β-mannanase activity than that of recombinant Saccharomyces cerevisiae or Aspergillus niger when cultivated in shake flasks, which is encouraging for the use of the enzyme in industrial processes such as extraction of vegetable oil from leguminous seeds and the reduction in viscosity of coffee extracts.     

Partial purification and characterization of extracellular cellulase from a strain ofTrichoderma viride isolated from forest soil

Partial purification of extracellular cellulase ofTrichoderma viride isolated from forest soil was done by ammonium sulfate precipitation of culture supernatant, centrifugation at higher speed, solubilization of protein in sodium acetate buffer and dialysis. The specific activity of cellulase in the culture supernatant, was 136 nkat/mg which was increased by 172% after the completion of final step (234 nkat/mg). The recovery of enzyme was 70%. The enzyme was characterized by demonstration of optimum activity at 55°C and pH 5.0 with 1% carboxymethyl cellulose as substrate.     

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C.

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3-7). Apparent Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mukat.mg-1, 36.5 mukat.mg-1 and 415 nkat.mg-1 for endopolygalacturonases I, II and C, respectively. K(m) values were < 0.15 mg.mL-1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes. Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated delta 4,5-unsaturated oligogalacturonates of degree of polymerization 4-8. Whereas endopolygalacturonase I showed a strong preference for generating the delta 4,5-unsaturated dimer, with endopolygalacturonase II the delta 4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the delta 4,5-unsaturated dimer and trimer were observed, although in different ratios.     

Purification and characterization of acetohydroxyacid reductoisomerase from spinach chloroplasts.

Acetohydroxyacid reductoisomerase was purified over 400-fold to a specific activity of 62 nkat.mg-1, with 2-aceto-2-hydroxybutyrate as substrate, from the stroma of spinach leaf chloroplasts. The enzyme was not intrinsically membrane bound. The native enzyme was a tetramer with a subunit Mr of 59,000. The activity was optimum between pH 7.5 and 8.5. The apparent Km for 2-acetolactate was 25 microM and for 2-aceto-2-hydroxybutyrate was 37 microM. The enzyme required Mg2+ and the Vmax. was attained at physiological Mg2+ concentrations. NADP+ competitively inhibited the reaction when NADPH was the varied substrate. The native enzyme eluted from Mono-Q ion-exchange resins as three distinct peaks of activity. This elution pattern was preserved when the peaks were combined, dialysed and re-chromatographed. Each form exhibited identical Mr of 59,000 after SDS/polyacrylamide gel electrophoresis (PAGE), whereas they were easily distinguishable from each other after PAGE under non-denaturing conditions. These results provide evidence for the existence of multiple forms of acetohydroxyacid reductoisomerase in chloroplasts isolated from spinach leaves.     

Influence of pH on the formation and location of β-glucosidase inAspergillus terreus grown on cellulose

Wild strainof Aspergillus terreus is very good producer of /gb-glucosidase. It produces 15 nkat/mL (0.9 IU/mL) of extracellular β-glucosidase at pH 5.0. The medium pH level strongly affects the production and binding of β-glucosidase on the cells and on residual cellulose. At pH 4.0 the rate of enzyme synthesis and the level of total activity is highest but 60—75 % of this activity is bound. At higher pH levels the enzyme is mainly released to the medium.     

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.     

Characterization of beta-galactosidase from a special strain of Aspergillus oryzae.

beta-Galactosidase [EC 3.2.1.23] was isolated from a partially purified preparation obtained from cultured cells of a special strain of Aspergillus oryzae, RT 102 (FERM-P1680). The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and was free from alpha-galactosidase, alpha- and beta-mannosidase, alpha- and beta-N-acetylhexosaminidase, and protease activities. The beta-galactosidase was capable of acting on aryl beta-galactosides, lactose, and lactosides. It also hydrolyzed beta-galactosyl linkages in urinary glycoasparagines and asialo alpha1-acid glycoprotein. The enzyme was rather stable in aqueous solution, retaining full activity at 4 degrees for at least several months. At pH 4.5, the optimum pH for the enzyme activity, and 37 degrees, full activity was maintained for several days.     

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

麻疯树叶中过氧化物酶JCP-1的分离纯化和几种特性的检测

麻疯树叶片蛋白粗提液经硫酸铵分级沉淀,强阴离子琼脂糖、强阳离子琼脂糖和交联葡聚糖层析,得到一个比活为4499U·mg-1（蛋白）过氧化物酶,命名为JCP-1。其分子量为49kDa,等电点为pH3.3,最适pH为5.0-6.0。以H2O2为底物的Km为2.14mmol·L-1。JCP-1具有宽泛的最适保存pH（7.0-11.0）和较高的耐热性（80℃高温处理15min,活性保持在90%以上）。30%PEG6000处理模拟干旱胁迫及50℃高温胁迫麻疯树苗,其叶片中JCP-1活性分别提高121%和155%。     

Production of high level of cellulase-free and thermostable xylanase by a wild strain of Thermomyces lanuginosus using beechwood xylan

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was studied for production of high level of cellulase-free thermostable xylanase at 50°C using xylan. Optimization of the medium composition was carried out on shake-flask level using Graeco-Latin square technique. This increased xylanase production from 527 nkat ml⁻¹ in the original medium to 9168–9502 nkat ml⁻¹ in the optimized medium under optimized culture conditions e.g. initial medium pH (6.0–6.5), culture temperature (50°C) and time (5–6 d). The lag phase was very much shorter in the laboratory reactor compared to which existed in the shake cultures and 7111 nkat of xylanase activity were obtained per ml of culture filtrate at 60 h of cultivation. With a 15 min reaction time, the optimal pH and temperature for the xylanase activity were at 6.5 and 65°C, respectively. The enzyme was almost stable over a broad range of pH 3–9 at 20°C, with an optimum stability at pH 6.5. After 51 h heating at 50°C the enzyme retained 60%, 100% and 90% activity at pH 5.0, 6.5 and 8.0, respectively. The crude enzyme could hydrolyse xylan effectively and in only 6 h 67.3%, 54.0% and 49.2% saccharifications were achieved for 2%, 5% and 10% substrate levels, respectively. The principal product of hydrolysis was xylobiose together with smaller amounts of xylooligosaccharides (degree of polymerization 3–7) and xylose.