Purification and characterization of Aspergillus ficuum endoinulinase

Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.     

Separation and identification of exo- and endoinulinase from Aspergillus ficuum

A new and convenient method was developed to separate and identify exo- and endoinulinase from Aspergillus ficuum by native polyacrylamide gel electrophoresis. Eight protein bands were obtained. Three bands were identified as exoinulinase, and two bands were endoinulinase, by using TLC and HPLC. The five bands were all detected as glycoproteins with the sensitive periodic acid-silver stain.     

Purification and characterization of xylanase from Aspergillus ficuum AF-98

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH₄)₂SO₄, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7 U/ mg protein) was a monomeric protein with a molecular mass of 35.0 kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45 °C and 5.0, respectively. The xylanase was activated by Cu²⁺ up to 115.8% of activity, and was strongly inhibited by Hg²⁺, Pb²⁺ up to 52.8% and 89%, respectively. The xylanase exhibited K_m and V_max values of 3.267 mg/mL, 18.38 M/min/mg for beechwood xylan and 3.747 mg/mL, 11.1 M/min/mg for birchwood xylan, respectively.     

Immobilization of Aspergillus ficuum phytase: product characterization of the bioreactor

Aspergillus ficuum phytase was covalently immobilized on Fractogel TSK HW-75 containing 2-oxy-l-alkylpyridinium salts. A packed-bed bioreactor was constructed with the immobilized phytase. An HPLC ion-exchange method was used to analyze the enzymatic products of the bioreactor. Immobilized fungal phytase was able to hydrolyze myo-inositol Hexa-, penta-, tetra-, tri-, and diphosphates. When the substrate solution was recirculated for 5 hr in the bioreactor about 50% inorganic orthophosphate was released and myo-inositol-diphosphate and mono-phosphate were the only remaining products.     

Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。     

Production and Partial Purification of Tannase from Aspergillus ficuum Gim 3.6

A novel fungal strain, Aspergillus ficuum Gim 3.6, was evaluated for its tannase-producing capability in a wheat bran-based solid-state fermentation. Thin-layer chromatography (TLC) analysis revealed that the strain was able to degrade tannic acid to gallic acid and pyrogallol during the fermentation process. Quantitation of enzyme activity demonstrated that this strain was capable of producing a relatively high yield of extracellular tannase. Single-factor optimization of process parameters resulted in high yield of tannase after 60 hr of incubation at a pH of 5.0 at 30°C, 1 mL of inoculum size, and 1:1 solid–liquid ratio in the presence of 2.0% (w/v) tannic acid as inducer. The potential of aqueous two-phase extraction (ATPE) for the purification of tannase was investigated. Influence of various parameters such as phase-forming salt, molecular weight of polyethylene glycol (PEG), pH, and stability ratio on tannase partition and purification was studied. In all the systems, the target enzyme was observed to preferentially partition to the PEG-rich top phase, and the best result of purification (2.74-fold) with an enzyme activity recovery of 77.17% was obtained in the system containing 17% (w/w) sodium citrate and 18.18% (w/w) PEG1000, at pH 7.0.     

无花果曲霉原生质体形成与再生条件的探讨

根据正交试验得出无花果曲霉原生质体形成的最佳条件,用１％的混合酶液（０．５％纤维素酶＋０．２５％蜗牛酶＋０．２５％溶菌酶）作用无花果曲霉菌体细胞,原生质体产量达３．２×１０７个·ｍｌ－１,渗透压稳定剂为０．６ｍｏｌ·Ｌ－１ＫＣｌ于０．２ｍｏｌ·Ｌ－１ＰＯ３＋４（ｐＨ５．８）中,酶解时间和酶解温度分别为３．０ｈ、３０℃．比较不同酶解时间、再生稳定剂和碳源等因素对原生质体再生的影响,可确定最佳再生条件,再生率达３０％以上．     

Supramolecular Organization of Inulinases from Aspergillus awamori,Aspergillus ficuum and Kluyveromyces marxianus: A Comparative Aspect

Biophysics - Computer models for the dimers of inulinases from Aspergillus awamori, Aspergillus ficuum and Kluyveromyces marxianus have been developed. The inulinases dimerization mechanisms from...     

PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases

PhyA gene products of Aspergillus ficuum (AF) and Peniophora lycii (PL) as expressed in industrial strains of Aspergillus niger and Aspergillus oryzae, respectively, were purified to homogeneity and then characterized for both physical and biochemical properties. The PL phytase is 26 amino acid residues shorter than the AF phytase. Dynamic light scattering studies indicate that the active AF phytase is a monomer while the PL phytase is a dimer. While both of the phytases retained four identical glycosylatable Asn residues, unique glycosylation sites, six for PL and seven for AF phytase, were observed. Global alignment of both the phytases has shown 38% sequence homology between the two proteins. At 58 degrees C and pH 5.0, the PL phytase gave a specific activity of 22,000 nKat/mg as opposed to about 3000 nKat/mg for AF phytase. However, the AF phytase is more thermostable than its counterpart PL phytase at 65 degrees C. Also, AF phytase is more stable at pH 7.5 than the PL phytase. The two phytases differed in K(m) for phytate, K(i) for myo-inositol hexasulfate (MIHS), and pH optima profile. Despite similarities in the active site sequences, the two phytases show remarkable differences in turnover number, pH optima profile, stability at higher temperature, and alkaline pH. These biochemical differences indicate that phytases from ascomycete and basidiomycete fungi may have evolved to degrade phytate in different environments.     

Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum

Two types of extracellular acid phosphatases are synthesized by Aspergillus ficuum NRRL 3135: a nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with an optimum pH of 2.0, and an enzyme with restricted specificity, a mesoinositol-hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with an optimum pH of 5.5. Although the pH 5.5 enzyme is termed a phytase, both enzymes hydrolyze phytin. Synthesis of the enzymes is repressed by high orthophosphate concentrations in the fermentation medium. The highest total level for each enzyme is synthesized in low orthophosphate medium. In high orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme. In low orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme during the early stages of growth, but the reverse occurs after 5 days. The enzymes are differentiated by heat denaturation at acid and alkaline pH levels. They are separated into two distinct fractions on Sephadex G-100 followed by carboxymethylcellulose column chromatography. This indicates that the two enzymes are structurally different. The K(m) for both enzymes is 1.25 mm when calcium phytate is the substrate. Orthophosphate competitively inhibits the pH 2.0 (K(i) = 1.1 x 10(-2)m) but not the pH 5.5 phosphatase. Neither enzyme is denatured by 50% (w/v) urea or inhibited by 0.01 m tartrate. Thus, they differ from human prostatic phosphatase.     

Cyclohexanedione modification of arginine at the active site of Aspergillus ficuum phytase.

Reaction of Aspergillus ficuum phytase with the arginine specific modifier 1,2-cyclohexanedione causes a rapid loss of activity. The inactivation can be partially reversed by 0.2 M hydroxylamine and exhibits pseudo-first order kinetics. The reaction order and second order rate constant of inactivation were 0.87 and 6.72 M-1 Min-1, respectively. Amino acid analysis of modified phytase indicates that about 7 arginine of the total 19 were modified. While the chymotryptic maps of treated and untreated phytase wer virtually identical, the tryptic maps had 4 peaks of altered mobility. An Arg containing tripeptide was identified in the phytase which is also present in other phosphohydrolases and may represent one of the labile Arg involved in the formation of the active site.     

Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum

A rapid purification scheme utilizing three chromatographic steps resulted in 6 fold purification of Aspergillus ficuum phytase (myo-inositol-hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8). At pH 5.0 and 60 degrees C the enzyme performed acceptably for 2.0 hr with only 30% diminished catalytic rate at the end. Substrate concentration exceeding 2mM was inhibitory. The inorganic orthophosphate, the product and a weak inhibitor, exhibited a Ki of 1.9 x 10(-3)M. The extracellular phytase has the potential for industrial use since it can be over produced, easily purified, remain catalytically active for a longer period and is not subjected to severe product inhibition.     

Purification,properties and comparison of invertase,exoinulinases and endoinulinases of Aspergillus ficuum

Summary One invertase (Inv), five exoinulinases (Exo I; II; III; IV; V) and three endoinulinases (Endo I; II; III) were isolated from a commercial inulinase preparation derived from Aspergillus ficuum using ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sephacel and DEAE-Trisacryl, gel filtration on Ultrogel and Fast Protein Liquid Chromatography on a Mono Q column. The invertase (Inv) had a molecular weight of 84000 and was much more active on sucrose than on inulin: the ratio of activity on inulin and sucrose (I/S ratio) was 0.01. The five exoinulinases show the same molecular weight of 74000 and I/S ratios in the range 0.16–0.36. The three endoinulinases had molecular weight of 64000 and I/S ratios in the range 0.86–2.92. All the -fructofuranosidases were glycoproteins with a high sugar content (from 22 to 41% w/w). A. ficuum is the first described organism containing the three activities: invertase, exo and endoinulinase.     

Electric field mediated stable transformation of carrot protoplasts with naked DNA

We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×10² transformants/10⁴ somatic embryos/g pTiC58 DNA was obtained.Abbreviations PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - MES morpholinoethane sulfonic acid - DMSO dimethyl sulfoxide - HSV Herpes Simplex virus - TK thymidine kinase     

Genetic transformation of green bean callus via Agrobacterium mediated DNA transfer

Summary Kanamycin resistant callus was produced from leaf disc or hypocotyl expiants of green bean (Phaseolus vulgaris L.) when cultured on a defined medium containing 50 mg/l kanamycin after 4 days of co-cultivation with Agrobacterium tumefaciens strain EHA101 containing the binary vector pKYLX71GUS. The presence of neomycin phosphotransferase II (NPT-II) in crude cellular extracts from the kanamycin resistant callus was confirmed by ELISA. The expression of the ß-glucuronidase (GUS) reporter gene was confirmed by histochemical and fluorimetric analyses. Southern blot border analysis confirmed the integration of the foreign DNA. In addition to the evidence obtained from Southern analysis, the absence of Agrobacterium in the transformed callus cultures was confirmed by microscopic observation and through test cultures. Using the above protocol, bean callus cultures were also transformed with a bean chalcone synthase promoter-GUS fusion. These cultures, when treated with the elicitor glutathione, showed higher levels of GUS expression than the unelicited callus clumps.     

Biological assay of prokaryotic genes in mouse cells following DNA mediated transformation

Summary Genes coding for leucine biosynthesis in Bacillus subtilis were introduced into mouse LTK^- cells by co-transformation with thymidine kinase⁺ (tk) DNA. Genomic DNA from the tk⁺ transformants was used to transform competent cultures of different B. subtilis leucine auxotrophs. Each auxotroph was transformed to prototrophy at a similar frequency and the number of leucine gene sequences per transformant genome as deduced by the B. subtilis bioassay strongly correlated with the number estimated by hybridization methods. Tk^- subclones were obtained by plating the transformants in 5-bromodeoxyuridine. One subclone still contained the non-selected leucine gene sequences and could transform auxotrophs of B. subtilis. No deletions or rearrangements in the linkage relationships of the leucine genes occurred in the LTK^- cells that inhibited transformation of B. subtilis.     

Extracellular PH 2.5 optimum acid phosphatase from Aspergillus ficuum: immobilization on modified fractogel

Aspergillus ficuum pH 2.5 optimum acid phosphatase (orthophosphoric monoesters phosphohydrolase, E.C.3.1.3.2) was covalently immobolized on 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP)-activated Fractogel TSK HW-50F. The catalytic parameters and stability of the immobilized enzyme were compared with those of the free enzyme. While the Km and the temperature optima were unchanged, the Ki for orthophosphate was changed from 185 microM to 422 microM and greater stability was observed against heat treatment.     

Expression of the INU2 gene for an endoinulinase of Aspergillus ficuum in Saccharomyces cerevisiae

The INU2 gene encoding an endoinulinase of Aspergillus ficuum was expressed by the Kluyveromyces marxianus INU1 promoter in a SUC2-deleted Saccharomyces cerevisiae to produce the endoinulinase preparation free of an exoinulinase and an extracellular invertase in the culture medium. A recombinant yeast strain produced the sufficient amount of the enzyme to make a halo around its colony, when inulin was included in the medium.