Structures of oligosaccharides on beta-galactosidase from Aspergillus oryzae

The carbohydrate portions of beta-galactosidase from Aspergillus oryzae were found to be composed of two types of sugar chains. They were released equally well with endo-beta-N-acetylglucosaminidase H, but were distinct in their chain length. The long sugar chains (fraction I), corresponding to 4% of the total carbohydrate chains, were composed of galactomannan-type oligosaccharides, which consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 30.0, 16.4, 1.4, and 2.1 per mol of aspartic acid, respectively. The short sugar chains (fraction II), corresponding to 96% of the total carbohydrate chains, consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 9.4, 0.6, 0.3, and 1.7 per mol of aspartic acid, respectively. Both types of sugar chains were fractionated into neutral and acidic subfractions. The neutral subfraction of fraction I (I-N), corresponding to 1% of the total carbohydrate chains, was very heterogeneous in length and was resistant to digestion with alpha-mannosidase and beta-galactosidase. The neutral subfraction of fraction II (II-N), corresponding to 91% of the total carbohydrate, was composed of a mixture of oligosaccharides with oligomanneoside chains (Mann GlcNAcol). The major components were similar to high mannose-type oligosaccharides of mammalian origin in their composition and size (n = 5-9). However, digestion of II-N with alpha 1,2-mannosidase produced considerable amounts of Man6GlcNAcol, an unusual product in the case of high mannose-type oligosaccharides of mammalian origin, in addition to the common one, Man5GlcNAcol.     

Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.     

Characterization of a neutral ceramidase orthologue from Aspergillus oryzae

Ceramide is an important molecule not only structurally but also regulationally as a modulator of various cellular events. Ceramidase (CDase) are classified into three different types (acid, alkaline, and neutral CDases). Neutral CDase could play an important role in the regulation of ceramide levels in the extracellular space. In this study, we describe the characterization of a neutral CDase orthologue from the filamentous fungus Aspergillus oryzae . The gene encoding the neutral CDase orthologue was cloned and overexpressed in A . oryzae . The purified recombinant enzyme was optimally active at pH 4.0–4.5 and 40 °C. The apparent K _m and V _max values of the enzyme for C12-NBD-ceramide were 3.32 μM and 0.085 μmol min⁻¹ mg⁻¹, respectively.     

Characterization of a novel lipolytic enzyme from Aspergillus oryzae

In this study, we report the characterization of a protein from Aspergillus oryzae, exhibiting sequence identity with paraben esterase from the genus Aspergillus. The coding region of 1,586 bp, including a 77-bp intron, encoded a protein of 502 amino acids. The gene without the signal peptide of 19 amino acids was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0–8.0 and 30 °C, respectively, and was stable at the pH range of 7.0–10.0 and up to 40 °C. The optimal substrate for hydrolysis by the purified recombinant protein, among a panel of α-naphthyl esters (C2–C16), was α-naphthyl butyrate (C4), with activity of 0.16 units/mg protein. The considerable hydrolytic activity of the purified recombinant enzyme toward tributyrin was determined. However, no paraben esterase activity was detected toward the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid. In addition, no activity was detected toward the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids that would indicate feruloyl esterase activity.     

Production of galacto-oligosaccharides from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth.

The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae beta-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased approximately 25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40 degrees C and 48 days at 50 degrees C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40 degrees C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported.     

Immobilization of enzymes on spongy polyvinyl alcohol cryogels: the example of beta-galactosidase from Aspergillus oryzae.

The catalytic properties of a beta-galactosidase from Aspergillus oryzae, entrapped into a spongy polyvinyl alcohol cryogel, were studied. This polymeric matrix was selected because of its mild conditions of preparation and its stability, biocompatibility, structural strength and diffusive properties. The enzyme was entrapped, in high percentage, into cryogel sponges and its activity and kinetic parameters were determined and compared with those of the free enzyme, using as substrates o-nitrophenyl-beta-galactopyranoside (ONPG) or lactose. The immobilized enzyme showed a reduced activity with ONPG and lactose, probably because of substrate diffusion limitations through the matrix, but it was more stable to temperature, pH and ionic strength than the free enzyme. Lactose hydrolysis under continuous experimental conditions was performed using the matrix-enzyme cited above.     

Immobilization/stabilization on Eupergit C of the beta-galactosidase from B. circulans and an alpha-galactosidase from Aspergillus oryzae

Two synthetically useful glycosidases, the beta-galactosidase from Bacillus circulans and an alpha-galactosidase from Aspergillus oryzae have been immobilized on Eupergit C. The immobilized enzymes retain high catalytic activity and show increased thermal stability compared with the free enzymes.     

一种米曲霉蛋白酶的酶学性质及其在酪蛋白磷酸肽制备中的应用

【目的】分离纯化米曲霉蛋白酶的主要组分,分析其酶学特性,并应用于酪蛋白磷酸肽(Casein phosphopeptides,CPPs)的制备。【方法】采用硫酸铵盐析、DEAE-Sepharose FF阴离子交换层析和Butyl-sepharose HP疏水层析对米曲霉蛋白酶进行分离纯化,SDS-PAGE检测分子量与纯度,MALDI-TOF-MS检测酶切位点。【结果】得到一种蛋白酶组分(命名为PE),分子量大小为58 kD左右。该酶最适反应条件为55 °C,pH 8.0,酶活被Fe3+抑制,被Mn2+激活。以酪蛋白为底物时,Km=0.36 g/L,最大反应速率Vm=18.18 mg/(L?min)。蛋白酶PE对牛胰岛素B链上-Leu-Cys-、-Val-Glu-、-Tyr-Leu-和-Arg-Gly-组成的肽键有较高的切割能力,酶切位点较多。利用其水解酪蛋白,通过钡-乙醇沉淀法得到CPPs,产率为15.87%,摩尔氮磷比r (N/P)为6.17,得到的CPPs可以使钙沉淀推迟35 min。【结论】利用米曲霉蛋白酶水解酪蛋白产生CPPs,为其在功能性食品加工方面的应用提供有利的参考。     

A novel heterofunctional epoxy-amino sepabeads for a new enzyme immobilization protocol: immobilization-stabilization of beta-galactosidase from Aspergillus oryzae

The properties of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the beta-galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12-fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).     

Preparation and properties of a new DNase from Aspergillus oryzae.

A DNase present in commercial preparations of Aspergillus oryzae alpha-amylase was purified 1550-fold in 25% yield by acetone precipitation and by chromatography on diethylaminoethyl- and carboxymethylcellulose. The enzyme was isolated free of contaminating RNases and DNases. The molecular weight of the enzyme determined by gel filtration on Sephadex G-100 was 48 000, while a molecular weight of 58 000 was determined for the single band observed upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the DNase is 9.2. The enzyme hydrolyzed only DNA with a pH optimum of 8.2 and was activated by Co2+, and to a lesser extent by Mg2+ and Mn2+. Native DNA was a better substrate than heat-denatured DNA. Enzymatic digests of calf thymus and E. coli DNA yielded oligomers of chain lengths ranging from 10 to 200, with mono- and small oligonucleotides (chain length less than 5) detected only when large (100 mg) amounts of DNA were fractionated by column chromatography on diethylaminoethyl-Sephadex A-25 in 7 M urea. The digestion products contained 5'-terminal phosphate groups and mostly adenosine at the 3' and guanosine and adenosine at the 5' ends.     

Cloning and characterization of a chitosanase gene from the koji mold Aspergillus oryzae strain IAM 2660

A genomic copy of the gene coding for chitosanase (csnA) was isolated from Aspergillus oryzae IAM 2660. A. oryzae csnA contains an open reading frame that encodes a polypeptide of 245 amino acids with a calculated molecular mass of 26,500 Da. The deduced amino acid sequence of A. oryzae csnA indicates extensive similarities to those of other fungal chitosanases.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

[This corrects the article on p. 507 in vol. 30.].     

Stability of ribonuclease T2 from Aspergillus oryzae.

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Preparation of przewalskinic acid A from salvianolic acid B using a crude enzyme from an Aspergillus oryzae strain

Przewalskinic acid A is a rare, water-soluble, and highly biologically active ingredient found, thus far, only in the Salvia przewalskii Maxim herb; however, the content in S. przewalskii herb is very low. In order to obtain useful quantities of przewalskinic acid A, the biotransformatin of salvianolic acid B from Salvia miltiorrhiza root (danshen in Chinese) into przewalskinic acid A was studied using a crude enzyme produced from Aspergillus oryzae D30s strain. The crude enzyme from the A. oryzae strain hydrolyzed salvianolic acid B into przewalskinic acid A and danshensu. The preparation afforded 31.3 g przewalskinic acid A (91.0 % purity) and 13.1 g danshensu (95 % purity) from 75 g salvianolic acid B. The preparation of przewalskinic acid A was therefore very successful with a yield of over 86 %, but the yield of danshensu was only 33 %. The product przewalskinic acid A was identified using ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) and NMR.