Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Galactofuranoic-oligomannose N-linked glycans of alpha-galactosidase A from Aspergillus niger.

Extracellular alpha-galactosidase A was purified from the culture filtrate of an over-producing strain of Aspergillus niger containing multiple copies of the encoding aglA gene under the control of the glucoamylase (glaA) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for approximately 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed of N-acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (beta-D-galactofuranose, Galf). At least 20 different N-linked oligosaccharides were fractionated by high-pH anion-exchange chromatography following release from the polypeptide by peptide-N-glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex(7-26)HexHA(c2). Indicating that oligosaccharides from GlcNA(c2)Man(7), increasing in molecular size up to GlcNA(c2)Man(24) were present. Each of these were additionally substituted with up to three beta-Galf residues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular-size N-linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein.     

Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.     

The kinetic of lactose hydrolysis for the beta-galactosidase from Aspergillus niger

The kinetics of glucose liberation from lactose by means of the beta-glactosidase from Aspergillus niger has been studied in a wide range of the main variables. The analysis shows that the kinetic models proposed so far are not adequate. The main finding is that the reaction rate is not linearly correlated to the enzyme concentration-it increase more than proportionally. This nonlinear relationship results because this lactase can distinguish between alpha-and beta-galactose alpha-Galactose acts as competitive and anticompetitive inhibitor while beta-galactose is a competitive one. The competitive inhibition of the alpha-anomer is approximately 12 times more sever than that of the beta-anomer. The kinetics, including a simplified model for the mutarotation of galactose is given for a temperature of 50 degrees C at a pH of 3.5-the most likely conditions for the application of this lactase in acid whey treatment.     

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.     

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding beta-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.     

beta-Galactosidase from Aspergillus niger. Separation and characterization of three multiple forms.

The enzyme beta-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, an hydrophobic chromatography. The isolated enzyme forms accounted for 83%, 8%, and 9% of the total beta-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124,000, 150,000 and 173,000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5%, 20.5% and 29% neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions. The beta-galactosidase forms were heat-stable up to about 60 degrees C. The Km values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30 degrees C for lactose and o-nitrophenyl-beta-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger beta-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.     

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.     

黑曲霉（Aspergillus niger）F044脂肪酶新型基因lipB的克隆、表达及酶学性质分析

摘要：【目的】本研究拟克隆新型的黑曲霉（Aspergillus niger）脂肪酶（EC 3.1.1.3）基因,实现其在大肠杆菌（Escherichia coli）的高效表达,并对表达产物进行系统的酶学性质分析,为该脂肪酶的工业化生产及应用奠定基础。【方法】通过PCR和RT-PCR克隆脂肪酶基因,并将其开放式阅读框（ORF）克隆入融合表达载体pET28a;表达产物经Ni-agarose纯化后对LipB进行酶学性质分析,并通过圆二色谱进行结构分析。【结果】成功地从A. niger F044中克隆了一个新型的脂肪酶基因lipB,获得了该基因的全基因组序列和cDNA序列（GenBank: FJ536287、FJ536288）,并实现了其在E. coli中的高效表达。LipB分子量约为43.0 kDa,最适底物为pNPC（C8）,酶学动力学常数Km=5.98 mmol/L,最适反应温度为50℃,最适pH为6.0;该酶能在40℃条件下保持稳定,在60℃条件下处理1 h后残余酶活仅为18.8%;该酶对Ca2+敏感,当脂肪酶经2 mmol/L Ca2+处理1 h后,酶活提高了2.6倍。圆二色谱分析表明该酶在Ca2+处理前后具有明显的结构变化。【结论】新型A. niger脂肪酶lipB基因的克隆不仅积累了脂肪酶基因资源,而且为高效基因工程菌的构建及规模化应用奠定基础;对LipB的酶学性质分析表明该酶在食品和油酯化工等领域具有广阔的应用前景。     

Cloning of two β-xylanase-encoding genes from Aspergillus niger and their expression in Saccharomyces cerevisiae

Two endo-β-1,4-xylanase-encoding genes were amplified from Aspergillus niger ATCC 90196 mRNA, inserted between the yeast ADH2 promoter and terminator sequences (genes designated XYN4 and XYN5) and expressed in Saccharomyces cerevisiae. The nucleotide sequences of the XYN4 and XYN5 genes revealed that both genes encode 211-amino acid proteins that are 92% identical to each other. Both the Xyn4 and Xyn5 enzymes have pH and temperature optima of pH 4 and 60°C, respectively. Autoselective S. cerevisiae strains were developed that allowed β-xylanase production and secretion in complex medium. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cloning of the xynNB gene encoding xylanase B from Aspergillus niger and its expression in Aspergillus kawachii

Aspergillus niger IFO 4066 produced two xylanases, xylanase A (XynNA) and xylanase B (XynNB), in culture medium, and these enzymes were purified. Acidophilic xylanase such as xylanase C (XynC) of white koji mold (Aspergillus kawachii IFO 4308) was not detected in A. niger cultures. However, results of Southern analysis using xynC cDNA of A. kawachii as a probe suggested that A. niger contained a gene homologous to xynC of A. kawachii. Therefore, we cloned this xylanase gene from A. niger. The predicted amino acid sequence of the cloned xylanase showed a homology to that of xynC of A. kawachii. However, a large number of amino acid substitutions were detected, especially in the N-terminal region. Both this cloned gene and xynC gene of A. kawachii had an intron at the same position in the coding region. The cloned gene was expressed in A. kawachii and a large quantity of xylanase was produced. The elution profile on an anion exchange chromatogram and the N-terminal amino acid sequence of the xylanase purified from the transformant were the same as those of XynNB. This confirmed that the cloned gene encoded XynNB.     

Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger

The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.     

Functional analysis of tunicamycin-inducible gene A polypeptide from Aspergillus niger.

Tunicamycin-inducible gene A polypeptide (TIGA) is a member of the protein disulfide isomerase (PDI) family and is suggested to facilitate the folding of nascent polypeptides. The functional properties of TIGA were investigated here. TIGA acted as an isomerase, catalyzing the refolding of denatured and reduced ribonuclease A. TIGA also exhibited chaperone activity in the refolding of denatured prochymosin but not in the refolding of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), indicating that it had substrate specificity with respect to chaperone activity. Detailed study with a series of thioredoxin-motif (trx-motif) mutants revealed that the 2 trx-motifs of TIGA were not equal in activity. The N-terminal trx-motif was more active than the C-terminal trx-motif, and the first cysteine in each trx-motif was necessary for isomerase activity.     

Cloning,sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.     

黑曲霉硫氧还蛋白的克隆表达纯化及其酶活位点特征分析

首次从黑曲霉Aspergillus niger全基因组中克隆出黑曲霉硫氧还原蛋白基因AnTrx,并对其编码蛋白的第33-37位保守区的活性位点实施定点突变C34S、C37S及C34S-C37S,获得相应的3个定点突变基因。将野生型AnTrx及其突变子分别在大肠杆菌Escherichia coli中诱导表达,比浊法测定纯化的各表达产物还原牛胰岛素α与β链之间二硫键的活性。结果表明,AnTrx的3个突变体都不表现明显催化活性。当突变型与野生型AnTrx等量混合后,发现突变型AnTrx-C34S可显著提高野生型AnTrx的催化效率,而突变型AnTrx-C37S却无此功能。由此证明,AnTrx活性结构域的第37位Cys残基上的巯基能参与攻击硫氧还蛋白和底物蛋白所形成的二硫键而释放被还原的底物蛋白,而第34位Cys残基同其他微生物的同一活性域一样参与硫氧化还蛋白与底物的结合。这一结果有助于认识真菌硫氧还蛋白第37位活性位点的作用。     

Purification and properties of a cellulase from Aspergillus niger.

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.     

Purification and characterization of a beta-glucuronidase from Aspergillus niger.

A beta-glucuronidase from Pectinex Ultra SP-L, a commercial pectolytic enzyme preparation from Aspergillus niger, was purified 170-fold by ion-exchange chromatography and gel filtration. Apparent M(r) of the purified enzyme, estimated by denaturing gel electrophoresis and size-exclusion chromatography, were 68,000 and 71,000, respectively, indicating that the enzyme is a monomeric protein. It released uronic acids not only from p-nitrophenyl beta-glucosiduronic acid (PNP-GlcA) but also from acidic galactooligosaccharides carrying either beta-D-glucosyluronic or 4-O-methyl-beta-D-glucosyluronic residues at the nonreducing termini through beta-(1-->6)-glycosidic linkages. The enzyme exhibited a maximal activity toward these substrates at pH 3.0. A regioisomer, 3-O-beta-glucosyluronic acid-galactose, was unsusceptible to the enzyme. The enzyme did act on a polymer substrate, releasing uronic acid from the carbohydrate portion of a radish arabinogalactan-protein modified by treatment with fungal alpha-L-arabinofuranosidase. The enzyme produced acidic oligosaccharides by transglycosylation, catalyzing the transfer of uronic acid residues of PNP-GlcA and 6-O-beta-glucosyluronic acid-galactose to certain exogenous acceptor sugars such as Gal, N-acetylgalactosamine, Glc, and xylose.