Starch-binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI > GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.     

Characterization of a glucoamylase G2 from Aspergillus niger

Peptide fragments were generated by enzymic or chemical degradation of the small form, G2, and the large form, G1, of Aspergillus niger glucoamylase (EC 3.1.2.3). The G2 form was either identical to residues Ala1-Pro512 or to Ala1-Ala514 of the G1 polypeptide chain containing 616 amino acid residues. Structural analysis of the O-linked carbohydrates from the 70-amino-acid-residues long extensively glycosylated segment of G2 revealed no significant differences in the contents of single mannose and oligosaccharide units in comparison to the corresponding region of G1. The results suggest that the present G2 form has been generated by limited proteolysis of the larger G1. In contradistinction to this, a recently reported splicing out of an intervening sequence from G1 mRNA leads to a smaller mRNA coding for a G2 protein product with a different COOH-terminal sequence than the G2 form described in the present work [Boel et al. (1984) EMBO J. 3, 1097-1102].     

Action of glucoamylase from Aspergillus niger on phosphorylated substrate

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger was purified to be free from alpha-amylase and phosphatase (glucose 6-phosphate as substrate). The phosphatase was well separated from the glucoamylase by phosphocellulose ion-exchange chromatography. The glucoamylase action was prevented by the esterified phosphate groups of the substrate. Thus, the extensive action of the glucoamylase on potato starch exposed the 6-posphorylglucosyl residue of the starch at the non-reducing terminal and large molecular weight limit dextrins remained. The concomitant action of the phosphatase was necessary for the complete degradation of the starch.     

Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.     

Molecular dynamics simulations of the unfolding of the starch binding domain from Aspergillus niger glucoamylase

The 600 ps molecular dynamics simulations to investigate the unfolding of the starch binding domain from Aspergillus niger glucoamylase were conducted in vacuum as well as in an external field with the dielectric constant of 80 with temperature jump technique. Electrostatic interactions play an important role in determining the stability of the beta-strands in this domain. The starch binding site 1 is less stable than site 2 since it is more exposed to the surface. The disulfide bond between C509 and C604 is unstable since these two residues are located near the flexible linker domain and in the mobile loop region between beta-strands 6 and 7, respectively. The melting temperature, at which the total residual beta-strand content is 50% that of the solution structure, is about 544K for the simulations with dielectric constant of 80, leading to the estimated unfolding timescale of 0.48 ms in vitro. In addition, the unfolding of the starch binding domain is proposed to initiate from the interior region by the lost of the integrity of the secondary structure.     

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.     

Chemical stabilization of glucoamylase from Aspergillus niger against thermal inactivation

The applicability of crosslinking an enzyme to an oxidized polysaccharide by reductive alkylation to enhance thermostability has been investigated for glucoamylase from Aspergillus niger. Direct covalent coupling of the enzyme to periodate-oxidized dextran in the presence of NaBH(3)CN results in a conjugate which has thermal properties similar to those of the native enzyme. Our working hypothesis postulates that enhancement of thermostability will result from rigidification of the protein's conformation subsequent to the formation of multiple covalent bonds between the protein and the support. On the basis of the known characteristics of glucoamylase from Aspergillus niger, it would seem necessary to introduce additional amino groups in the polypeptide chain of the protein. The incorporation of new amino groups was performed in two phases. First, the glycosidic part of glucoamylase was oxidized by periodate and the resulting aldehyde groups were reductively aminated by a diaminoalkane and NaBH(3)CIM. Secondly, additional amino groups were introduced on carboxyl functions into the previously aminated glucoamylase by a diaminoalkane and a water-soluble carbodiimide in the presence of maltose to protect the active site. The final derivative was then coupled to periodate-oxidized dextran T-70 in the presence of NaBH(3)CN. Starting with native glucoamylase, three successive operations give rise to a conjugate which retained 27% of the initial activity when measured with soluble starch and 39% when measured with maltopentaose. Using substrates of various sizes, it was observed that steric hindrance at the active site may result from covalent coupling to dextran T-70. It was demonstrated in heat inactivation experiments that the thermostability of the conjugate was in all cases superior to that of the native enzymes. Finally, it was observed that the operational stability of the conjugate was at least twice that of native glucoamylase at 70 degrees C on 18% maltodextrin. Additional experiments rule out the possibility that thermosta-bilization of the complex is due to other reasons than the increase in the amino content of the protein prior to crosslinking. Neither chemical modification, reticulation nor change in the net charge of the protein resulted in a derivative of glucoamylase which presented enhanced thermostability after conjugation. We conclude that for enzymes which have a low content of available amino groups, the thermostabilization method proposed previously by the present authors may still be applicable if additional amino groups are introduced into the protein prior to its crosslinking to an oxidized polysaccharide. This new example reinforces the generality of this method of stabilization.     

产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

Structural studies on the O-glycosidically linked carbohydrate chains of glucoamylase G1 from Aspergillus niger

Glucoamylase G1 from Aspergillus niger contains an unusual type of carbohydrate-protein linkage, involving mannose O-glycosidically linked to serine and threonine. The majority of the neutral oligosaccharides of glucoamylase G1 are located in a region of about 70 amino acid residues which carries about 35 oligosaccharide units [(1983) Carlsberg Res. Commun. 48, 517-527]. Structural analysis was performed on the O-linked carbohydrates of a tryptic fragment from glucoamylase G1 comprising the segment characterized by a high degree of glycosylation. The carbohydrate structures released by trifluoroacetolysis were elucidated using sugar analysis, methylation analysis, mass spectrometry, chromium trioxide oxidation, digestion with alpha-mannosidase and 1H-NMR spectroscopy. The following structures could be identified. (formula; see text)     

Effect of maltose on glucoamylase formation by Aspergillus niger

Low levels of glucoamylase are produced when Aspergillus niger is grown on sorbitol, but substitution of the latter by glucose, maltose, or starch results in greater formation of glucoamylase as measured by enzymatic activity. Both glucoamylase I and glucoamylase II are formed in a yeast extract medium; however, glucoamylase I appears to be the only form produced when ammonium chloride is the nitrogen source. Maltose or isomaltose (1.4 x 10(-4)m), but no other disaccharides or monosaccharides, dextrins, dextrans, or starches, stimulated glucoamylase formation when added to mycelia pregrown on sorbitol-ammonium salts. The induction of glucoamylase by maltose was independent of sulfate concentration but showed a dependency on low pH and the absence of utilizable carbon sources.     

The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase

High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg x L(-1) and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x mL(-1) for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.     

Interaction of radical anion probes with glucoamylase I from Aspergillus niger.

The radical anions (SCN)2.- and Br2.- produced during a pulse radiolysis of the respective potassium salts have been used to study the tryptophan residues of the glucoenzyme, glucoamylase I (EC 3.2.1.3.). At neutral pH, Br2.- reacted with the tryptophan residues of glucoamylase I as expected from previous studies of proteins and free amino acids. However, (SCN)2.- at neutral and high pH was surprisingly unreactive towards the native enzyme. Reaction did occur, however, between (SCN)2.- and glucoamylase from which one-third of the covalently bound carbohydrate had been removed, producing a tryptophyl radical. Reaction also occured between (SCN)2.- and glucoamylase I inactivated by treatment with sodium dodecyl sulphate, but the tryptophan residues were not involved. It is concluded from the results that two 'types' of tryptophan residues are found in glucoamylase I; both are attacked by Br2.- but only one type is attacked by (SCN)2.-.     

Directed evolution of Aspergillus niger glucoamylase to increase thermostability

Using directed evolution and site‐directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2‐1 . CR2‐1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2‐1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2‐1 GA has a catalytic efficiency (k_cat/K_m) at 35°C and a specific activity at 50°C similar to that of wild‐type GA. Irreversible inactivation tests indicated that CR2‐1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol^?1 compared with that of wild‐type GA. Thus, CR2‐1 is more thermostable (by 5 kJ mol^?1 at 80°C) than the most thermostable A. niger GA variant previously described, THS8 . In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol^?1, respectively, at 80°C.     

Production and properties of inulinase from Aspergillus niger

Summary A thermostable inulinase was identified in a strain of A. niger. The highest activity was observed at 50 °C (50 Lml^–1) and 77% and 34% of this was retained at 60° and 65°C, respectively. pH stability, the effect of thermal stabilizers such as Propylene glycol (10%) and Sorbitol (10%) and effects of different cations were investigated. It was found that the activity was completely inhibited by Ag⁺ and Hg²⁺, while Na⁺ had an activator effect.     

Thermodynamic effects of disulfide bond on thermal unfolding of the starch-binding domain of Aspergillus niger glucoamylase

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with beta-cyclodextrin at 25 degrees C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t1/2 of the two mutant proteins decreased by about 10 degrees C as compared to the wild-type protein at pH 7.0. At t1/2 of the wild-type protein (52.7 degrees C), the mutant proteins destabilized by about 10 kJ mol(-1) in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.     

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger

Abstract The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10–20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase ( gla A), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any gla A coding sequence. The portion of gla A used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.