Native Glucoamylase Research Articles

Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

Glucoamylases from Penicillium verruculosum and Myceliophthora thermophila: analysis of differences in activity against polymeric substrates based on 3D model structures of the intact enzymes.

Two glucoamylases, a recombinant enzyme from Penicillium verruculosum (PvGla) heterologously expressed in Penicillium canescens RN3-11-7 (niaD-) strain and a native glucoamylase from Myceliophthora thermophila (MtGla), were purified and their properties were studied. MtGla displayed 2-5-fold higher specific activities against soluble starch, amylose and amylopectin than PvGla. MtGla also provided higher glucose yields in extended hydrolysis of the polymeric substrates. Analysis of 3D model structures of the intact PvGla and MtGla, which were built using the 2vn7.pdb crystal structure of the intact Trichoderma reesei glucoamylase (TrGla) as a template, showed that the reason for lower hydrolytic performance of PvGla in comparison to MtGla may be less strong interactions between the enzyme domains as well as a longer (by 17 residues) linker in the first enzyme.

Recombinant hepatitis B surface antigen production in Aspergillus niger: evaluating the strategy of gene fusion to native glucoamylase

This study demonstrates the potential of Aspergillus niger as a candidate expression system for virus-like particle production using gene fusion. Hepatitis B surface antigen (HBsAg) production, targeted through the secretory pathway in A. niger, resulted in completely assembled and properly folded HBsAg. This was achieved by implementing a gene fusion strategy using the highly expressed catalytic domain of the native glucoamylase gene (GlaA ( G2 )) fused to the HBsAg S gene. The inducible glucoamylase promoter (GlaA ( p )) was used to control transcription in the A. niger D15 host. The gene fusion strategy was designed for cleavage of the fused product by the KEX2-like protease, resulting in intracellular accumulation of HBsAg and extracellular secretion of glucoamylase. Immunodetection using a monoclonal HBsAg antibody could not detect the fused GlaA ( G2 ) ::S product in intracellular and extracellular fractions, indicating that full assembly and maturation of HBsAg occurred after cleavage of the fused product in the Golgi complex. Several breakdown products showing an immunoreactive response to the glucoamylase polyclonal antibody indicated a level of intracellular degradation. The choice of carbon source used in cultivation significantly affected HBsAg production levels through induction of the glucoamylase promoter. The highest specific HBsAg production was observed during growth on inducing substrates of starch and its degradation products (maltodextrin and maltose), although residual glucose accumulation in the mid-exponential phase reduced HBsAg production. HBsAg production in starch-based cultures may be improved further by optimization of the rates of starch hydrolysis by glucoamylase and subsequent glucose consumption by the host.

Isolation of a strain of Trichoderma reesei with improved glucoamylase secretion by flow cytometric sorting

A single copy of the Renilla reniformis green fluorescent protein (GFP) gene under control of the cellobiohydrolase I ( cbh1) promoter was inserted into a strain of Trichoderma reesei at the pyr4 locus by homologous recombination. The parent strain contained two copies (a tandem repeat) of the native glucoamylase (GA) gene integrated at a different locus, and controlled by a separate copy of the cbh1 promoter. A large positive correlation ( r = 0.54) was observed between GFP expression and GA activity in ∼1900 randomly mutagenised germlings cultured in microtiter plates. The GFP-expressing strain was randomly mutagenised and screened using fluorescence activated cell sorting. Single germinating spores expressing GFP under conditions of carbon catabolite repression were sorted into individual wells of ten 96-well microtiter plates, cultured for 6 days, and assayed for GA activity. The strain producing the highest titres of GA from each of three rounds was re-screened by FACS. A variant (R3-14) was isolated that produced about 35% more total protein at 70% greater specific productivity in 14L fed-batch fermentation. The genome of strain R3-14 was analysed using comparative genome hybridization and large duplications totalling more than 750 kbp were identified on scaffolds 13, 2, 33 and 38 comprising 189 open reading frames. These included several putative transcriptional regulators, carbohydrate hydrolysing enzymes, and sugar transporters.

Effect of aniline coupling on kinetic and thermodynamic properties of Fusarium solani glucoamylase

Purified glucoamylase (GA) from Fusarium solani was chemically modified by cross-linking with aniline hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 1 [aniline-coupled glucoamylase-1 (ACG-1)], 7 (ACG-7), and 13 min (ACG-13). The aniline coupling of GA had a profound enhancing effect on temperature, pH optima, and pK (a)'s of active site residues. The specificity constants (K (cat)/K (m)) of native, ACG-1, ACG-7, and ACG-13 were 136, 244, 262, and 208 at 55 degrees C for starch, respectively. The enthalpy of activation (DeltaH*) and free energy of activation (DeltaG*) for soluble starch hydrolysis were lower for the chemically modified forms compared to native GA. Proteolysis of ACGs by alpha-chymotrypsin and subtilisin resulted in activation.

Characterization of humanized antibodies secreted by Aspergillus niger.

Two different humanized immunoglobulin G1(kappa) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex(6)GlcNAc(2) to Hex(15)GlcNAc(2). An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

The role of the Aspergillus niger furin-type protease gene in processing of fungal proproteins and fusion proteins : Evidence for alternative processing of recombinant (fusion-) proteins

We have characterized growth and protein processing characteristics of Aspergillus niger strains carrying a disrupted allele of the previously cloned and characterized kexB gene [Appl. Environ. Microbiol. 66 (2000) 363] encoding a furin-type endoprotease. Deletion of the single-copy gene confirms it to be non-essential but disruptant strains exhibit a morphologically distinct phenotype characterized by hyperbranching. Processing of homologous pro-proteins and fusion proteins comprised of a heterologous protein fused down-stream of glucoamylase and separated at the fusion junction by an endoproteolytic cleavage site was compared in wildtype and mutant strains of A. niger. We show that maturation of the native glucoamylase requires KexB, whereas maturation of aspergillopepsin does not. The processing of fusion proteins carrying Lys–Arg requires KexB, although alternative endoproteases are capable of cleaving protein fusions at sites adjacent to Lys–Arg.

Cloning and expression of fungal phytases in genetically modified strains of Aspergillus awamori.

In an effort to produce phytases cost-effectively, and to determine the efficiency of a novel expression system, the genes for Aspergillus awamori ( phyA) phytase and Aspergillus fumigatus ( phyA) phytase (a putative thermostable enzyme) were cloned and overexpressed in A. awamori. Regulation of phytase expression was achieved by separately placing the genes under the transcriptional control of the glucoamylase A ( glaA) promoter of A. awamori. A gene fusion strategy was employed that involved the insertion of a hexapeptide Kex-2 protease cleavage site between the native glucoamylase and heterologous proteins and allowed for the efficient secretion and processing of the resultant chimeric proteins produced in this system by an endogenous Kex-2 protease. The genes for both of the above-mentioned phytases have already been cloned; however, this is the first report of either of the two phytases being fused with the glucoamylase gene, placed under the transcriptional control of the glaA promoter and overexpressed in A. awamori. Following transformation of A. awamori with separate expression vectors (one for each phytase), induction of phytase expression in submerged culture was effected by utilisation of a starch-containing medium. Optimisation of heterologous protein production in small shake-flask cultures involved changes in medium constituents. Maximum phytase expression levels of 200 phytase units (PU) ml(-1) and 62 PU ml(-1) for recombinantly expressed phytases from A. awamori and A. fumigatus, respectively, were obtained in crude fermentation extracts. Subsequent process scale-up to 4 l batch fermentation yielded phytase production levels comparable to those obtained on small scale. The enzyme yields herein reported demonstrate that the expression system developed and the host strain utilised were capable of expressing phytase at levels comparable to, or exceeding, previously reported data.

Ethanol and lactic acid production as affected by sorghum genotype and location

Genotype, environment, location, and their interactions have a significant effect on end-use quality characteristics of grain sorghum ( Sorghum biocolor (L.) Moench). The objective of this research was to study the effect of sorghum genotype and production environment on ethanol and lactic acid production. Eight sorghum varieties from two locations were used. Whole sorghum grain was ground, liquefied, saccharified, and fermented to ethanol using Saccharomyces cerevisiae ( S. cerevisae, ATCC 24860). For lactic acid fermentation, whole ground sorghum grain was liquefied and fermented to lactic acid with Rhizopus oryzae NRRL 395; saccharification depended upon native gluco-amylase. Results with this limited number of sorghum varieties and locations showed that both sorghum genotype and location had a significant effect on ethanol and lactic acid yields. Variations of 5 and 15% in ethanol and lactic acid yields were observed among the 16 sorghum samples. The effect of location on the fermentation yields was as much as 5% for ethanol and 10% for lactic acid. The effects of variety and location on ethanol and lactic acid production are strongly related to chemical composition and physical properties of grain sorghum samples. Ethanol and lactic acid production increased as starch content increased, whereas the ethanol and lactic acid production decreased as protein content increased. Chemical composition had a greater effect on the ethanol and lactic acid yields than physical properties of the sorghum kernels. The effect of physical properties on ethanol and lactic acid yields was not significant ( P>0.05).

Use of immobilised glucoamylase G2 for separation of enantiomers

The aim of this study was to compare the isolated glucoamylase G2 domain as chiral selector with previously presented data obtained using immobilised native glucoamylase as chiral stationary phase. The glucoamylase G2 was isolated from a commercial preparation ofAspergillus niger glucoamylase and immobilised onto silica particles. Enantioselectivity was tested for several amino alcohols including the β-receptor blocking agents metoprolol and alprenolol. Separation factors greater than 2 were observed. Mobile phase pH was varied to optimise the enantioselective recognition and several different uncharged additives were included to examine their effect on retention. For some of the tested solutes increased retention times were observed when increasing the content of uncharged modifier. Increased mobile phase concentration of several of the tested organic modifiers resulted in increased separation factors. The effect of column temperature was studied. Retention and enantioselectivity increased at higher temperatures. Addition to the mobile phase of acarbose, an inhibitor that binds to the catalytic site of G2 with picomolar affinity, resulted in total loss of enantioselectivity. Comparison of the present results with those obtained previously with the glucoamylase G1 form also containing a starch binding domain, shows that the catalytic domain is essential for chiral recognition of amino alcohols.

The Effect of Organic Nitrogen Sources on Recombinant Glucoamylase Production by Aspergillus niger in Chemostat Culture

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 ± 0.01 h−1, pH 5.4). In cultures supplemented with l-alanine, l-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA.

Evidence for a polysaccharide-binding domain in Hormoconis resinae glucoamylase P: effects of its proteolytic removal on substrate specificity and inhibition by beta-cyclodextrin.

The hydrolysis of soluble starch, raw starch and pullulan with recombinant glucoamylase P from Hormoconis resinae was competitively inhibited by beta-cyclodextrin with apparent Ki values of 190 microM, 13 microM and 1.4 microM, respectively. Inhibition of dextran hydrolysis was partial: a maximum inhibition of 22% was achieved with a dextran concentration of 0.3 x Km and up to 4 mM beta-cyclodextrin. Hydrolysis of short oligosaccharides was not inhibited by beta-cyclodextrin at levels up to 20 mM. The enzyme bound to raw starch at pH 4.3 and 4 degrees C with an association constant of 3.4 x 10(5) M-1. Sequence alignment studies showed raw-starch-binding consensus amino acids in the C-terminal part of glucoamylase P. Partial hydrolysis with papain resulted in degradation of deglycosylated glucoamylase P into three fragments of 53, 51 and 14 kDa, respectively, as estimated by SDS-PAGE. The amino-terminal sequences of the 51 and 53 kDa fragments were identical with that of native glucoamylase P. The amino terminus of the 14 kDa fragment (Ser-Ser-X-Gln-Val-Ser-), corresponded to the sequence starting at residue 474 of intact glucoamylase P. Kinetic measurements of truncated glucoamylase P showed changes in the Km values of larger polysaccharides, but no changes in kcat values compared to the intact enzyme. It was concluded that glucoamylase P contains a catalytic core domain and a raw-starch-binding domain involved in inhibition of polysaccharide hydrolysis by beta-cyclodextrin.

Physiological optimization of secreted protein production by Aspergillus niger

Physiological factors affecting hen eggwhite lysozyme and native glucoamylase production by Aspergillus niger have been examined in batch culture. Expression of the genes encoding both proteins was controlled by the glucoamylase promoter. In standard expression medium (ACMS/N/P), secreted lysozyme yields were found to be maximal at 20–25°C (8–10 mg l −1) and markedly reduced at 30–37° C (3–5 mg l −1). Production of lysozyme exhibited similar induction or repression profiles to that of endogenous glucoamylase such that secreted lysozyme yields could be ordered with respect to growth on the following carbon sources: soluble starch > maltose > glucose > > xylose. Significantly higher yields of up to 30–60 mg l −1 were obtained in a richer medium containing soya milk, although in contrast to growth in ACMS/N/P, the highest levels of secreted lysozyme were achieved at 37°C. This improvement is attributed partly to an increase in culture biomass concentration and to a reduction in medium acidification. Growth in this medium produced a markedly different pellet morphology.

Purification and Properties of the Raw Starch Digesting Amylase from Penicillium brunneum No. 24

AbstractRaw sago starch digesting amylase was obtained from Penicillium brunneum No. 24. The crude enzyme from this strain contains carboxymethylcellulase (CMC‐ase), avicelase, α‐amylase and α‐glucosidase. Affinity chromatography (α‐cyclodextrin‐Sepharose 6B) of the enzyme after ammonium sulfate fractionation, Toyopearl HW‐55F gel filtration, DEAE‐Sephadex A‐50 and DEAE‐cellulose chromatographies fractionation steps, resulted in a homogeneous glucoamylase. SDS‐polyacrylamide gel electrophoresis of purified enzyme showed a single band, and a molecular weight of 80,000 for the native glucoamylase from Penicillium brunneum No. 24 was observed. After modification of the native glucoamylase with subtilisin, the molecular weight was reduced to 76,000. It lost the ability to digest and adsorb onto raw starches. However, its ability to digest gelatinized starches was preserved.

Elucidation of the role of sugar chains in glucoamylase using endo-β- N-acetylglucosaminidase from Flavobacterium sp.

Endo-β- N-acetylglucosaminidase (glycopeptide- d-mannosyl- N 4-( N-acetyl- d-glucosaminyl) 2-asparagine 1,4- N-acetyl-β-glucosaminohydrolase, EC 3.2.1.96), homogenized from the culture filtrate of Flavobacterium sp., could liberate about 50% of the sugar chains from the glucoamylase of Rhizopus niveus. The native and carbohydrate-depleted glucoamylases were compared in their various enzymatic properties. It was found that they were identical in their catalytic activities. However, the carbohydrate-depleted glucoamylase was less thermally stable than the native glucoamylase. Moreover, the carbohydrate-depleted glucoamylase was more sensitive to proteinases such as pronase, subtilisin and trypsin. These results suggest that the sugar chains of the glucoamylase contribute to the high stability of the enzyme. However, circular dichroism spectra of the native and carbohydrate-depleted glucoamylase were found to be identical.

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.

Glucoamylase produced by Rhizopus and by a recombinant yeast containing the Rhizopus glucoamylase gene.

Recombinant Rhizopus glucoamylase produced by a yeast containing the Rhizopus glu-coamylase gene was purified, characterized, and compared with native Rhizopus glucoamylase. The recombinant glucoamylase was similar to Gluc 1, one of three forms of Rhizopus native glucoamylase, but it degraded raw starch more efficiently than the native glucoamylase. Recombinant glucoamylase and Gluc 1 adsorbed to gelatinized soluble starch as well as raw starch. These findings suggested that Rhizopus glucoamylase consists of two domains; one for causing adsorption to the starch and the other for catalyzing degradation of the starch molecule.

Action pattern of .ALPHA.-amylase immobilized on granular polyacrylonitrile by amidination reaction.

An α-amylase from Bacillus subtilis (liquefying type) was immobilized on partially imido esterized granular polyacrylonitrile (PAN) by an amidination reaction . The action patterns in the hydrolysis of soluble starch were compared between the native and the immobilized α-amylase . The hydrolysis character of immobilized α-amylase was not basically changed except for retardation in the production of oligosaccharides due to diff usional limitation and the deficiency of maltohexaose production potency. The amount of glucose production by a combination of the enzymatic reactions by the immobilized α-amylase with the native glucoamylase was also studied at 40°C. Approximately the same glucose amount was attained between the native and the immobilized α-amylase in the successive hydrolysis by glucoamylase.

粒状固定化酵素カラムによる可溶性でんぷんの加水分解

Guucoamylase was immobilized by amidination reaction on the granular polyacrilonitrile resin which had been preliminarily treated with dry HCl in methanol. The granular polyacrilonitrile resin was made by drop-wise addition of the dimethylf ormamide solution of polyacrilonitrile to methanol. Continuous hydrolysis of soluble starch was operated in a column which was packed with the immobilized glucoamylase (granularity : 1 mmφ). Estimated values of apparent MICHAELIS constant (Km) with the immobilized glucoamylase depended on flow rates in the column and were found to be higher than that with the native glucoamylase in the batch system. The immobilized glucoamylase column was connected in a series with a column of the immobilized a-amylase (granularity : 30-200 μmφ). And, a soluble starch solution was firstly passed through the immobilized α-amylase column and then through the immobilized glucoamylase column. The effect of the pretreatment of soluble starch in the immobilized α-amylase column on the rate of hydrolysis was recognized with regard to the balance between amount of immobilized α-amylase and the degree of the hydrolysis in terms of reducing sugar unit. A similar effect was observed in the pretreatment of soluble starch with native α-amylase in a batch system. The molecular weight distribution of the reaction product of α-amylase processing was analyzed by the gel filtration (Sephadex G-75)method for the higher molecular weight fraction and by the thin layer chromatographic method for the lower molecular weight fraction up to DP 12. On the basis of these results, the pretreatment with α-amylase was thought to be significant particularly in that the effective concentration of the substrate was increased through the fission of the glucoside chains. The endurance test of the immobilized glucoamylase column was pursued by the continuous operation for two weeks with a flow rate of 0.5 ml/min at room temperature and 40°C. Similar situations were referred to in the previous study was obtained.

Preparation and General Properties of Glucoamylase Bound to Halogenacetyl Cellulose

The coupling reaction of glucoamylase and halogenacetyl cellulose (HAC) without pretreating with organic solvent led to form large particles of immobilized glucoamylase and the activity and the specific activity of the preparation were very low. However, the coupling reaction with HAC pretreated with organic solvents allowed to form very fine particles and the activity was increased by five times. The latter contained 3~6% of enzyme protein and the specific activity to maltose reached to 80~90% of native glucoamylase. Since the specific activity of the preparation was presumed to be much influenced by the particle size, the specific activity and general properties of different particle sizes were compared with those of native enzyme. The specific activities of particles of 0~15μ, 15~55μ, 70~190μ and 130~270μ showed 82%, 33%, 27% and only 7% of native enzyme, respectively. Km values of native form, 0~15μ, 15~55μ and 70~190μ particles were 0.90×10−3m, 1.35×10−3m, 1.60×10−3m and 2.1×10−3m in the case of malt...