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.     

Purification and properties of the raw-starch-digesting glucoamylases from Corticium rolfsii

Corticium rolfsii AHU 9627, isolated from a tomato stem, is one of the strongest producers of a raw-starch-digesting amylase. The amylase system secreted by C. rolfsii AHU 9627 consisted of five forms of glucoamylase (G1–G5) and a small amount of α-amylase. Among these amylases, G1, G2 and G3 were able to hydrolyze raw starch. Five forms of glucoamylase were separated from each other and purified to an electrophoretically homogeneous state. The molecular masses were: G1 78 kDa, G2 78 kDa, G3 79 kDa, G4 70 kDa, and G5 69 kDa. The isoelectric points were: G1 3.85, G2 3.90, G3 3.85, G4 4.0, and G5 4.1. These glucoamylases showed nearly identical characteristics except that G4 and G5 were unable to hydrolyze raw starch. Received: 16 December 1997 / Received last revision: 6 May 1998 / Accepted: 1 June 1998     

Interaction of antidiabetic α‐glucosidase inhibitors and gut bacteria α‐glucosidase

Carbohydrate hydrolyzing α‐glucosidases are commonly found in microorganisms present in the human intestine microbiome. We have previously reported crystal structures of an α‐glucosidase from the human gut bacterium Blaubia (Ruminococcus) obeum (Ro‐αG1) and its substrate preference/specificity switch. This novel member of the GH31 family is a structural homolog of human intestinal maltase‐glucoamylase (MGAM) and sucrase–isomaltase (SI) with a highly conserved active site that is predicted to be common in Ro‐αG1 homologs among other species that colonize the human gut. In this report, we present structures of Ro‐αG1 in complex with the antidiabetic α‐glucosidase inhibitors voglibose, miglitol, and acarbose and supporting binding data. The in vitro binding of these antidiabetic drugs to Ro‐αG1 suggests the potential for unintended in vivo crossreaction of the α‐glucosidase inhibitors to bacterial α‐glucosidases that are present in gut microorganism communities. Moreover, analysis of these drug‐bound enzyme structures could benefit further antidiabetic drug development.     

Chemical modification results in hyperactivation and thermostabilization of Fusarium solani glucoamylase

Chemical modification of carboxyl groups of glucoamylase from a mesophilic fungus, Fusarium solani, was carried out using ethylenediamine as nucleophile in the presence of water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. Modification brought about a dramatic enhancement of catalytic activity and thermal stability of glucoamylase. Temperature and pH optima of ethylenediamine-coupled glucoamylase (ECG) increased as compared with those of native enzyme. The specificity constant (k(cat)/K(m)) of native, ECG-2, ECG-11, and ECG-17 was 136, 173, 225, and 170, respectively, at 55 degrees C. The enthalpy of activation (Delta H*) and free energy of activation (Delta G*) for soluble starch hydrolysis were lower for the chemically modified forms. All of the modified forms were stable at higher temperatures and possessed high Delta G* against thermal unfolding. The effects of alpha-chymotrypsin and subtilisin on the modified forms were activating as compared with native. Moreover, denaturation of ECG-2, ECG-11, and ECG-17 in urea at 4 mol x L(-1) also showed an activation trend. A possible explanation for the thermal denaturation of native and increased thermal stability of ECG-2, ECG-11, and ECG-17 at higher temperatures is also discussed.     

Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.     

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].     

High Level Secretion of Calf Chymosin Using a Glucoamylase-prochymosin Fusion Gene in Aspergillus oryzae

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1–603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1–511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.     

表达淀粉酶、糖化酶葡糖酸醋杆菌工程菌的构建

采用基因融合技术,将葡糖酸醋杆菌Gluconacetobacter hansenii ATCC23769分泌蛋白CMCax的信号肽序列分别与来源于枯草芽胞杆菌的淀粉酶基因、黑曲霉的糖化酶基因融合构建融合蛋白,连入能在G.hansenii ATCC23769自主复制的载体pbs-H1S中,电击转入G.hansenii ATCC23769,构建能内源表达淀粉酶、糖化酶,以及淀粉酶-糖化酶的葡糖酸醋杆菌。淀粉平板透明圈检测结果和DNS测酶活结果显示,构建的3种工程菌能成功表达并分泌淀粉酶和糖化酶。     

Synthesis of novel heterobranched beta-cyclodextrins from alpha-D-mannosylmaltotriose and beta-cyclodextrin by the reverse action of pullulanase, and isolation and characterization of the products

Alpha-D-mannosyl-maltotriose (Man-G3) were synthesized from methyl alpha-mannoside and maltotriose by the transfer action of alpha-mannosidase. (Man-G3)-betaCD and (Man-G3)2-betaCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M betaCD at 50 degrees C for 4 days. The reaction products, (Man-G3)-betaCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)2-betaCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-betaCDs was identified as 6-O-alpha-(6(3)-O-alpha-D-mannosylmaltotriosyl)-betaCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)2-betaCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by alpha-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-betaCDs. The structures of three main components of (Man-G3)2-betaCDs were identified as 6(1),6(2)-, 6(1),6(3)- and 6(1),64-di-O-(63-O-alpha-D-mannosyl-maltotriosyl)-betaCD.     

蓝光诱导促进黑曲霉产糖化酶的研究

考察了蓝光对黑曲霉产糖化酶的影响并采用扫描电镜观察蓝光下黑曲霉形态发育过程,结果表明,与黑暗对照组相比,蓝光处理使菌丝粗壮,孢囊增大,分生孢子发育提前,黑曲霉糖化酶活力增加,孢子发育和产糖化酶的进程有一定的对应性。黑曲霉在黑暗下生长至36h时,经蓝光诱导糖化酶产量提高更为明显,提示了黑曲霉存在一个对蓝光反应产生最适光感应的发育阶段,对于光调节黑曲霉产糖化酶来说,蓝光诱导的光强由弱到强,比持续蓝光培养或采用较高光强诱导效果更好,表明黑曲霉产糖化酶存在一种光适应机制,能够感应和适应光强度变化,调节其自身代谢。从抑制性扣除杂交实验和蓝光光强变化对差异基因表达的分析来看,糖化酶基因以及呼吸链中部分氧化还原酶基因在蓝光诱导下表达皆有增强,蓝光信号转导影响了核基因编码的线粒体呼吸链相关酶基因表达水平,交替氧化酶可能参与了蓝光信号途径,影响了黑曲霉产糖化酶和孢子发育。研究结果可为在现有水平上应用蓝光调节提高糖化酶产量找到新的技术突破口和提供新思路。     

The starch-binding domain from glucoamylase disrupts the structure of starch

The full-length glucoamylase from Aspergillus niger, G1, consists of an N-terminal catalytic domain followed by a semi-rigid linker (which together constitute the G2 form) and a C-terminal starch-binding domain (SBD). G1 and G2 both liberate glucose from insoluble corn starch, although G2 has a rate 80 times slower than G1. Following pre-incubation of the starch with SBD, the activity of G1 is uniformly reduced with increasing concentrations of SBD because of competition for binding sites. However, increasing concentrations of SBD produce an initial increase in the catalytic rate of G2, followed by a decrease at higher SBD concentrations. The results show that SBD has two functions: it binds to the starch, but it also disrupts the surface, thereby enhancing the amylolytic rate.     

Small angle X-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution

The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.     

Studies on carbohydrate moieties of the glycoprotein,glucoamylase II ofAspergillus niger: Nature of carbohydrate-peptide linkage and structure of oligosaccharides

Electrophoretically homogeneous type 1 (GP-C₁ and GP-C₂), type 2 (GP-C_3a and GP-C3b,) and type 3 (GP-D₁, and GP-D₂) glycopeptides fromAspergillus niger glucoamylase II (Manjunath and Raghavendra Rao, preceding paper) were separately treated with alkaline borohydride. The (\-eliminated oligosaccharides were subjected to single and sequential digestion with specific glycosidases and the products analysed by gas liquid chromatography. The studies revealed that carbohydrate moieties were present as mannose, Man-Man-, and trisaccharide structures, namely, (a) GIc-Man-Man-, (b) Gal-Man-Man, (c) Man-Man-Man-, (d) GlcNAc-Man-Man-, and (e) Xyl-Man-Man. None of the glycopeptides contained all the trisaccharide structures (a) to (e). Type 1 glycopeptide contained structures (a), (b) and (c); type 2, (a) and (d) and type 3, (a), (b) and (e). The number of carbohydrate units (mono-, di-and trisaccharides) present in the major glycopeptides was determined and tentative structures for the glycopeptides proposed. Carbohydrate units appeared to occur in clusters of 4 to 7 in each glycopeptide, a structure unique to the carbohydrate moiety inAspergillus niger glucoamylase. Based on carbohydrate analysis and yields of glycopeptide, the number of units of each type of glycopeptide present in glucoamylase II was tentatively calculated to give two of type Man:Glc:Gal = 12–15:l:l, one of type Man:Glc:GlcN = 10-l1:1:2 and one of type Man :GIc :Gal:Xyl = 4–8:0.1:0.5-0.8:0.3-1 glycopeptides.     

Isolation and Sequencing of a New Glucoamylase Gene from an Aspergillus niger Aggregate Strain (DSM 823) Molecularly Classified as Aspergillus tubingensis

Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues. The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92% An erratum to this article is available at .     

The effect of point amino acid substitutions in an internal α-helix on thermostability of Aspergillus awamori X100 glucoamylase

Conformational flexibility of α-helices in glucoamylase of the fungus Aspergillus awamori was studied by molecular dynamics methods. Several amino acid substitutions (G127A, P128A, I136L, G137A, and G139A) optimizing intrinsic interactions in one of the α-helices (D) within the hydrophobic core of this protein were constructed and studied. It was found that these point mutations had different effects on the glucoamylase thermal inactivation constant. Unlike amino acid substitution P128A and substitutions G137A and A246C, I136L and G139A displayed a pronounced additive thermostabilizing effect.