Isopullulanase Research Articles

The side chain of a glycosylated asparagine residue is important for the stability of isopullulanase.

N-glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N-glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N-glycan in IPU, we focused on an N-glycosylated residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5-8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molecules, Mol-A, B, C and D, in the asymmetric unit. The conformation of a loop composed of amino acid residues 435-455 in Mol-C was identical to wild-type IPU, whereas the conformations of this loop in Mol-A, Mol-B and Mol-D were different from each other. These results suggest that the Asn448 side chain is primarily important for the stability of IPU. Our results indicate that mutation of only N-glycosylated Asn residue may lead to incorrect conclusion for the evaluation of the function of N-glycan. Usually, the structures of N-glycosylation sites form an extended configuration in IPU; however, the Asn448 site had an atypical structure that lacked this configuration.

Crystal Structure of Aspergillus niger Isopullulanase, a Member of Glycoside Hydrolase Family 49

An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes α-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H f to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-Å resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 β-strands and forms a β-sandwich. Domain C, where the active site is located, forms a right-handed β-helix, and the lengths of the pitches of each coil of the β-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites + 2 and + 3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the β-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.

Insights into the reaction mechanism of glycosyl hydrolase family 49

Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite -1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and beta-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities.

Construction of an efficient expression system for Aspergillus isopullulanase in Pichia pastoris, and a simple purification method.

Aspergillus niger ATCC 9642 isopullulanase (IPU) was heterologously expressed by Pichia pastoris GS115 under three different signal sequences of Saccharomyces cerevisiae acid phosphatase, S. cerevisiae alpha-factor prepro peptide, and A. niger isopullulanase. One-step purification using lectin Con A affinity chromatography yielded recombinant IPU (IPU-PP) with high purity. IPU-PP had a higher carbohydrate content than native IPU and IPU-AO expressed in A. oryzae M-2-3. IPU-PP hydrolyzed various substrates containing the structure of panose, which indicated a strict subsite recognition of the panose motif.

Deglycosylated Isopullulanase Retains Enzymatic Activity.

Isopullulanase (IPU) from Aspergillus niger ATCC 9642 is a cell-bound glycoprotein that hydrolyzes pullulan into isopanose. The sugar content of the recombinant enzyme expressed in Aspergillus oryzae M-2-3 decreased from 33.8 to 2.1% by 13 h-treatment with endoglycosidase H (Endo H), and deglycosylated rec-IPU had 65% of the original activity. 3 Deg-IPU (prepared by 3 h-treatment of Endo H; 6.8% sugar) showed the same substrate specificities, optimum pH and optimum temperature as native IPU and rec-IPU, while its kinetic parameters, ko and ko/Km values for pullulan, and Km, ko and ko/Km values for panose decreased with deglycosylation, except Km for pullulan. The oligosaccharide chains of rec-IPU were typed using lectin-peroxidase reagents and classified as hybrid- and/or high-mannose types.

プルラン分解酵素の構造と機能に関する研究

α-Amylases from Thermoactinomyces vulgaris R-47 (TVA I and TVA II) hydrolyze pullulan to produce panose, and isopullulanase (IPU) from Aspeygillus niger ATCC 9642 hydrolyzes Pullulan to produce isopanose. The structure/function relationships of these pullulan-hydrolyzing enzymes were studied. The crystal structure of TVA II has been determined. TVA II is composed of domain A/B, which has a (β/α) 8 barrel structure with a small component called domain B; domain C, which contains the C-terminus; and domain N, which contains the N-terminus. Although the role of domain N for the enzyme activity has been unclear, it is unique in the structures of the α-amylase family. We modified some amino acid residues in region II, one of the four regions conserved in α-amylase family enzymes, of the TVA I by means of site-directed mutagenesis. The action pattern of the mutated enzyme for pullulan was greatly altered and it produced maltotriose from pullulan. The ipuA gene encoding IPU has been isolated. Although IPU does not hydrolyze dextran, IPU showed a high amino acid sequence similarity with dextranases. The ipuA gene was expressed in Aspeygillus oryzae, and the substrate properties of the recombinant IPU were identical with those of the native enzyme.

Two Components of Cell-bound Isopullulanase fromAspergillus nigerATCC 9642—Their Purification and Enzymatic Properties

Cell-bound isopullulanase (pullulan 4-glucanohydrolase: EC 3.2.1.57, IPU) from Aspergillus niger ATCC9642 [Y. Sakano et al., Denpun Kagaku, 37, 39-41 (1990)] was separated into two active components, IPU F1 (pI = 5.0) and IPU F2 (pI = 4.9), using a Mono-P HR 5/20 column. The substrate specificity on pullulan and panose, specific activity, optimum pH, pH stability, and susceptibility to certain chemical reagents were similar between IPU F1 and IPU F2. IPU F1 and F2 had an identical N-terminal amino acid sequence, A-V-T-A-D-N-S-Q-L-L. However, IPU F1 contained more total carbohydrate (15.3%) than IPU F2 (12.4%). SDS-polyacrylamide gel electrophoresis showed that the molecular weight of IPU F1 (71,000) was greater than that of IPU F2 (69,000). After deglycosylation of IPU F1 and F2 with peptide-N-glycosidase F, the molecular weights of IPU F1 and F2 became 59,000.

Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ菌体結合型と細胞外イソプルラナーゼの比較

Aspergillusniger菌体結合型イソプルラナーゼを液体培養で得た菌体から,1)Novozyme234で菌体から酵素を可溶化し,2)ρ-セルロース(pH3.8)とDEAE-セルロース(pH5.5)のクロマトグラフィー,3)クロマトフォカシング(pHを6 .2から4.0に変化させる)および,4)バイオゲルP-100によるゲル濾過で精製した.精製酵素はPAGEで1本の糖タンパクのバンドを,TSK-ゲル(G-3000Swxz)のHPLCで1本のピークを示した.プルラン,パノース(Glcp(α1→6)Glcp(α1→4)Glc),62-α-マルトシルマルトース(Glcp(α1→4)Glcp(α1→6)Glcp(α1→4)Glc)に対する作用は既報の菌体外酵素と同じであった.至適pHと温度は両者ほぼ同じであったが,分子量および等電点では異なり,それぞれ62,000と4.8(菌体結合型酵素),74,000と4.65(細胞外酵素)であった.

α‐１，４‐：‐１，６グルカンの分解と合成に関する微生物酵素の研究

(1) Isopullulanase [EC. 3. 2. 1. 57 pullulan 4-glucanohydrolase] from Aspergillus niger was highly purified by acetone-precipitation, ion-exchange chromatography and gel-filtration. This enzyme hydrolyzed α-1, 4-glucosidic linkages adjacent to α-1, 6-linkages of pullulan to produce only isopanose (6-maltosylglucose). Its substrate specificity was different from those of pullulanase [EC. 3. 2. 1. 41] and isoamylase [EC. 3. 2. 1. 68]. From the results, it was expected that isopullulanase would be used as a suitable enzyme for the structural analysis of α-1, 4-:-1, 6-glucooligosaccharide. (2) Cell free extract of Aureobasidium pullulans synthesized pullulan from UDPG in the presence of ATP. It was suggested that pullulan Would be synthesized through a sugarlipid intermediate. (3) Thermoactinomyces vulgaris α-amylase hydrolyzed α-1, 6-linkages in partial hydrolyzates of pullulan as well as α-1, 4-linkages in starch and pullulan. Pseudomonas stutzeri maltotetraose-forming amylase hydrolyzed starch from non-reducing end to produce α-anomer of maltotetraose, whereas glucoamylase and β-amylase hydrolyze starch from non-reducing end to produce β-anomer. (4) Large scale preparation of isopanose and panose from pullulan was facilitated by the use of special enzymes, isopullulanase and T. vulgaris α-amylase.

Enzymic preparation of panose and isopanose from pullulan

The trisaccharides panose and isopanose were prepared in good yield from enzymic hydrolyzates of pullulan. Pullulan was hydrolyzed by the purified alpha amylase preparation of Thermoactinomyces vulgaris R-47. The digest was applied to a carbon-Celite column and eluted with a linear gradient of 1-propanol from 0 to 5%. From the trisaccharide fractions eluted, panose was prepared in about 70% yield. Pullulan was also hydrolysed by purified isopullulanase (EC 3.2.1.57 pullulan 4-glucanohydrolase) of Aspergillus niger ATCC-9642, and isopanose was prepared in about 90% yield by using the same technique as that for the preparation of panose.

プルランを分解する酵素

Many pullulan-hydrolyzing enzymes have been purified from several microorganisms and higher plants since 1961 when Bender and Wallenf els found pullulanase (EC 3.2.1.41 pullulan 6-glucanohydrolase) in culture filtrate of Aerobacter aerogenes. They are classified into four groups according to their substrate specificities, that is, the types of glucoamylase (EC 3.2 .1.3 1, 4-α-glucan glucanohydrolase), pullulanase, isopullulanase (EC 3.2. 1.54 pullulan 4-glucanohydrolase) and the Thermoactinontyces vulgaris α-amylase that produces panose from pullulan. Three kinds of enzymes described above, except glucoamylase, are considered to be the specific enzymes acting on α-1, 6-glucosidic linkages or α-1, 4-glucosidic linkages adjacent to α-1, 6-glucosidic linkages of starch, glycogen and oligosaccharides. In this paper, the progress of researches concerning pullulanase, isopullulanase and the Thernmactinomyces α-amylase is summarily described and, in particular, rough estimation of subsites of two pullulanases and isopullulanase was performed on the basis of data that have hitherto been reported. It is suggested that Streptococcus mitis pullulanase, A. aerogenes pullulanase and Aspergillus niger isopullulanase have at least seven, six and four subsites, respectively.

Structural Uniformity of Pullulan Produced by Several Strainsof Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.

Pullulan 4-glucanohydrolase from Aspergillus niger

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 6 3-α-glucosylmaltotriose, 6 2-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.

Hydrolysis of the interchain linkages in glycogen-type polysaccharides by a plant enzyme

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 63-α-glucosylmaltotriose, 62-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.