The enzymatic reaction for the production of panose and isomaltose by glucosyltransferase from Aureobasidium

Glucosyltransferase from Aureobasidium produced 212 mg ml^-1 of glucosyl-oligosaccharides (panose: Glcα1→6Glcα1→4Glc 189 mg ml^-1 and isomaltose: Glcα1→6Glc 23 mg ml^-1) from maltose: Glcα1→4Glc at a high concentration (500 mg ml^-1) and the efficiency of production was 42-4%. The enzymatic reaction from maltose to panose is reversible but that from panose to isomaltose is not.     

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan.

The action of neopullulanase from Bacillus stearothermophilus on many oligosaccharides was tested. The enzyme hydrolyzed not only alpha-(1----4)-glucosidic linkages but also specific alpha-(1----6)-glucosidic linkages of several branched oligosaccharides. When pullulan was used as a substrate, panose, maltose, and glucose, in that order, were produced as final products at a final molar ratio of 3:1:1. According to these results, we proposed a model for the pattern of action of neopullulanase on pullulan as follows. In the first step, the enzyme hydrolyzes only alpha-(1----4)-glucosidic linkages on the nonreducing side of alpha-(1----6) linkages of pullulan and produces panose and several intermediate products composed of some panose units. In the second step, taking 6(2)-O-alpha-(6(3)-O-alpha-glucosyl-maltotriosyl)-maltose as an example of one of the intermediate products, the enzyme hydrolyzes either alpha-(1----4) (the same position as that described above) or alpha-(1----6) linkages and produces panose or 6(3)-O-alpha-glucosyl-maltotriose plus maltose, respectively. In the third step, the alpha-(1----4) linkage of 6(3)-O-alpha-glucosyl-maltotriose is hydrolyzed by the enzyme, and glucose and another panose are produced. To confirm the model of the pattern of action, we extracted intermediate products produced from pullulan by neopullulanase and analyzed the structures by glucoamylase, pullulanase, and neopullulanase analyses. The experimental results supported the above-mentioned model of the pattern of action of neopullulanase on pullulan.     

Kinetics of the hydrolysis of di- and trisaccharides with Aspergillus niger glucoamylases I and II

Near-homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were used to hydrolyze a number of di- and trisaccharides linked by alpha-D-glucosidic bonds. Maximum rates and Michaelis constants were obtained at various temperatures and pH values with glucoamylase I for the disaccharides beta,alpha-trehalose, kojibiose, nigerose, maltose, and isomaltose and the trisaccharides panose and iso-maltotriose, and with glucoamylase II for maltose, maltotriose, and isomaltotriose. Maximum rates were highest and energies of activation were lowest for maltose, maltotriose, and panose, the only three substrates containing alpha-D-(1, 4)-glucosidic bonds. Michaelis constants were lowest and standard heats of binding were most negative for maltose and maltotriose. The variation of maximum rates and Michaelis constants with varying pH values suggested that two carboxyl groups were involved in substrate binding.     

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.     

一种新型淀粉酶的鉴定及其产酶菌株的筛选

对筛选到的菌株ZX99产生的一种新型淀粉酶（异麦芽低聚糖酶）进行了分析鉴定,ZX99菌株能产生一种胞外淀粉酶,该酶能催化淀粉的降解产生异麦芽低聚糖,对原产酶菌株ZX99多次进行紫外线照射诱变后,获得了优良,稳定的变异菌株RB3．232,其产酶水平为原株的160％,产物薄层层析证明,该酶能催化淀粉的降解,产生异麦芽糖,潘糖,异麦芽三糖和异麦芽四糖等低聚糖,但对普鲁兰基本不起作用,由此证明它是一种不同于新型普鲁兰酶（nepullulanase)和 传统淀粉酶（amylase)的一种新型淀粉酶。     

Kinetics, equilibria, and modeling of the formation of oligosaccharides from D-glucose with Aspergillus niger glucoamylases I and II

Near-homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were incubated with D-glucose at a number of temperatures and pH values. Kinetics and equilibria of the formation of alpha,beta-trehalose, kojibiose, nigerose, maltose, isomaltose, panose, and isomaltotriose, which with isomaltotetraose were the only products formed, were determined. There was no difference in the abilities of GA I and GA II to form these products. Activation energies for the formation of maltose and panose were lower than those of the other Oligosaccharides. Relative rates of oligosaccharide production based on glucoamylase hydrolytic activity did not vary significantly between pH 3.5 and 4.5 but were lower at pH 5.5. Maltose was formed much faster than any other product. Equilibrium concentrations at higher dissolved solids concentrations decreased in the order isomaltose, isomaltotriose, kojibiose, nigerose, maltose, alpha, beta-Mrehalose, panose, and isomaltotetraose. They were not appreciably affected by changes in temperature or pH. A kinetic model based on adsorption of D-glucose and the seven di- and trisaccharides by the first three glucoamylase subsites was formulated. Oligosaccharide formation was simulated with the model, using equilibrium data gathered for this article and subsite binding energies and kinetic parameters for oligosaccharide hydrolysis measured earlier. Agreement of simulated and actual oligosaccharide formation data through the course of the reaction was excellent except at very high solid concentrations.     

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, 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.     

Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[¹⁴C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)-α- and (1 → 6)-α-d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.     

Metabolism of the reserve polysaccharide of Streptococcus mitis. Properties of alpha-(1-->6)-glucosidase, its separation from transglucosylase, and the action of the two enzymes on branched oligosaccharides

1. An α-(1→6)-glucosidase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by adsorption of the latter on retrograded amylose. 2. The enzyme was detected in five of the six strains of S. mitis that were studied; α-(1→6)-glucosidase was not found in strain RB1633, a strain that did not store polysaccharide. 3. The glucosidase could act on compounds in which α-glucose is joined through an α-(1→6)-bond to either a maltosaccharide or an isomaltosaccharide. 6²-α-Glucosylmaltose (panose) and 6³-α-glucosylmaltotriose were hydrolysed more rapidly and isomaltodextrins more slowly than isomaltose. 4. Transferring activity towards isomaltose and panose was appreciable when the concentration of substrate was 2% or higher. 5. The enzyme had no action on α-(1→4)-glucosidic linkages. 6-α-Maltodextrinylglucoses were hydrolysed only after transglucosylase action had attenuated them to isomaltose.     

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 °C and pH 1–2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 °C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 °C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 °C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 °C for both P. torridus and T. acidophilum, and 20 h at 90 °C for P. oshimae. The enzyme system of T. acidophilum has a lower K _m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na⁺, Mg⁺⁺, Ca⁺⁺, Ni⁺⁺, Zn⁺⁺, Fe⁺⁺, EDTA and DTT. This revised version was published online in August 2006 with corrections to the Cover Date.     

Engineering of Escherichia coli to facilitate efficient utilization of isomaltose and panose in industrial glucose feedstock

Industrial glucose feedstock prepared by enzymatic digestion of starch typically contains significant amounts of disaccharides such as maltose and isomaltose and trisaccharides such as maltotriose and panose. Maltose and maltosaccharides can be utilized in Escherichia coli fermentation using industrial glucose feedstock because there is an intrinsic assimilation pathway for these sugars. However, saccharides that contain α-1,6 bonds, such as isomaltose and panose, are still present after fermentation because there is no metabolic pathway for these sugars. To facilitate more efficient utilization of glucose feedstock, we introduced glvA, which encodes phospho-α-glucosidase, and glvC, which encodes a subunit of the phosphoenolpyruvate-dependent maltose phosphotransferase system (PTS) of Bacillus subtilis, into E. coli. The heterologous expression of glvA and glvC conferred upon the recombinant the ability to assimilate isomaltose and panose. The recombinant E. coli assimilated not only other disaccharides but also trisaccharides, including alcohol forms of these saccharides, such as isomaltitol. To the best of our knowledge, this is the first report to show the involvement of the microbial PTS in the assimilation of trisaccharides. Furthermore, we demonstrated that an l-lysine-producing E. coli harboring glvA and glvC converted isomaltose and panose to l-lysine efficiently. These findings are expected to be beneficial for industrial fermentation.

     

Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195

A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0, and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35 degrees C and the enzyme exhibited stability up to 50 degrees C. The enzyme hydrolyzed cycloalternan [CA; cyclo(-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(-->3)-alpha-d-Glcp-(1-->)] as the best substrate, to produce only isomaltose via an intermediate, alpha-isomaltosyl-(1-->3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates, such as panose, alpha-isomaltosyl-(1-->4)-maltooligosaccharides, alpha-isomaltosyl-(1-->3)-glucose, and alpha-isomaltosyl-(1-->3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1-->4)- or (1-->3)-linkages. The K(m) value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The k(cat) value on panose (14.4s(-1)) was not significantly different from that of cycloalternan (10.8 s(-1)). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195, its role being to hydrolyze cycloalternan inside the cells.     

An improved synthesis of 6-azathymidine

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.     

A novel alpha-glucosidase from the moss Scopelophila cataractae

Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.     

Enzymatic synthesis and characterization of oligosaccharides structurally related to the repeating unit of Pullulan

A trisaccharide (Glcalpha1-4Glcalpha1-6Glc) and a tetrasaccharide (Glcalpha1-4Glcalpha1-4Glcalpha1-6Glc) the structures of which are related to that of repeating unit of pullulan have been obtained, exploiting the transglycolytic activity of Aspergillus niger cyclodextrin glucanotransferase. Both products were obtained in one-pot reaction using as a donor the alpha-cyclodextrin and as an acceptor the disaccharide isomaltose. The regioselectivity of the reaction was 85% for the tetrasaccharide and 80% for the trisaccharide. The yield of reaction resulted to be 42% for the synthesis of trisaccharide and 25% for that of tetrasaccharide. Purification of products was performed by size exclusion chromatography and by semipreparative reverse phase HPLC after reversible derivatization with 2-aminopyridine. Structural characterization was performed by capillary electrophoresis, ion-spray mass spectrometry, and by 13C-NMR spectroscopy. A comparison of these results with those obtained by using alpha-D-glucosidase, which had been effective for the synthesis of the disaccharide isomaltose, is reported.     

Purification and Properties of Wall-bound α-Glucosidase from Suspension-cultured Rice Cells

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroma-tography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64,000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mm. The enzyme produced panose as the main α- glucosyltransferred product from maltose.     

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1.

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Effects of mutation of Asn694 in Aspergillus niger α-glucosidase on hydrolysis and transglucosylation

Aspergillus niger α-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of α-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of α-(1→6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(α1–4)Glc] yields both α-(1→6)- and α-(1→4)-glucosidic linkages, the latter constituting ~25% of the total transfer reaction product. The maltotriose [Glc(α1–4)Glc(α1–4)Glc], α-(1→4)-glucosyl product disappears quickly, whereas the α-(1→6)-glucosyl products panose [Glc(α1–6)Glc(α1–4)Glc], isomaltose [Glc(α1–6)Glc], and isomaltotriose [Glc(α1–6)Glc(α1–6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the α-(1→4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).

     

Characterization of Two Novel α-Glucosidases from Bifidobacterium breve UCC2003

Two α-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the α-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His₁₂ sequence was incorporated (Agl1_His and Agl2_His, respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers—palatinose, trehalulose, turanose, and maltulose—while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1_His exhibits higher thermo and pH optima than Agl2_His. The two purified α-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.