Hans Bender

Pullulan, ein extracelluläres Glucan von Pullularia pullulans

The formation is described of a extracellular polysaccharide by a yeast-like fungus, morphologically identical with Aureobasidium pullulans syn. Pullularia pullulans. The polymer is formed in a yield of 20 to 22% in Czapek-Dox medium with glucose, saccharose and fructose as a source of carbon. Thiamine has a stimulating effect increasing the yield to 32%. However, the production during optimal growth on maltose was low. The polymerisation product has a optical activity [α]D20 in H2O of + 168°. Chromatography showed glucose to be present as sole product after hydrolysis. After partial hydrolysis isomaltose and a small percentage of maltose were identified. The infra-red spectrum suggested a predominant percentage of α-I,4-linkages. The similarity to bacterial dextrans is discussed.

The enzymic synthesis, by transglucosylation of a homologous series of glycosidically substituted malto-oligosaccharides, and their use as amylase substrates

Abstract (1 → 4)-α- d -Glucan 4-glucosyltransferase (EC2.4.1.19) of klebsiella pneumoniae transforms maltose (G2) into d -glucose (G1) and a mixture of malto-oligosaccharides (G 2 —G 9 ), and maltotriose (G3) into G l —G ll in addition to cyclo-hexa-, -hepta-, and -octa-amyloses (cG 6—8 ). It produces a similar mixture, but with higher amounts of G 2 —G 11 , by transfer from cyclohexaamylose to G 1 . By using p -nitrophenyl α- and β- d -glucosides, 4 methylumbelliferyl α- d -glucoside, and strophanthyl α- d -glucoside as acceptors and cyclohexaamylose as donor, a homologous series of substituted malto-oligosaccharides having chain lengths of up to 12 d -glucose residues was produced. High-pressure liquid chromatography on B io-Gel P 2 permitted separation of these products of transferase activity on analytical and preparative scales. By the same technique, the nitration product of phenyl hepta- O -acetyl-α-maltoside, after deacetylation, was separated into about equal amounts of the o - and p -isomers. The synthetic p -nitrophenyl α-maltoside ( p NPG 2 ) was used to identify the first member of the series of biochemical transfer-products. p -Nitrophenyl maltotrioside ( p NPG 3 ) and maltotetraoside ( p NPG 4 ) were shown to be the higher homologues. They are very good substrates for human and pig-pancreatic alpha amylase. This substrate behavior may be measured conveniently in the case ofpNPG3 by the rapid liberation of nitrophenolate; the enzyme used p NPG 4 only on addition of α- d -glucosidase. Human-parotis amylase of equal starch-splitting activity as the pancreatic enzyme acts upon p NPG 3 and p NPG 4 but about 100 times more slowly.

Molecular cloning, nucleotide sequence and expression in Escherichia coli of the β-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8

SummaryThe β-cyclodextrin glycosyltransferase (β-CGTase) gene was isolated from a λ-library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant β-CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme.

Can cyclodextrin glycosyltransferase be useful for the investigation of the fine structure of amylopectins?: Characterisation of highly branched clusters isolated from digests with potato and maize starches

Abstract Maize and potato amylopectin (57 and 64%, respectively) were recovered as non-cyclic products from 4-h digests of the starches with cyclodextrin glycosyltransferase {(1→4)-α- d -glucan:[(1→4)-α- d -glucopyranosyl]transferase (cyclising), EC 2.4.1.19} from Klebsiella pneumoniae M 5 al. Besides smaller saccharides, highly branched fragments of different sizes (average d.p. 40–140) were obtained by fractionation. The extents of beta-amylolysis varied between 24 and 37%, indicating that the clusters were not equally susceptible to attack by cyclodextrin glycosyltransferase. The fragments of potato amylopectin still contained larger amounts of material of high molecular weight. Accordingly, part of the longer B-chains of the basic structure were protected from the enzymic attack, presumably because of interchain branches. By debranching with pullulanase, it was evident that the beta-limit dextrins of the fragments of potato amylopectin were composed of longer B-chains (average chainlength 17.8) than those of maize amylopectin (average chain-length 14.1). The A/B-chain ratios, which were calculated from h.p.l.c. data for the debranched beta-limit dextrins, were 1.22 (maize) and 1.06 (potato). Some structural differences between potato and maize amylopectin are discussed.

[95] Pullulanase (an amylopectin and glycogen debranching enzyme) from Aerobacter aerogenes

Publisher Summary This chapter discusses the preparation of pullulanase from Aerobacter aerogenes. Pullulan is a linear α-glucan produced by the yeastlike fungus Pullularia pullulans, in which about, 480 maltotriose units are linked by 1→ 6 glycosidic bonds. By pullulanase it is exclusively degraded to maltotriose units. The assay method for pullulanase is based on the determination of maltotriose split off from pullulan by the enzyme. The increase in reducing power is measured with Nelson reagent. The pullulanase unit is defined as that amount of enzyme, which in 1 minute at 30 ° and a pH of 5.0 liberates 1 micromole of maltotriose. The cultivation of Aerobacter and preparation of the pullulanase is also described in the chapter. Batchwise-cultivated Aerobacter excretes the pullulanase into the culture filtrate while continuously cultivated cells contain the enzyme cell-bound. Pullulanase produced under the conditions of continuous cultivation is tightly bound. Even prolonged shaking of the resuspended cells in water, different buffers, or hypertonic solutions removed only one-third of the cell-bound activity.

Studies of the mechanism of the cyclisation reaction catalysed by the wildtype and a truncated α-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al, and the β-cyclodextrin glycosyltransferase from Bacillus circulans strain 8

The actions of the wildtype and a truncated alpha-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al on malto-oligosaccharides showed no significant differences, and there was marked dependence of the kinetic parameters on the chain lengths of the substrate. The action of the beta-cyclodextrin glycosyltransferase from Bacillus circulans was less dependent on the chain length of the substrate, but Vmax of the initial cyclisation with the longer malto-oligosaccharides was only 28% of that determined for the enzyme of K. pneumoniae. The rate parameters suggested that the active site of each enzyme spans nine glucosyl residues, and that the catalytic sites are situated between subsites three and four for the K. pneumoniae enzymes and between subsites two and three for the B. circulans enzyme. The molecular binding affinities and the affinities of the 9th subsite were calculated from the rate parameters. The primary and tertiary structures of alpha-amylases and cyclodextrin glycosyltransferases are compared in the context of the reaction mechanism of the latter enzymes.

Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

Abstract1.When growing with cyclodextrins, Klebsiella pneumoniae M 5 al produces extracellular cyclodextrin glucanotransferase in amounts comparable to those obtained during the growth with potato starch.2.Intracellular cyclodextrin glucanotransferase-activity was demonstrated to be present in the homogenates of cells grown with cyclodextrins. In addition, an amylomaltase-like enzyme and the maltodextrin phosphorylase could be pointed out. The cyclodextrins are metabolized to glucose-1-phosphate and glucose by the concerted actions of these three enzymes. paraGlucose-1-phosphate is liberated from cyclohexaamylose by the actions of purified cyclodextrin glucanotransferase and purified maltodextrin phosphorylase. The liberation of the sugar phosphate is increased fivefold by addition of glucose as an acceptor. This sugar, however, retards the formation of glucose-1-phosphate from the cyclic compound by the enzymes of the cell extract: In the presence of glucose the amylomaltase is incapable of synthesizing substrates for the phosphorylase from maltose. This experimental result clearly demonstrates that the amylomaltase is involved in the disproportionation of maltosaccharides arising from the cyclodextrins.3.A NADP+-specific glucose dehydrogenase was demonstrated to be present in the cell extracts. This enzyme, which is activated by ADP, may control the energy-depending pool of free glucose. Glucose originates from the disproportionation of maltosaccharides catalyzed by the glucanotransferases.4.A glucose-1-phosphate-hydrolysing phosphatase, which is shown to be present in the cell extract, seems to be without physiological significance for the metabolism of the cyclodextrins.5.Preliminary permeation studies make it probable that the cyclodextrins are transported into the cells as such and degraded only within the cells.6.A scheme for the metabolism of cyclodextrins in Klebsiella pneumoniae M 5 al is proposed.Zusammenfassung1.Klebsiella pneumoniae M 5 al wächst mit Cyclodextrinen gleich gut wie mit Glucose. Die mit den cyclischen Kohlenstoffquellen synthetisierte Menge an extracellulärer Cyclodextrin-Glucanotransferase entspricht derjenigen, wie sie bei Wachstum mit Kartoffelstärke produziert wird.2.Im Zellextrakt der mit Cyclodextrinen gewachsenen Zellen kann intracelluläre Cyclodextrin-Glucanotransferase festgestellt werden. Daneben lassen sich ein amylomaltase-ähnliches Enzym und die Maltodextrin-Phosphorylase nachweisen. Diese drei Enzyme sind im wesentlichen für den Abbau der Cyclodextrine verantwortlich.Gereinigte Cyclodextrin-Glucanotransferase und gereinigte Maltodextrin-Phosphorylase setzen aus Cyclohexaamylose Glucose-1-Phosphat frei. Während in diesem System die Geschwindigkeit der Glucose-1-Phosphat-Freisetzung durch Zugabe des Akzeptors Glucose verfünffacht wird, verzögert Glucose die Bildung von Glucose-1-Phosphat durch die Enzyme des Zellextraktes: Die Amylomaltase ist in Gegenwart von Glucose unfähig, Substrate für die Phosphorylase aus Maltose aufzubauen. Aufgrund dieser experimentellen Befunde kann die Beteiligung des Enzyms an der Disproportionierung der durch Linearisierung der Cyclodextrine entstehenden Maltosaccharide als sicher gelten.3.Das Zellhomogenat enthält eine durch ADP aktivierbare NADP+-spezifische Glucose-Dehydrogenase. Dieses Enzym könnte den energie-abhängigen Pool an freier Glucose kontrollieren. Glucose entsteht bei der Disproportionierung der Maltosacchaide durch die Glucanotransferasen.4.Eine im Zellextrakt nachzuweisende Glucose-1-Phosphat spaltende Phosphatase scheint keine physiologische Bedeutung beim Metabolismus der Cyclodextrine zu haben.5.Orientierende Permeationsuntersuchungen machen es wahrscheinlich, daß die Cyclodextrine als solche in die Zelle transportiert und erst innerhalb der Zelle linearisiert werden.6.Anhand der experimentellen Befunde wird ein Schema für den Metabolismus der Cyclodextrine durch Klebsiella pneumoniae M 5 al gegeben.

(1→4)-α-d-Glucopyranosyltransfer-produkte aus cyclohexaamylose☆

Abstract At sufficiently high concentrations of the substrate cyclohexaamylose and low concentrations of the acceptors maltose or d -glucose, the cyclodextrin glycosyl-transferase (EC 2.4.1.19) from Klebsiella pneumoniae M 5 al catalyses (1→4)-α- d -glucopyranosyl-transfer reactions giving amylose-like (1→)-α- d -glucopyranosyl chains ( d.p. 45) up to 28.7% of the total carbohydrate content. Increase of the acceptor concentration reduces the proportion of the longer chains and the average chain-length. The proportions of cyclooctaamylose, of cyclohexaamylose, and of maltooligosaccharides depend on the acceptor concentration. Significantly larger proportions of the longer chains, and of cycloheptaamylose are formed with the acceptor maltose. Even at equimolar concentrations of substrate and acceptor, larger proportions of (1→4)-α- d -glucopyranosyl chains ( d.p. 16) are formed in the early phase of the transfer reaction, 2.6 times as much with maltose as with d -glucose. Correspondingly, much more cycloheptaamylose is present if the initial acceptor is maltose. The relationship between the d -glucopyranosyl chains of d.p. 16 and the formation of cycloheptaamylose, and between the chains of d.p. > 20 and the formation of cyclooctaamylose is discussed.

Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp.

A cell-bound cyclodextrin-degrading enzyme with a relative molecular mass (Mr) of around 62 000 and an isoelectric point (pI) near 8.0 was isolated and purified to 94% homogeneity from Flavobacterium sp. The enzyme hydrolysed maltooligosaccharides and cyclodextrins to glucose, maltose, and maltotriose. Less glucose, but larger amounts of the line of maltooligosaccharides from maltose to (in case of cyclodextrins) the linearized substrates were found in short-term digests. Digestion of maltotriose yielded glucose, maltose, and some maltotetraose to maltohexaose, i.e. the enzyme catalysed both hydrolysis and transglycosylation. Starch was a poorer substrate, and was hydrolysed to mainly glucose and maltose, presumably by a kind of exo-attack. Pullulan was slightly digested, the products being glucose, panose/isopanose, and larger saccharides containing α-1,6-glucosidic bonds. Since maltohexaose to maltooctaose were hydrolysed at higher rates than the cyclodextrins of corresponding lengths, the enzyme of Flavobacterium sp. was proposed to be classified as a decycling maltodextrinase.

Kinetische untersuchungen der durch die cyclodextrin-glycosyltransferase katalysierten (1→4)-β-d-glucopyrano-syltransferreaktionen, insbesondere der zyklisierungs-reaktion, mit (1→4)-α-D-glucopyranosylketten “durchschnittlicher polymerisationsgrad von 16” als substrat☆

Abstract The transfer reactions, particularly the cyclization reaction, catalysed by the cyclodextrin glycosyltransferase {(1→4)-α- D -glucan:[(l→4)-α- D -glucopyranosyl]-transferase (cyclizing), EC 2.4.1.19; CGT} from Klebsiella pneumoniae M 5 al were studied with (1→4)-α- D -glucopyranosyl chains (d.p. 16). The initial rate of the cyclization reaction with substrate concentrations from 1 up to 16m M indicated a V of 6.2 kat &∣ kg -1 of protein and a molar catalytical activity of 421.6 kat &∣ mol -1 of enzyme. K m was found to be 1.03m M . In addition to the cyclization, CGT simultaneously catalysed a disproportionation of the substrate, yielding shorter maltooligosaccharides and (1→4)-α- D -glucopyranosyl chains which were significantly longer than the substrate itself. Cyclohepta- and cycloocta-amylose were accumulated in the course of longer incubation. They arose mainly from coupling reactions with the initially formed cyclohexaamylose and corresponding disproportionation of these transfer products. The extremely low formation rates of the higher cyclodextrins point to a “mistake” of the enzyme, when cyclizing to cyclohepta- and cyclooctamylose.