Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae.

Some strains of Klebsiella pneumonia secrete pullulanase, a debranching enzyme which produces linear molecules (maltodextrins, amylose) from amylopectin and glycogen. pulA, the structural gene for pullulanase, was introduced into Escherichia coli, either on a multiple-copy-number plasmid or as a single copy in the chromosome. When in E. coli, pulA was controlled by malT, the positive regulatory gene of the maltose regulon. Indeed, pulA expression was undetectable in a malT-negative mutant and constitutive in a malTc strain. Furthermore, the plasmid carrying pulA titrated the MalT protein. When produced in E. coli, pullulanase was not localized in the same way as in K. pneumoniae. In the latter case it was first exported to the outer membrane, with which it remained loosely associated, and was then released into the growth medium. In E. coli the enzyme was distributed both in the inner and the outer membranes and was never released into the growth medium.     

Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production byBacillus stearothermophilus G-82

Cell growth and extracellular pullulanase production ofBacillus stearothermophilus G-82 were investigated in batch culture using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell growth in batch culture was better under oxygen unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation took place in the late growth phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of oxygen limitation.     

Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

1. 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.

     

Synthesis of phenyl 2-acetamido-2-deoxy-4-O-α-l-fucopyranosyl-β-d-glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-6-O-α-l-fucopyranosyl-β-d-glucopyranoside

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.     

Effect of different substrates and their preliminary treatment on cyclodextrin production

Summary Various kinds of substrates were tested for cyclodextrin production with cyclodextrin glucanotransferase (CGTase) from Bacillus megaterium. The enzyme formed cyclodextrin from different kinds of starch, dextrins, amylose, and amylopectin. However, the highest degree of conversion was obtained from starch. Corn starch appeared to be the best substrate – the cyclodextrin yield was 50.9%. The effect of molecular mass and preliminary treatment of starch with α-amylase on the CD yield was investigated. It was proved that CGTase preferred native starch with high molecular mass and low dextrose equivalent. The preliminary treatment with α-amylase occurred to be inefficient and unnecessary since it did not lead to an increase in the CD yield. Some of the substrates were treated with pullulanase. The effect of debranching was highest in the case of corn starch: the cyclodextrin yield increased by 10%.     

Co-expression of saccharifying alkaline amylase and pullulanase in Micrococcus halobius OR-1 isolated from tapioca cultivar soil

Bacterial isolates from Tapioca cultivar soil were systematically identified. The effect of culture conditions and medium components on the production of extracellular amylase and pullulanase by Micrococcus halobius OR-1 were investigated. Amylase and pullulanase activity in the cell-free supernatant reached a maximum of 8.6 U/ml and 4.8 U/ml after 48 h, respectively. The enzyme converted the complex polysaccharides starch, dextrin, pullulan, amylose and amylopectin predominantly into maltotriose. Saccharification of 15% cereal, tuber starches and root starches with the whole cultured cells (WCC) or cell-free supernatant (CFS) showed comparable and complete saccharification within 90 min. These saccharifying enzymes had a pH optimum of 8.0 and were stable over a broad pH range of 6–12. Thus the coexpressed physicochemically compatible extracellular amylase and pullulanase produced by the Micrococcus halobius OR-1 strain might have important value in the enzyme-based starch saccharification industry.     

Pullulanase synthesis in Klebsiella (Aerobacter) aerogenes strains growing in continuous culture

1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.     

Kinetic studies of the (1 linked to 4)-alpha-D-glucopyranosyltransferase reaction catalyzed by cyclodextrin glycosyltransferase, particularly the cyclization with amylose, amylopectin and total starch as substrate

The time course of the (1 leads to 4)-alpha-D-glucopyranosyltransfer reactions catalyzed by the cyclodextrin glycosyltransferase ((1 leads to 4)-alpha-D-glucan: [(1 leads to 4)-alpha-D-glucopyranosyl]transferase (cyclizing), EC 2.4.1.19, CGT) from Klebsiella pneumoniae was studied with several commercial amyloses, potato starch, and amylopectin, respectively. Amyloses were poor substrates for the cyclization reaction. In the initial phase of the transfer reactions, the CGT catalyzed a rapid shortening of the amylose chains. The rate of this shortening reaction was significantly accelerated by addition of maltooligosaccharides. Maximum rate of cyclohexaamylose formation was reached with amylose chains sufficiently short (less than Glc100) for the cyclization reaction. Cyclohexaamylose was formed with maximum rate from amyloses containing amylopectin impurities in the initial phase of the transfer reactions, suggesting that the non-reducing ends of the outer amylopectin chains serve as acceptors for the disproportionation of the amylose. Accordingly, water-soluble, high-molecular-weight products containing higher percentages of lengthened outer-chains were obtained from potato starch or amylopectin. In the course of the transfer reactions, only traces of smaller maltooligosaccharides were detected chromatographically.     

Synthesis of Maltosyl(α1→6)cyclodextrins through the Reverse Reaction of Thermostable Bacillus acidopullulyticus Pullulanase

Maltosyl(α1→6)α-, β or γ-cyclodextrin was synthesized from maltose and α-, β- or γ- cyclodextrin, respectively, using Bacillus acidopullulyticus pullulanase (EC 3.2.1.41). More than 40% of each cyclodextrin substrate was converted to the corresponding maltosyl(α1→6)cyclodextrin under the conditions given below; the combined concentration of maltose and cyclodextrin was 70 ~ 75 % (w/w), the molar ratio of maltose to cyclodextrin was 9~18, and the amount of pullulanase was 100~200units/g of cyclodextrin. The optimum pH and temperature for the formation of maltosyl(α1→6)cyclodextrins were 4.0—4.5 and 60~70°C, respectively. Each maltosyl(α1→6)-cyclodextrin produced was separated from noncyclic saccharides, maltose and branched tetraose, by methanol and ethanol precipitations. The maltosyl(α1→6)cyclodextrins were further purified by gel filtration on a Toyopearl HW 40 S column and crystallization from aqueous (for maltosyl(α1→6)β-cyclodextrin) or methanol (for maltosyl(α1→6)β-cyclodextrin) solution. From 10 g each of the corresponding cyclodextrin, the yields of the purified maltosyl(α1→6)α-, β- and γ-cylcodextrins were 3.0 ~ 3.6 g, 2.5 ~ 2.8g and 2.2 ~ 2.5 g, respectively. Identification of the maltosyl(α1-6)cyclo-dextrins was performed by means of hydrolysis with Klebsiella pneumoniae pullulanase, methyla- tion analysis and ¹³C-NMR analysis.     

Klebsiella pneumoniae strain K21: evidence for the rapid secretion of an unacylated form of pullulanase

Klebsiella pneumoniae strain PAP996 was previously shown to secrete fatty acylated, aggregated (micellar) pullulanase only after the end of exponential growth. Here we show that the closely related strain K21 secretes large amounts of unacylated, non-aggregated (monomeric) pullulanase during exponential growth. Only a small amount (less than 10%) of the secreted pullulanase was initially retained by the exponentially growing cells to be subsequently secreted in a fatty acylated, aggregated form. Despite the absence of fatty acids in secreted monomeric pullulanase, the effects of the antibiotic globomycin on pullulanase maturation indicated that all of the enzyme synthesized by strain K21 is processed by lipoprotein signal peptidase.     

Starch degradation by the mould Trichoderma viride I. The mechanism of starch degradation

The mechanism of starch degradation by the fungus Trichoderma viride was studied in strain CBS 354.44, which utilizes glucose, starch and dextrins but is unable to assimilate maltose. It was shown that the amylolytic enzyme system is completely extracellular, equally well induced by starch, amylose or amylopectin and that it consists mainly of enzymes of the glucoamylase type which yield glucose as the main product of starch hydrolysis. Small amounts of -amylase are produced also. The enzymes produced in starch cultures degrade starch, amylose and amylopectin equally well.Enzyme synthesis in starch media takes place to a considerable extent after exhaustion of the carbon source when maximum growth has been attained.Low-molecular dextrins are degraded by extracellular enzymes of the glucoamylase type. These enzymes are produced in media containing starch or dextrins. Maltotriose is consumed for only one third leaving maltose in the culture filtrate. Maltose is hardly attacked and hardly induces any amylolytic enzyme activity. No stable -glucosidase appears to be produced.     

Export and secretion of the lipoprotein pullulanase by Klebsiella pneumoniae

Pullulanase, a secreted lipoprotein of Klebsiella pneumoniae, is initially localized to the outer face of the outer membrane, as shown by protease and substrate accessibility and by immunofluorescence tests. Freeze-thaw disruption of these cells released both membrane-associated and apparently soluble forms of pullulanase. Membrane-associated pullulanase co-fractionated with authentic outer membrane vesicles upon isopycnic sucrose-gradient centrifugation, whereas the quasi-soluble form had the same equilibrium density as inner membrane vesicles and extracellular pullulanase aggregates. The latter also contained outer membrane maltoporin, but were largely devoid of other membrane components including LPS and lipids. K. pneumoniae carrying multiple copies of the pullulanase structural gene (pulA) produced increased amounts of cell-associated and secreted pullulanase, but a large proportion of the enzyme was neither exposed on the cell surface nor released into the medium, even after prolonged incubation. This suggests that factors necessary for pullulanase secretion were saturated by the over-produced pullulanase. When pulA was expressed under lacZ promotor control, the pullulanase which was produced was not exposed on the cell surface at any time, suggesting that pullulanase secretion genes are not expressed constitutively, and raising the possibility that they, like pulA, may be part of the maltose regulon.     

A new pullulanase from a hyperthermophilic archaeon for starch hydrolysis

A crude preparation of thermostable pullulanase from Thermococcus hydrothermalis produced glucose and maltose syrups from starches. The use of pullulanase reduced the saccharification reaction time up to 37.5%. In the case of maltose syrup production, the addition of pullulanase to - amylase led to an almost total hydrolysis of the substrate (dextrins) which is translated into a rise in the yield of the whole sugars from 6.5 to 14%.     

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.     

Formation of Isoamylase by Pseudomonas

We have isolated a Pseudomonas sp. (strain SB15) which produces an isoamylase (EC 3.2.1.9). Highest yields of this enzyme were obtained when the bacterium was grown in shaken culture in a medium containing maltose, dextrin, starch, or isomaltose. Specific carbon and nitrogen sources were required for growth. The most satisfactory medium consisted of 2% maltose, 0.4% sodium glutamate, 0.3% diammonium hydrogen phosphate, and other inorganic salts. The optimal pH for enzyme production was 5 to 6. The enzyme is stable between pH 3 and 6 but is extremely labile above pH 7. It splits amylopectin completely by combined action with beta-amylase but does not attack pullulan.     

Metabolism of the reserve polysaccharide of Streptococcus mitis. Some properties of a pullulanase

1. A pullulanase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by fractionation with ammonium sulphate. 2. Pullulanase was produced in the absence of inducers, and addition of glucose or maltose to the broth did not increase the yield of enzyme. 3. The pullulanase acted rapidly on alpha-(1-->6)-bonds in substrates having the structure alpha-maltodextrinyl-(1-->6)-maltodextrin, but had no action on isomaltose, 6-alpha-glucosylmaltodextrins or 6-alpha-maltodextrinylglucoses. 4. 6-alpha-Maltotriosylmaltodextrins were hydrolysed over 10 times faster than 6-alpha-maltosylmaltodextrins. 5. The branch linkages of amylopectin phosphorylase limit dextrin, glycogen phosphorylase limit dextrin and glycogen beta-amylase limit dextrin were hydrolysed. The action of pullulanase on amylopectin and glycogen was accompanied by a rise in the iodine stain of 50% and 30% respectively. 6. A reversal of pullulanase action occurred on incubation with high concentrations of maltotriose. Condensation of maltosyl units to form a branched tetrasaccharide occurred less readily. 7. S. mitis pullulanase was rapidly inactivated at temperatures higher than 40 degrees , and the enzyme did not recover activity on storage at room temperature.     

Crystal structure of the branching enzyme I (BEI) from Oryza sativa L with implications for catalysis and substrate binding

Starch-branching enzyme catalyzes the cleavage of α-1, 4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central α-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the α-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 ? by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the α-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase.     

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R

Background  

The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related α-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans.     

Pullulanase is a characteristic of manyKlebsiella species and functions in the degradation of starch

Summary The type strainsKlebsiella pneumoniae NCTC 9633,K.ozaenae NCTC 5050 andK.rhinoscleromatis NCTC 5046, representative for all members of the genusKlebsiella, were found to produce pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41). In addition, 58 fresh isolates ofKlebsiella sp. of human origin were screened for growth on a defined solid medium with either maltose, maltodextrin mixture, soluble starch, glycogen, or pullulan as the sole carbon source. All of the strains showed luxurious growth on maltose and maltodextrins, seven strains grew poorly or not at all on the polymeric substrates, soluble starch, pullulan or glycogen. Three fresh isolates out of the 51 strains which did grow on each carbon source tested were examined in more detail with respect to a possible involvement of pullulanase in the utilization of -glucans. The production of pullulanase was inducible by growth of the cells on -glucans, whereas cultivation on glycerol, D-glucose or lactose did not lead to enzyme formation. The level of pullulanase activity in the three strains varied under otherwise comparable culture conditions, as did the level of a co-inducible -amylase. Comparative growth experiments on linear or branched -glucans allow the conclusion that the cooperation of hydrolases specific for 1,4--glucosidic linkages (-amylase) and for 1,6--linkages (pullulanase) is an obligatory requirement for the effective utilization of starch and glycogen.     

Isolation and Chemical Characterization of a Mating Pheromone Produced by Saccharomyces kluyveri

The pullulanase gene (pul) of Klebsiella aerogenes was transferred in vivo to Escherichia coli by using RP4:: Mu cts. The pul gene was expressed in E. coli, although the level of pullulanase activity in E. coli was lower than that in K. aerogenes, and the Pul⁺ transconjugants were relatively unstable in an unselective medium. Production of pullulanase, which is used to make maltose from starch, was induced in E. coli by pullulan, waxy maize amylopectin, soluble starch and maltose. When the transconjugant cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Retransfer of the pul_k gene from E. coli to K. aerogenes by conjugation resulted in an increase of the production of extracellular pullulanase.