Pullulans produced by strains of Cryphonectria parasitica—I. Production and characterisation of the exopolysaccharides

Studies published during the last four decades indicated that pullulan has mainly -(1→6) maltotriose subunits, accompanied by -(1→6) maltotetraose subunits and other anomalies in the polymer chain. The presence and the extent of these anomalies depend on the strain of Aureobasidium pullulans used for obtaining the pullulan as exocellular material, and on the culture conditions as well. Besides A. pullulan, other fungi produce this polysaccharide. In all cases, the maximum amount of -(1→6) maltotetraose subunits was believed to be 7%. This paper reports the characterization of pullulan samples, obtained by liquid cultures of a virulent strain (CP159) and two hypovirulent strains (CP263 and CP102) of Cryphonectria parasitica. It was found that in all cases pullulan was much richer in -(1→6) maltotetraose subunits than the pullulan(s) so far investigated. Chestnut blight caused by C. parasitica is a disease of worldwide importance in chestnut production. The structure of pullulan could be correlated with the pathogenesis and symptoms expressed by infected plants.     

Observations on the structure of pullulan

An extracellular α-glucan, pullulan, elaborated by a strain of Pullularia pullulans, contains 0.6% of a maltotetraose subunit, as well as the already known major component, maltotriose. The majority, at least, of the maltotetraose is contained within the polymeric chains and is linked, through its terminal glucose units, by α-1→6— bonds.     

Purification and characterization of a soluble,cytoplasmic decycling maltodextrinase from aLactobacillus species,strain 26X,isolated from kitchen waste water

A soluble, cytoplasmic decycling maltodextrinase with a relative molecular mass (M _r) of around 62 000, and an isoelectric point (pI) of about 4 was purified to 93% homogeneity from aLactobacillus species, strain 26X, that was isolated from the waste water of a kitchen and resembledLactobacillus plantarum in its physiological and biochemical characteristics, but differed from this species in producing exclusively L( + )-lactic acid. The enzyme exhibited higher activities with maltotetraose to maltoheptaose than with cyclodextrins, in which cyclomaltohexaose was hydrolysed fastest, and the amounts of the linearized cyclic substrates were in the order cyclomaltohexaose < cyclomaltoheptaose < cyclomaltooctaose. The substituents of cyclomaltoheptaose derivatives considerably blocked the enzymic activity, and the accessibility to enzymic attack depended on the degree of substitution. The main product of hydrolysis proved to be maltose. The maltodextrinase exhibited, above all with maltotetraose, some transglycosylation activity. Except for maltopentaose, maltosyl transfer predominated. Starch and pullulan were degraded at low rates, the main products from starch being glucose and maltose, and from pullulan a branched trisaccharide, presumably panose.     

Pullulans produced by strains of Cryphonectria parasitica—II. Nuclear magnetic resonance evidence

The structure of pullullan-like polysaccharides produced as exocellular material by different strains of Cryphonectria parasitica, the fungus responsible for chestnut tree cankers, was investigated with nuclear magnetic resonance (NMR) techniques. ¹³C, mono- and bidimensional ¹H, and ¹H–¹³C heteronuclear correlated NMR spectra (HSQC and HMBC) were recorded. Advanced analysis of the NMR spectra allowed the main resonance of the atoms in the maltotriose and in the maltotetraose repeat units of pullulan-like polysaccharides from C. parasitica to be recognised with confidence. In all cases investigated, the presence of large amounts of -(1→6) maltotetraose subunits was evidenced, in addition to the -(1→6) maltotriose subunits, corresponding to the repeating unit of pullulan produced by Aureobasidium pullulans and other fungi. The results were in agreement with other data from this laboratory, obtained with independent techniques. The belief that in ‘pullulans’ the maximum amount of -(1→6) maltotetraose subunits is about 7% can thus be considered as definitely outdated.     

Limited Hydrolysis of Ricin D with Trypsin in the Presence of Sodium Dodecyl Sulfate

An α-amylase which produces both maltotetraose and maltopentaose from starch as the main products was found in the culture filtrate of a strain of Bacillus circulans which was newly isolated from soil. The enzyme was purified to be almost homogeneous on disc electrophoresis in polyacrylamide gel by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 gel filtration.

The optimum pH and temperature of the enzyme were around pH 7.0 and around 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Fe²⁺ and Co²⁺ strongly inhibited the enzyme activity. The molecular weight was about 45,000. The yields of maltotetraose and maltopentaose from potato starch were 30 ~ 40% and 20 ~ 30%, respectively.     

Pullulan production by a non-pigmented strain of Aureobasidium pullulans using batch and fed-batch culture

The production of pigment-free pullulan by Aureobasidium pullulans in batch and fed-batch culture was investigated. Batch culture proved to be a better fermentation system for the production of pullulan than the fed-batch culture system. A maximum polysaccharide concentration (31.3 g l⁻¹), polysaccharide productivity (4.5 g l⁻¹ per day), and sugar utilization (100%) were obtained in batch culture. In fed-batch culture, feed medium composition influenced the kinetics of fermentation. For fed-batch culture, the highest values of pullulan concentration (24.5 g l⁻¹) and pullulan productivity (3.5 g l⁻¹ per day) were obtained in culture grown with feeding substrate containing 50 g l⁻¹ sucrose and all nutrients. The molecular size of pullulan showed a decline as fermentation progressed for both fermentation systems. At the end of fermentation, the polysaccharide isolated from the fed-batch culture had a slightly higher molecular weight than that of batch culture. Structural characterization of pullulan samples (methylation and enzymic hydrolysis with pullulanase) revealed the presence of mainly α-(1→4) (∼66%) and α-(1→6) (∼31%) glucosidic linkages; however, a small amount (<3%) of triply linked (1,3,4-, 1,3,6-, 1,2,4- and 1,4,6-Glc p) residues were detected. The molecular homogeneity of the alcohol-precipitated polysaccharides from the fermentation broths as well as the structural features of pullulan were confirmed by ¹³C-NMR and pullulanase treatments followed by gel filtration chromatography of the debranched digests.     

Structure of Dextran Synthesized by Dextrin Dextranase from Acetobacter capsulatus ATCC 11894

The structure of dextran synthesized from maltotetraose by dextrin dextranase (EC 2.4.1.2) from Acetobacter capsulatus ATCC 11894 was analyzed. When the Acetobacter dextran (AD) was acetolyzed, glucose and maltose were produced. AD was allowed to react with α-amylases. AD was digested by bacterial saccharifying α-amylase and bacterial liquefying α-amylase, and glucose, maltose, and maltotriose were produced. The structure of the fraction obtained from dextranase-digested AD by activated charcoal chromatography, which did not contain glucose, isomaltose, and isomaltotriose, was investigated by methylation analysis, and the ratio of 2,3,4,6-tetra-O-methyl-: 2,3,4-tri-O-methyl-: 2,3,6-tri-O-methyl-: 2,3-di-O-methyl-alditol acetate was estimated as 22.9:46.8:15.5:14.8. This result indicated the existence of α-1,4 branches and that of α-1,4 linkages in α-1,6 glucosyl linear chains. Native AD was calculated to be constructed with 6.23 branching points and 6.53 α-1,4 linked glucosyl residues per 100 glucosyl units. Though AD was digested slightly by rat intestinal acetone powder, high molecular weight polymers remained. Therefore AD could be used as a dietary fiber.     

A New Fungal α-D-glucan,elsinan, elaborated by Elsinoe leucospila

A new α-D-glucan, designated elsinan, has been isolated from the culture filtrate of Elsinoe leucospila grown in potato extract-sucrose medium. Acid hydrolysis of the methylated polysaccharide gave 2,3,6- and 2,4,6-tri-O-methyl-D-glucose, in the ratio of 2.5:1.0, together with small proportions of 2,3,4,6-tetra- (0.7%) and 2,4-di-O-methyl-D-glucose (0.5%), indicating that the glucan is an essentially linear polymer containing (1→4)- and (1→3)-α-D-glucosidic linkages. Periodate oxidation, followed by borohydride reduction and mild hydrolysis with acid (mild Smith degradation) yielded 2-O-α-D-glucosyl-D-erythritol and erythritol, in the molar ratio of 1.0:1.4, and a trace of glycerol. Partial acid hydrolysis, and also acetolysis, of elsinan gave nigerose, maltose, O-α-D-glucopyranosyl-(1→3)-O-α-D-glucopyranosyl (1→4)-D-glucopyranose, O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→3)-D-glucopyranose, maltotriose, and a small proportion of maltotetraose. It is concluded that elsinan is composed mainly of maltotriose residues joined by α-(1→3)-linkages, in the sequence →3)-α-D-Glcp-(1→4)-α-D-Glcp-(1→.The unique structural features of elsinan are discussed in comparison with other glucans.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.     

Starch metabolism in Pseudomonas stutzeri. I. Studies on maltotetraose-forming amylase.

The extracellular maltotetraose-forming amylase of Pseudomonas stutzeri was purified to homogeneity by a combination of affinity and hydroxyapatite chromatography. Sodium dodecyl sulfate-gel electrophoresis indicated that the oligomeric enzyme contains two different subunits with molecular weights of 48 000 and 58 000. Cross-linking studies using dimethyl suberimidate have demonstrated that the native enzyme consists of dimers. Seven isozymes of the amylase have been identified after polyacrylamide gel electrophoresis and amylose-digestion zymograms. The amylase of Ps. stutzeri is known to produce maltotetraose from linear and branched alpha-glucans by an exomechanism. The relatively high conversion rate of starch (75% hydrolysis), and the hydrolysis of cross-linked blue starch by this amylase indicate that the enzyme can cleave its substrates also by an endomechanism. Further strong evidence for an endomechanism was obtained from the action of the amylase on maltotetraose units which are located within the pullulan molecule. Dextran, pullulan, and maltotetraose are compeititve inhibitors. EDTA caused reversible inactivation. Amylase activity could be restored by addition of Ca2+. Heavy metals are inhibitory.     

Fractionation of the Leuconostoc mesenteroides NRRL B-1299 Dextran and Preliminary Characterization of the Fractions

The extracellular dextran elaborated by Leuconostoc mesenteroides NRRL B-1299, which was shown to be heterogeneous, was separated into five fractions by assorted fractionation methods. Each fraction turned out to be homogeneous by ultracentrifugation and/or electro-phoretic analysis. Gel filtration analysis suggested that these fractions were divided into two groups on the basis of their average molecular weights, one of which had comparatively low molecular weight of 150,000~200,000 (BPS and CPS) and the other had high molecular weight of about 2,000,000 (BPP, CPP and CS). From the results of periodate oxidation, each fraction was shown to contain 41~46% of 1,6-glucosidic linkage (including non-reducing terminal group) as well as 1,4- like (1,4- and/or 1,2-) and 1,3-like linkages. Partial acid hydrolysis of each fraction yielded a series of α-1,6-linked oligosaccharides and acetolysis gave koji-biose and nigerose. Moreover, these fractions gave characteristic precipitation patterns, when incubated with concanavalin A. On the basis of these results, the structural features of the fractions were discussed.     

Asymmetric Reduction of Iminium Salt with Chiral Dihydropyridines

A gram positive bacterium (strain No. 109) isolated from soil as a producer of cyclodextrinase was identified as Bacillus coagulans. The cyclodextrinase from B. coagulans was purified to a homogeneous state by disc-electrophoresis after Streptomycin treatment, DEAE-Sephadex column chromatography, Ultrogel AcA44 gel filtration and hydroxyapatite column chromatography. The molecular weight of the enzyme was determined to be 6.2}10⁴ by sodium dodecyl-sulfate gel electrophoresis. The isoelectric point of the enzyme was pH 5.0. The enzyme was most active at pH 6.2 and 50°C, and stable up to 45°C at pH 7.0 and in the range of pH 6.0 ~ 7.3 at 40°C on 2 hr incubation. This enzyme hydrolyzed linear maltooligosaccharides (such as maltotetraose (G4), maltopentaose (G5) and maltohexaose (G6)) and α-, β- and α-cyclodextrins (CDs) faster than maltotriose (G3) and short chain amylose ( 18), but did not hydrolyze maltose. The rates of hydrolysis for polysaccharides (such as starch, amylose and amylopectin) were below 1 % as compared to that for β-CD. The Km values for G3, G4, G5, G6, short chain amylose ( 18) and α, β- and γ-CD were 4.5, 4.0,2.3,1.5,1.5,10,2.8 and 0.47 mM, respectively. The products with this enzyme had the α-configulation.     

Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1

Exopolysaccharide produced by a new novel colour variant strain of Aureobasidium pullulans FB-1 was purified by cell harvesting and precipitation of the polymer. Various organic solvents were screened for pullulan precipitation. Isolation and purification of pullulan from fermentation broth was carried out using single-step purification strategy by isopropyl alcohol precipitation. Ratio of culture supernatant to isopropyl alcohol and time of precipitation were optimized for pullulan precipitation. Maximum yield (4.47%, w/v) of polysaccharide was obtained when two volumes of ice-cold isopropyl alcohol were added to one volume of supernatant with precipitation time of 12 h. IR spectra as well as carbon-13 and proton NMR spectra in aqueous solution of intact polysaccharide obtained from A. pullulans FB-1 and commercially available pullulan (Sigma, USA) revealed solely α-(1 → 6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Maximum hydrolysis (94.25%) of purified pullulan at 50 °C by pullulanase was achieved under agitation (150 rpm) after 360 min.     

PURIFICATION AND CHARACTERIZATION OF PULLULANASE FROM Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co²⁺ and completely inhibited by Hg²⁺. The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

Novel teichulosonic acid from cell walls of some representatives of the genus Kribbella

Cell walls of each of five bacterial strains belonging to the genus Kribbella (family Nocardioidaceae, order Actinomycetales) contain a neutral polysaccharide (mannan) and teichulosonic acid of novel structure in different proportions. The novel teichulosonic acid found in strains VKM Ac-2500, VKM Ас-2568, VKM Ас-2572, and VKM Ас-2575 is a heteropolymer with an irregular structure where fragments I (predominant) alternate with fragments II (minor):The teichulosonic acid from Kribbella sp. VKM Ac-2527 has in general a structure similar to that above with the exception that the Pse residue is randomly glycosylated at O-4 with β-l-Rhap (along with α-d-Galp3OMe or α-d-Galp2,3OMe). The strain VKM Ac-2572 contained additionally teichuronic acid with the disaccharide repeating unit consisted of aminomannuronic acid and 2,3-diacetamido-2,3-dideoxy-α-glucopyranose. The mannan, a polysaccharide common to all of the strains, is built of (1→6)-linked α-d-mannopyranose substituted with α-d-mannopyranose at O-2. The structures of all the glycopolymers were established by a combination of chemical and NMR spectroscopic methods.     

Action of Rice α-Glucosidase on Maltose and Starch

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3~2.6 times in both enzymes at the concentration of 50 mM of potassium chloride. Such activation was also caused by mono and di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-glucosidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.     

Cloning,purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1

Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 °C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications.     

Synthesis of N-Carbamyl-d-2-thienyl-glycine and d-2-Thienylglycine by Microbial Hydantoinase

A debranching enzyme purified from germinating rice endosperm hydrolyzed oligosaccharides having maltosyl or maltotriosyl branches (B₄-B₆) moderately. Hydrolysis of maltosylmaltose by a “pullulanase” of higher plant origin has been scarcely reported, while our enzyme debranched maltosylmaltose like microbial pullulanase. Additionally, the enzyme slowly hydrolyzed isopanose to glucose and maltose.

Gel-filtration analyses of hydrolysis products of polysaccharides with the enzyme suggested that while it hydrolyzed α-1,6-linkages of pullulan at random, it hydrolyzed amylopectin and glycogen at the outer α-1,6-linkages preferentially In the hydrolysis products of glycogen with the enzyme for a longer incubation time, large molecular-weight glucans still remained. This indicated that the enzyme was able to hydrolyze a few of the α-1,6-linkages of glycogen.     

Functional expression and enzymatic characterization of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> cyclomaltodextrinase catalyzing novel acarbose hydrolysis

Cyclomaltodextrinases (CDases) belong to Glycoside Hydrolases (GH) family 13, which show versatile hydrolyzing and/or transglycosylation activity against cyclodextrin (CD), starch, and pullulan. Especially, some CDases have been reported to hydrolyze acarbose, a potent α-glucosidase inhibitor, and transfer the resulting acarviosine-glucose to various acceptors. In this study, a novel CDase (LPCD) gene was cloned from Lactobacillus plantarum WCFS1, which encodes 574 amino acids (64.6 kDa) and shares less than 44% of identities with the known CDase-family enzymes. Recombinant LPCD with C-terminal six-histidines was produced and purified from Escherichia coli. It showed the highest activity on β-CD at 45°C and pH 5.0, respectively. Gel permeation chromatography analysis revealed that LPCD exists as a dodecameric form (~826 kDa). Its hydrolyzing activity on β- CD is almost same as that on starch, whereas it can hardly attack pullulan. Most interestingly, LPCD catalyzed the unique modes of action in acarbose hydrolysis to produce maltose and acarviosine, as well as to glucose and acarviosineglucose.