ISOLATION AND CHARACTERISATION OF THE AMYLOTIC SYSTEM OF SCHWANNIOMYCES CASTELLII

The amylolytic system of Schwanniomyces castellii has been isolated and purified by means of ultrafiltration followed by polyacrylamide gel electrophoresis. Both α-amylase and glucoamylase were purified. α-Amylase activity was stable from pH 5·5 to 6·5 and glucoamylase activity was stable at a more acidic range of pH 4·2 to 5·5. The optimal temperature of α-amylase activity was between 30 and 40°C with rapid deactivation at 70°C. The optimal temperature of glucoamylase was 40 to 50°C with rapid decline of activity at 60°C. The K_m of α-amylase with soluble starch as the substrate was 1·15 mg/ml and the K_m of glucoamylase with the same substrate was 10·31 mg/ml. Glucoamylase was able to hydrolyze α-1, 4 and α-1,6 glucosidic linkages, as demonstrated by its ability to hydrolyse maltose and isomaltose respectively, whereas α-amylase could hydrolyse α-1,4 glucosidic linkages only. α-Amylase was shown to be a glycoprotein, whereas no carbohydrates were associated with glucoamylase.     

Purification and Characterization of a Maltotriogenic α-Amylase I and a Maltogenic α-Amylase II Capable of Cleaving α-1,6-Bonds in Amylopectin

Extracellular α-amylases I and II, produced by a facultative thermophile Bacillus thermoamyloliquefaciens KP 1071 capable of growing at 30–66°C, were purified to homogeneity. α-Amylase I consisted of a single polypeptide with methionine residue at the NH₂-terminus. α-Amylase II consisted of two equivalent polypeptides each comprising a methionine at the NH₂-terminus. α-Amylase I hydrolyzed endotypically α-1,4-bonds in glycogen, amylopectin and β-limit dextrin, but not their α-1,6-bonds. α-Amylase II degraded amylopectin and β-limit dextrin in exo-fashion by cleaving preferentially α-maltose units from the non-reducing ends and hydrolyzing their α-1,6-branch points. α-Amylase II hydrolyzed maltotriose, phenyl-α-maltoside, α- and β-cyclodextrins and pullulan, whereas α-amylase I had no activity for all these sugars. α-Amylases I and II hydrolyzed maltotetraose, maltopentaose, α-limit dextrin and amylose, but they were inactive for maltose, isomaltose and panose. It was suggested that α-amylase I is the most thermostable type of hitherto known maltotriogenic endo-acting α-amylases, and α-amylase II is the first maltogenic exo-acting α-amylase able to split α-1,6-bonds in amylopectin.     

Die Wirkung von α-Amylasen auf β-Cyclodextrin und der Nachweis von α-Amylasefremdaktivitäten in ausgewählten Enzympräparaten

The action of α-amylases on β-cyclodextrin and the evidence of foreign activity of α-amylase in selected preparations of enzymes The interaction between cyclodextrins and α-amylases taken from different sources is discribed contradictious in the literature. Some α-amylases e.g. isolated from Aspergillus oryzae, porcine pancreas and saliva hydrolized cyclodextrins to glucose. The hydrolysis of cyclodextrins catalysed by α-amylase from Bacillus species have been described conflicting. In this paper the action of hydrolysis of different preparations of α-amylases on β-cyclodextrin have been investigated. It has been shown that Rohalase M3 (α-amylase from Aspergillus niger) cleaves the ring of β-cyclodextrin. 2 α-amylases from Bacillus subtilis are not able to hydrolyse β-cyclodextrin. The reasons for the different actions of hydrolysis have been discussed with size and structure of the active centre of the enzymes. Moreover, different preparation of hydrolysis have been tested on secondary activity of α-amylase. 2 glucoamylases from Aspergillus oryzae have been shown secondary activity of α-amylase. With the hydrolases α-glucosidase from fungies, β-amylase from malt, saccharase from yeast, invertase from S. cerevisiae and pullulanase from Aerobacter aerogenes no cleavage of the ring of β-cyclodextrin could be detected.     

PRODUCTION OF AMYLOLYTIC ENZYMES BY SEVERAL YEAST SPECIES

Several amylolytic yeast species, endomycopsis spp, schwanniomyces spp, pichia spp and saccharomyces spp have been compared for their ability to synthesise α-amylase (E.C. 3.2.1.1), glucoamylase (E.C. 3.2.1.3) and pullulanase (E.C. 3.2.1.9). Endomycopsis fibuligera strain 240 possessed the highest glucoamylase activity (208 nmoles glucose/min) and second highest α-amylase activity (128 nmoles maltose/min) and produced the largest amount of biomass. Schwanniomyces spp were the only yeast species studied which exhibited significant debranching activity. It was found that in endomycopsis spp, schwanniomyces spp and pichia spp, glucose, at a concentration above 3.0 × 10^?3 m , appeared to repress α-amylase activity. However, glucoamylase activity was not repressed at that glucose concentration.     

Some Methods for the Characterisation of Natural and Dyed Amyloses as Potential Substrates for the Assay of α-Amylase

Methods based on ion exclusion and gel permeation chromatography and enzymic degradation are reported for the assessment of the character, purity and amylopectin contents of amylose samples and their suitability for conversion by dyeing with Cibacron® Blue 3 G-A to give coloured substrates for the assay of α-amylase. The methods were also applicable to the assessment of the dyed materials. A new method for the estimation of α-(1→6) linkages in starch samples, based on exhaustive hydrolysis with β-amylase is reported. The susceptibilities of the free dye and dyed soluble amylose to acid and alkali are described along with their application to the measurement of the dye loading of dyed amyloses.     

Production of Branched Cyclodextrins by Reverse Reaction of Microbial Debranching Enzymes

Formation of maltosyl cyclodextrins from mixtures of maltose and cyclodextrins by reverse reactions of Flavobacterium isoamylase and Klebsiella pullulanase was experimented and it was found that Klebsiella pullulanase produced 50.4mg maltosyl α-cyclodextrin, 35.0mg maltosyl β-cyclodextrin and 55.4mg maltosyl γ-cyclodextrin per ml of reaction mixture, whereas Flavobacterium isoamylase did not form maltosyl cyclodextrins. Optimum conditions for formation of maltosyl β-cyclodextrin by Klebsiella pullulanase were pH 4, 50°C reaction temperature and proportion of substrate 100mg β-cyclodextrin/600mg maltose per ml.     

Kombinierte enzymatische Stärkehydrolyse

Combined Enzymatic Starch Hydrolysis. From researches so far there comes out that glucoamylase AMG 300 L® and pullulanase Promozyme 200 L® when used in quantities the same as in preparation of Dextrozyme 225/75 L® Novo at an action on liquified starch by means of α-amylase after 48 h of saccharification already (similarly like Dextrozyme®) are able to get up to 98 DE. Chromatographic analysis proved that glucoamylase AMG 300 L Novo® and succouring it pullulanase Promozyme 200 L® are working most effectively when both enzymes are added to the liquified starch medium simultaneously. From this comes out that pullulanase hydrolyzes better α-1,6 bonds in lowmolecular dextrins than in oligosaccharides G₄ to G₇ formed at previous action of glucoamylase. At an optimum ratio of glucoamylase and pullulanase in relation to the dissolved starch after 8 h of the hydrolysis there are neither iso-sugars (isomaltose, panose), no oligosaccharides higher than G₅ and no dextrins. At the solution of the starch by α-amylase and its hydrolysis by enzymatic preparation Fungamyl 800 L Novo®, at doses 0,02–0,08% to d. s. of starch, already after 8 h the reaction of hydrolysis contents of 36–62% maltose in dry substance of hydrolyzates are reached with only traces of glucose.     

Molecular cloning and characterization of a thermostable α-amylase exhibiting an unusually high activity

An α-amylase gene was cloned from the thermophilic bacterium Bacillus subtilis isolated from Indonesian oil palm shell waste. The gene expressed an extracellular enzyme. Optimal hydrolysis conditions for the enzyme were 70°C and pH 6.0. The specific activity of the enzyme was 16.0 kU per mg of protein, which was higher than for other thermostable amylases. Hydrolytic products of the enzyme using starch and glycogen were mainly maltohexaose and maltopentaose. The enzyme had a K _m value of 0.099 mg/mL for amylopectin, more than 10 times lower than for amylose. The catalytic efficiency of the enzyme using amylopectin was 39,200 mL/mg·s and was 3,270 mL/mg·s using amylose. The enzyme liquefied corn starch at pH 5.0, which was successfully converted to glucose using commercial glucoamylase and pullulanase without pH adjustment. The enzyme has advantages for industrial applications.     

COMPUTER SIMULATION OF THE β-AMYLOLYSIS OF STARCH COMPONENTS*

In an attempt to model quantitatively the product distributions arising from the degradation of starch by the conjoint action of α- and β-amylase, such as occurs in the commercial mashing of malted cereal, a computer program has been written to simulate the amylolysis of amylose or amylopectin or a mixture of these components. Using Monte-Carlo techniques the program simulates the action of either α-amylase or β-amylase or both enzymes acting conjointly. The program was tested for pure β-amylolysis. Parameters were estimated by a non-linear regression technique and predicted product concentrations were compared with relevant experimental data for amylose and amylopectin substrates and for mixtures of these starch components. The simulation model accurately predicted the results of amylolysis except at high product concentrations where the deviations between predicted and experimental values ranged from 1% to 4%.     

Digestibility of Amylose-Lipid Complexes in-vitro and in-vivo

Amylose from potatoes was complexed with lysolecithin and oleic acid. The degradation of complexed amylose by hog pancreatic α-amylase in-vitro was studied, as well as the in-vivo absorption in the rat. The presence of a bacterial thermostable α-amylase in the gelatinization step increased the result of a starch analysis using glucoamylase. Complexed amylose displayed a substantially reduced susceptibility to α-amylase in-vitro. However, when adding a large excess of enzyme, the complex was completely hydrolyzed after 3 h. Amylose-lysolecithin complex disappeared from the gastrointestinal tract within 120 min. The complexed amylose was hydrolyzed and adsorbed to the same extent as free amylose in-vivo but somewhat slower.     

Properties of Residual Starches of Sugary-2 and Sugary-2 Opaque-2 Maize Following Amylase Hydrolysis

Properties of residual starches of sugary-2 opaque-2 and sugary-2 maize starch granules hydrolyzed with glucoamylases were investigated. A crude and two crystalline glucoamylases were used. The amylopectin fractions of both starches hydrolyzed easier than that of amylose with all enzymes. Residual starches hydrolyzed by the crude glucoamylase accumulated low-molecular weight materials, which was not observed in residual starches attacked by crystalline glucoamylase. It was suggested that in the crude enzyme the contaminating α-amylase caused the accumulation of the minified fraction. It is also suggested that the crystalline region of sugary-2 opaque-2 starch may consist of a mixture of A-type and B-type patterns. Evidence for this was from observation of the changes in X-ray diffraction patterns of residual starch following amylase and acid hydrolysis.     

Studies on the Starches of Barley Genotypes: The Waxy Starch

Two varieties of waxy barley originating from Japan, Sumiremochi and Mochimugi, have been characterized in detail for the first time. These two glutinous starches were found to be essentially identical. Both contained less than 0.5% of amylose, had a β-amylolysis limit of 53% conversion into maltose, and an average length of unit-chain of about 23 glucose units. The weight average molecular weight was 300 × 10⁶. These properties are characteristic of amylopectin. The iodine binding capacity of six other varieties of Japanese waxy barley has been measured and apparent amylose contents of 3 to 13% were obtained. Possible reasons for the presence of this amount of amylose are discussed.     

The Interaction of the Amylose-Extender and Waxy Mutants of Maize (Zea Mays L.) Fine Structure of Amylose-Extender Waxy Starch

Investigations into the nature of the effect of the amylose-extender (ae) mutant of maize (Zea mays L.) on amylopectin structure were conducted by studying the fine structure of amylose-extender waxy (ae wx) starch. Approximately 59.6% of the starch from ae wx endosperms was converted to maltose by β-amylase. This starch contained 21% apparent amylose and had a λmax of 580 for the iodine-starch complex. Fractionation of ae wx starch on Sepharose 4B-200 gave an elution profile with a single peak characteristic of amylopectin. From these observations we concluded that ae wx starch consisted of an altered amylopectin, with iodine binding properties such that an apparent amylose content of 21% was measured. The fine structures of ae wx and waxy (wx) starches were determined. Pullulanase-debranched chains of whole starches and β-limit dextrins were fractionated by gel permeation. The amylopectin of ae wx was shown to be a loosely branched amylopectin with an average internal chain length of 52 glucose units compared with a length of 30 glucose units for wx. The ae wx outer chains were longer than those of wx and fewer in number per mg of starch. These characterizations demonstrate that ae wx starch has a unique structure which is similar to the anomalous amylopectin reported in ae starch.     

Comparative Studies on Glucoamylases Isolated from a Strain of Aspergillus

Two glucoamylase fractions have been isolated from an Aspergillus culture preparation by anion-exchange chromatography and characterised by physico-chemical and biochemical methods. The fractions were homogeneous on electrophoresis, molecular sieve chromatography and ultracentrifugation with molecular weights of 81,000. Both fractons were shown to be devoid of endo-amylase activity and to possess similar activities towards wheat amylopectin (15–17 IU mg⁻¹) and maltose (3–4 IU mg⁻¹) at the optimum pH of 4.6. The fractions catalysed extensive, but not complete, conversion of wheat amylopectin to D -glucose which was liberated with β-anomeric configuration.     

Acceptor Molecule of Granular-bound Starch Synthase from Sweet-potato Roots

In order to elucidate acceptor molecule of starch synthase bound to starch granules, the enzyme from sweet-potato roots was reacted with ADP- and UDP-[¹⁴C]-glucose, and the reaction products were structurally characterized. The radioactivity of the products was released as [¹⁴C]-maltose by β-amylase. The [¹⁴C]-glucose incorporation was found to be mostly in amylopectin component. However, approximately 30 and 20% of total radioactivity transferred from ADP- and UDP-[¹⁴C]-glucose, respectively, were also present in a molecule with a lower molecular weight than that of amylose component. The radioactivity was incorporated into the much longer unit-chains of amylopectin than into the usual chains. These results indicate that starch synthase bound to sweet-potato starch granules transfers glucose from ADP- and UDP-glucose into specific outer chains of amylopectin component, and that the incorporation of the glucose into a short-chain amylose molecule is simultaneously catalyzed by the starch synthetase.     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

Immobilised β-Amylase in the Production of Maltose Syrups

In order to have a full continuous starch hydrolysates production process, the use of continuous conversions catalysed by immobilised enzymes is necessary. With respect to this, immobilisation of β-amylase on anion exchange resins and continuous conversion of maltodextrins to maltose syrups is investigated in packed bed column reactors at lab-scale. It is observed that the type of carrier and the source of the β-amylase are important parameters for the required capacity of the continuous process. Results show that, from the investigated enzymes, the commercial Spezyme BBA 1500L® β-amylase preparation immobilised on Duolite A568® as preferred carrier gives the best results in terms of initial performance and stability under realistic conversion conditions (50°C, pH 4.5, 50% d.s. substrate). On basis of the present results, the process is promising in terms of applicability at plant-scale after optimisation.     

Characterization of Starch from Sagittaria trifolia L. var. sinensis Makino

The physicochemical properties of starch from the corm of Sagittaria trifolia L. var. sinensis Makino (arrowhead) were investigated. The starch contained 31.65% of amylose and 0.0897 mg/g of phosphorus. It had a gelatinization temperature range of 56.1–61.7–64.9°C, a mixed type of Brabender viscosity pattern, a one-stage swelling pattern, 99.6% water binding capacity, low solubility in dimethyl sulfoxide, and high a-amylase susceptibility. The amylose was found to be a branched molecule of DP 2202 and was hydrolyzed 86.6% with β-amylase. Its amylopectin had an average chain length of 24.5 and was hydrolyzed 65.5% with β-amylase. The characteristics of this starch were different from those of typical corm, bulb and tuber starches.     

The Interaction Between Cibacron® Blue 3G-A Dyed Amyloses and Microcrystalline Cellulose

It has been shown with the aid of chemical analysis, gel permeation chromatographic analysis of malto-oligosaccharide mixtures produced by the action of α-amylase (1, 4,-α-D-glucan glucanohydrolase, E. C. 3. 2. 1. 1.) and photomicroscopy, that dyed amylosic fragments, produced by the conventional action of α-amylase on Cibacron® Blue 3G-A dyed amylose substrates, can become adsorbed by microcrystalline cellulose. The adsorption occurs between the aromatic residues of the dye molecule and the surface of microcrystalline cellulose, and only when the amount of solubilisation of the dyed amylose substrate is high. Thus, under these circumstances erroneously low values for α-amylase activity will be obtained when using such a chromogenic substrate due to adsorption of soluble dyed fragments onto the surface of microcrystalline cellulose, thereby escaping detection. When α-amylase levels are low, thereby causing little solubilisation of the dyed substrate, as is frequently the case in clinical chemistry, the amount of absorption cannot be detected and therefore the use of microcrystalline cellulose as a tablet binder to facilitate the routine use of these dyed amylose chromogenic substrates is permissible.     

Physicochemical Property of Huaishan (Rhizoma Dioscorea) and Matai (Eleocharis dulcis) Starches

The physicochemical properties of starch, the main component in tuberous root of Huaishan (Rhizoma Dioscorea) and corm of Matai (Eleocharis dulcis) was investigated and compared with those of a Japanese yam, “Yamanoimo” starch, and potato starch. Mean particle sizes of Huaishan and Matai starches were 24 ± 5 μm and 12 ± 5 μm, respectively. X-Ray diffraction pattern suggests that Huaishan starch was B-type or C-type just close to B-type and Matai starch was A-type or C-type just close to A-type. Apparently, the intermediate component (IntCom) of Huaishan starch, which was obtained by fractionation of the starch into amylose and amylopectin, may still contain amylose and amylopectin. IntCom of Matai starch has an intermediate nature between amylose and amylopectin. It is concluded that the amylopectin molecules of Huaishan starch contain a larger amount of longer branch-ed chains and those of Matai starch contain a larger amount of shorter branched chains. Amylograms of Matai and Huaishan starches suggest that the gelatinized starches are difficult to retrograde. Digestibility of Huaishan starch by an α-amylase was the highest among the tested starches.