Co-immobilization of amyloglucosidase and pullulanase for enhanced starch hydrolysis

Summary Amyloglucosidase and pullulanase were co-immobilized using a hydrophilic polyurethane foam (Hypol^® 2002). The combined amyloglucosidase and pullulanase activity of the immobilized enzyme was 32.2% ± 1.7% relative to the non-immobilized enzyme. The co-immobilized enzymes were capable of using a variety of glycogen and starch substrates. Co-immobilization of amyloglucosidase and pullulanase increased the glucose yield 1.6-fold over immobilized amyloglucosidase alone. No decrease in activity was observed after 4 months storage for the co-immobilized enzymes. The results suggest that co-immobilization of amyloglucosidase and pullulanase in polyurethane foams is a potentially useful approach for commercial starch hydrolysis. Offprint requests to: K. B. Storey     

Starch Measurement in Plant Tissue Using Enzymatic Hydrolysis

This work explored completeness of starch hydrolysis in situ in relation to degree of gelatinization, starch content of tissue, evailable enzyme activity, and time allowed for hydrolysis. Maximum hydrolysis of starch in lyophilized red oak (Quercus rubra L.) root tissue with purified Diazyme (amyloglucosidase) or Clarase (Takadiastase) required high enzyme activity (2.4 U Diazyme or 48 U Clarase per mg starch). Results suggested that at least 70 U Clarase or 5 U Diazyme should be used per mg starch in routine analyses. Neither prolonging gelatinization (more than 15 min) nor hydrolysis (more than 24 to 48 lh) offset inadequate starch hydrolysis caused by insufficient enzyme activity. Starch was completely hydrolyzed in situ after 48 h without gelatinization by 5 U of Diazyme per mg starch. Tissue weight (5 to 100 mg) had no effect on starch hydrolysis by sufficient enzyme. Methanol: chloroform: water (12:5:3 by volume) freed tissues of solubles before starch hydrolysis. No interference with the glucose oxidase analysis of hydrolysates was encountered. In addition, the pigment free methanol–water fractions (soluble sugars, amino acids, organic acids) and chloroform fractions (lipids and pigments) were available or further analysis. Results obtained with red oak were verified with issue from other species such as jack pine (Pinus banksiana lamb.) and white spruce (Picea glauca (Moench) Voss). The resulting technique simply and reliably measured less than 5% starch in 5 mg lyophilized tissue, with a minimum of sample manipulation.     

Comparison of Immobilized and Free Amyloglucosidase Process in Glucose SyrupsProduction from White Sorghum Starch

Optimum conditions for glucose syrups production from white sorghum were studied through sequential liquefaction and saccharification processes. In the liquefaction process, a maximum dextrose equivalent (DE) of 10.98 % was achieved using 30 % (w/v) of starch and Termamyl ɑ-amylase from Bacillus licheniformis. Saccharification was performed by free and immobilized amyloglucosidase from Rhizopus mold at 1 % (w/v). DE values of 88.32 % and 79.95 % were obtained from 30 % (w/v) of starch with, respectively, free and immobilized enzyme. The immobilized Amyloglucosidase in calcium alginate beads showed reusable capacity for up to 6 cycles with 46 % of the original activity retained. The kinetic behaviour of immobilized and free enzyme gives K_m value of 22.13 and 16.55 mg mL⁻¹ and V_max of 0.69 and 1.61 mg mL⁻¹ min⁻¹, respectively. The hydrolysis yield using immobilized amyloglucosidase were lower than that of the free one. However, it is relevant to reuse enzyme without losing activity in order to trim down the overall costs of enzymatic bioprocesses as starch transformation into required products in industrial manufacturing. Hydrolysis of sorghum starch using immobilized amyloglucosidase represents a promising alternative towards the development of the glucose syrups production process and its utilization in various industries.     

Syntheses of hydroxybenzyl-α-glucosides by amyloglucosidase-catalyzed transglycosylation

Glycosides were synthesized from soluble starch and hydroxybenzylalcohols (o, m and p) by the transglycosylation activity of amyloglucosidase from Rhizopus sp. in a water system. The synthesized glycosides were identified as hydroxybenzyl--glucosides, of which one molecule of hydroxybenzylalcohol was bound to 1-OH of glucose. The transglycosylation reaction was performed in a reaction system containing 50 mg starch, 50 mg hydroxybenzylalcohol, and 10 units of enzyme in pH 5.0 at 45°C. The glycoside was rapidly hydrolyzed to glucose and hydroxybenzylalcohol by the -glucosidase.     

An enzymic microassay for starch

Abstract Conditions are described for measuring the starch content of plant tissues or extracts as glucose over the range from 10^?7 mol to 10^?14 mol. The method is based on the hydrolysis of gelatinized starch by amyloglucosidase; the glucose released is measured by reduction of NADP⁺ by coupled enzymic reactions. The NADPH is determined directly either spectrophotometrically or fluorimetrically, or after enzymic amplification. Amyloglucosidases were tested for contaminating enzymes which might degrade glucans other than starch, and a commercial preparation from Rhizopus niveus was found to be suitable for use without pretreatments. Glucose present in tissues and extracts may be measured and subtracted from starch values using appropriate blanks, or first destroyed by dilute alkali and heat. Addition of α-amylase to amyloglucosidase during starch hydrolysis was not found to increase percentage hydrolysis from the normal range of 86–99% from starches of different sources. The procedures described are rapid and several orders of magnitude more sensitive than current methods, and can be used to measure the starch content of single cells.     

Production of ethanol from raw cassava starch by a nonconventional fermentation method

Raw cassava root starch was transformed into ethanol in a one-step process of fermentation, in which are combined the conventional processes of liquefaction, saccharification, and fermentation to alcohol. Aspergillus awamori NRRL 3112 and Aspergillus niger were cultivated on wheat bran and used as Koji enzymes. Commercial A. niger amyloglucosidase was also used in this experiment. A raw cassava root homogenate–enzymes–yeast mixture fermented optimally at pH 3.5 and 30°C, for five days and produced ethanol. Alcohol yields from raw cassava roots were between 82.3 and 99.6%. Fungal Koji enzymes effectively decreased the viscosity of cassava root fermentation mashes during incubation. Commercial A. niger amyloglucosidase decreased the viscosity slightly. Reduction of viscosity of fermentation mashes was 40, 84, and 93% by commercial amyloglucosidase, A. awamori, and A. niger enzymes, respectively. The reduction of viscosity of fermentation mashes is probably due to the hydrolysis of pentosans by Koji enzymes.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

A one-step process for the production of single-cell protein and amyloglucosidase

Summary A new Rhizopus species was isolated from traditional Indonesian food, tempeh. The newly isolated species was similar in its morphological characteristics to Rhizopus oligosporus UQM 145F, but grew faster on potato-dextrose agar as well as in submerged culture. The new isolate was found to convert ground cassava tuber directly into single cell protein without pretreatment due to its high amyloglucosidase formation.From 100 g ground tuber, a dry biomass of 33.75 g containing 26.48% true protein together with 60 ml of highly active amyloglucosidase (282 units) was obtained in 12 h. The amyloglucosidase was recovered by ultrafiltration, releasing 26.226 millimol glucose/l/min from soluble starch. The crude enzyme exhibited a pH optimum between 4.6 and 5.0, a temperature optimum between 55 and 60° C and an apparent Km of 3.125 g/l. High substrate concentrations and ammonium sulphate are inhibitory to the enzyme.     

Co-encapsulation of amyloglucosidase with starch and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as basis for a long-lasting CO<Subscript>2</Subscript> release

CO₂ is known as a major attractant for many arthropod pests which can be exploited for pest control within novel attract-and-kill strategies. This study reports on the development of a slow-release system for CO₂ based on calcium alginate beads containing granular corn starch, amyloglucosidase and Saccharomyces cerevisiae. Our aim was to evaluate the conditions which influence the CO₂ release and to clarify the biochemical reactions taking place within the beads. The amyloglucosidase was immobilized with a high encapsulation efficiency of 87% in Ca-alginate beads supplemented with corn starch and S. cerevisiae biomass. The CO₂ release from the beads was shown to be significantly affected by the concentration of amyloglucosidase and corn starch within the beads as well as by the incubation temperature. Beads prepared with 0.1 amyloglucosidase units/g matrix solution led to a long-lasting CO₂ emission at temperatures between 6 and 25?°C. Starch degradation data correlated well with the CO₂ release from beads during incubation and scanning electron microscopy micrographs visualized the degradation of corn starch granules by the co-encapsulated amyloglucosidase. By implementing MALDI-ToF mass spectrometry imaging for the analysis of Ca-alginate beads, we verified that the encapsulated amyloglucosidase converts starch into glucose which is immediately consumed by S. cerevisiae cells. When applied into the soil, the beads increased the CO₂ concentration in soil significantly. Finally, we demonstrated that dried beads showed a CO₂ production in soil comparable to the moist beads. The long-lasting CO₂-releasing beads will pave the way towards novel attract-and-kill strategies in pest control.     

Purification and Properties of α-Glucosidase and Glucoamylase from Lentinus edodes (Berk.) Sing.

An α-glucosidase and a glucoamylase have been isolated from fruit bodies of Lentinus edodes (Berk.) Sing., by a procedure including fractionation with ammonium sulfate, DEAE-cellulose column chromatography, and preparative gel electrofocusing. Both of them were homogeneous on gel electrofocusing and ultracentrifugation. The molecular weight of α-glucosidase and glucoamylase was 51,000 and 55,000, respectively. The α-glucosidase hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose, and soluble starch, but did not act on sucrose. The glucoamylase hydrolyzed maltose, maltotriose, phenyl α-maltoside, soluble starch, amylose, amylopectin, and glycogen, glucose being the sole product formed in the digests of these substrates. Both enzymes hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. The glucoamylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen, converting them almost completely into glucose. It was found that β-glucose was liberated from amylose by the action of glucoamylase, while α-glucose was produced by the α-glucosidase.

Maltotriose was the main α-glucosyltransfer product formed from maltose by the α-glucosidase.     

Studies on α-Glucosidase in Rice

Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.

Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).

Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.

Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II.     

A thermophilic amyloglucosidase fromHalobacterium sodomense,a halophilic bacterium from the Dead Sea

Halobacterium sodomense, a halophilic bacterium from the Dead Sea, degraded starch to glucose by means of an extracellular amyloglucosidase with a temperature optimum of around 65°C in the presence of 1.4 M NaCl, and around 75°C in the presence of 3.9 M NaCl. The enzyme required salt concentrations higher than 1 M for optimal activity, NaCl, KCl, and MgCl₂ being equally suitable as activators. The optimum pH was 7.5.H. sodomense culture supernatants showed only a very low maltose degrading activity. H. sodomense excreted amyloglucosidase constitutively, and relatively high activities were found in cultures grown in the absence of starch; when glucose was added to the growth medium, the amount of enzyme excreted into the medium decreased.     

Influence of culture conditions on the cell yield and amylases biosynthesis in continuous culture by Schwanniomyces castellii

Schwanniomyces castellii excreted -amylase and amyloglucosidase into the medium in the presence of starch. The biosynthesis and the rate of excretion were influenced by dissolved oxygen (specially for -amylase), pH of the culture and dilution rate. The cell yield observed (0.59) remained constant up to D=0.35h^-1 with starch as substrate. But in the case of growth on glucose, the yield observed was equal to 0.62 up to a dilution rate of D=0.18 h^-1. Beyond this value Y x/s decreased and ethanol was produced. The onset of fermentation dependend partly on the nature of the substrate and not only on the environment in particular on the quantity of dissolved oxygen present.     

Two Forms of Glucoamylase from Mucor rouxianus

Both of the two forms of glucoamylase (glucoamylases I and II) from the wheat bran culture of Mucor rouxianus hydrolyzed amylopectin, amylose, glycogen, soluble starch, maltotriose, and maltose, but did not act on isomaltose and isomaltotriose. Phenyl α-maltoside was hydrolyzed into glucose and phenyl α-glucoside by both glucoamylases. Maltose was hydrolyzed about one-fifth as rapidly as amylopectin. Both enzymes produced glucose from amylopectin, amylose, glycogen, soluble starch in the yields of almost complete hydrolysis. They hydrolyzed amylose with the inversion of configuration, producing the β-anomer of glucose. Glucoamylase II hydrolyzed raw starch at 3-fold higher rate than glucoamylase I. The former hydrolyzed rice starch almost completely into glucose, whereas the latter hydrolyzed it incompletely (nearly 50%).     

Immobilization of amyloglucosidase on poly [(glycidyl methacrylate) Co(ethylene dimethacrylate)] carrier and its derivatives

Immobilization of amyloglucosidase (EC 3.2.1.3) from Endomycopsis bispora and Aspergillus niger was followed with carriers containing epoxide, aldehyde, and primary amino groups. Determinations of stability of bound enzyme showed that the most active and stable preparations were obtained by application of the carrier with amino groups activated with glutaraldehyde (activity half-life 96.6 days). Optimum pH and and temperature were found for cleavage of starch solution and K_M(app) and V_max(app) values were determined for all prepared samples.     

Production and Starch Saccharification by a Thermostable and Neutral Glucoamylase of a Thermophilic Mould Thermomucor Indicae-Seudaticae

The present investigation was aimed at producing a thermostable and neutral glucoamylase (amyloglucosidase, EC 3.2.1.3) by a thermophilic mould, Thermomucor indicae-seudaticae in submerged cultivation and testing its applicability in starch saccharification. Parametric optimization resulted in the secretion of 30,000 U/l of glucoamylase in a synthetic medium (5% soluble starch, 0.1% yeast extract, 0.05% K₂HPO₄ and 0.01% MgSO₄· 7H₂O) using 5 × 10⁶ spores/50 ml of a 3-day-old inoculum at 40 °C and 250 rev/min in shake flasks in 48 h. The enzyme secretion was not affected to any significant extent by the tested additives and detergents. A 1.7-fold increase in glucoamylase secretion was attained when T. indicae-seudaticae was grown in a laboratory fermenter. The enzyme alone catalysed the hydrolysis of soluble starch to an extent of 65%. A prior treatment of starch with thermostable α-amylase and amylopullulanase, followed by glucoamylase, resulted in a greater extent of hydrolysis, 79 and 91%, respectively.     

Effect of starch application into the proximal duodenum of ruminants on starch digestibility in the small and total intestine

Four Slovakian Black‐and‐white bulls (LW 410 ± 12 kg; Exp. 1) and four Slovakian Black‐and‐white non lactating dairy cows (LW 475 ± 14 kg; Exp. 2) with permanent ruminal cannulas, duodenal T‐cannulas and ileal re‐entrant cannulas were used in a 4 × 4 Latin square design to determine the postruminal capacity of starch digestion.

In Exp. 1 bulls received 5.4 kg DM from corn silage and 3.6 kg DM from alfalfa hay, in Exp. 2 cows consumed only 2.1 kg DM corn silage and 1.9 kg DM alfalfa hay. Additionally, either 750 or 1500 g (Exp. 1) or resp. 1000 or 2000 g (Exp. 2) gelatinized corn or wheat starch per animal and day were applied as pulse doses or as infusion into the proximal duodenum.

In both experiments the duodenal and ileal nutrient flow, as well as the faecal excretion without starch application, were measured in a pre‐period. After starting starch application ileal digesta and faeces were sampled over 120 h after 9 or 23 days of adaptation respectively. Cr₂O₃ was used as a flow marker.

It was shown, that the capacity of starch utilisation in the small intestine was limited. The effect of different doses of bypass‐starch was more pronounced than the effect of different starch sources. Starch digestibility decreased with increasing amounts of starch in the intestine (Exp. 1: corn starch: from 74.3 to 68.0%, P < 0.001; wheat starch: from 76.7 to 67.4%, P <0.001; Exp. 2: corn starch: from 71.4 to 50.3%, P <0.001; wheat starch: from 73.8 to 53.1%, P <0.001). Corn starch was 0.6 to 2.4% units (P <0.05) and 2.4 to 2.8% units (P < 0.001) less digested than wheat starch in Exp. 1 and Exp. 2, respectively.

The decreased starch digestibility in the small intestine with increasing amounts of starch at the duodenum was not totally compensated in the large intestine. The starch digestibility in the total intestine for the low and high amounts of applied starch was: 83.7 and 81.0% (P < 0.001, corn starch, Exp. 1), 86.0 and 81.7% (P < 0.001, wheat starch, Exp. 1), 95.5 and 79.1% (P < 0.001, corn starch, Exp. 2), 99.8 and 81.7% (P < 0.001, wheat starch, Exp. 2).

Corn starch was 0.7 to 2.3% units (P <0.001) and 2.6 to 4.3% units (P <0.001) less digested than wheat starch in Exp. 1 and Exp. 2, respectively.

Model calculations were used to quantify the efficiency of starch utilisation. The recommended maximal amount of bypass‐starch is supposed to be 1.3 to 1.8 kg per animal and day.     

Efficiency of genetically engineered yeast in the production of ethanol from dextrinized cassava starch

Summary The ability of a polyploid/aneuploidSaccharomyces diastaticus spheroplast fusion product and a diploidSaccharomyces diastaticus hybridization product, to produce ethanol from dextrinized cassava starch with varying amounts of supplemented glucoamylase (amyloglucosidase), was investigated. It was found that the added glucoamylase could be reduced by over 50% using these glucoamylase producing strains as compared to a commercially availableSaccharomyces cerevisiae strain commonly used in ethanol producing industries.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

Thermostable α‐Amylase and Glucoamylase from Thermomyces lanuginosus F1

An α‐amylase and a glucoamylase produced by Thermomyces lanuginosus F₁ were separated by ion‐exchange chromatography on Q‐Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G‐100 and Phenyl‐Sepharose CL‐4B.The molecular weights and isoelectric points of the enzymes were 55,000 Da and pHi 4.0 for α‐amylase and 70,000 Da and pH_i 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60 °C for α‐amylase, and 6.0 and 70 °C for glucoamylase,respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90 °C (1 h), the α‐amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α‐amylase had highest affinity for soluble starch with K_M values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α‐amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α‐amylase and glucoamylase increased in the presence of Mn²⁺, Co²⁺, Ca²⁺, Zn²⁺ and Fe²⁺, but were inhibited by guanidine‐HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.