Amyloglucosidase Research Articles

A Flow Injection System for the Determination of Starch in Starch from Different Origins with Immobilized α‐Amylase and Amyloglucosidase Reactors

AbstractA heat stable α‐amylase (Termamyl) was immobilized on controlled pore glass (CPG) with a pore size of 1 489Å and amyloglucosidase (AMG) was immobilized on a ceramic silica support (Micropil A) with a pore size of 300Å. These enzyme supports were packed into two separate immobilized enzyme reactors (IMERs) which together with a third reactor containing co‐immobilized glucose dehydrogenase/mutarotase were incorporated into a flow injection (FI) system for the determination of the total glucose content of starch related poly‐ and oligosaccharides. Samples of maltose, a maltooligosaccharide mixture, three soluble starches, three amylopectins, two amyloses, glycogen, and native starches from different origins were injected. The degree of hydrolysis was determined by comparing the produced amount of glucose in the FI system with the calculated amount of glucose in the samples and with samples to which had been added and let to react for six hours soluble α‐amylase and AMG before injected into the FI system also containing the IMERs. Virtually complete conversion to glucose was obtained for maltose, maltooligosaccharides, two soluble starches and native potato starch. Maximum enzymatic degradation by the starch hydrolyzing enzyme reactors was obtained in most instances except for glycogen (96%), native wheat (88%), rice (93%), and corn starch (83%).

A kinetic model and simulation of starch saccharification and simultaneous ethanol fermentation by amyloglucosidase and Zymomonas mobilis

A mathematical model is described for the simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and Zymomonas mobilis. By introducing the degree of polymerization (DP) of oligosaccharides produced from sago starch treated with α-amylase, a series of Michaelis-Menten equations were obtained. After determining kinetic parameters from the results of simple experiments carried out at various substrate and enzyme concentrations and from the subsite mapping theory, this model was adapted to simulate the SSF process. The results of simulation for SSF are in good agreement with experimental results.

Enzyme and protein mass transfer coefficient in aqueous two-phase systems—II. York-Scheibel extraction column

Fractional dispersed phase hold-up and dispersed side mass transfer coefficients for bovine serum albumin (BSA) and amyloglucosidase (AMG) were measured in 50 mm i.d. York-Schiebel columns using salt-polyethylene glycol (4000), potassium phosphate-PEG and sodium sulphate-PEG systems. The effect of column height and the effect of phase compositions of the aqueous phase system were studied. The critical impeller speed for flooding and the critical superficial dispersed phase velocity for flooding were measured. Empirical correlations have been developed for fractional dispersed phase hold-up and dispersed side mass transfer coefficients. The performance of the York-Scheibel column has been compared with that of the spray column.

Multi-enzyme electrodes for the determination of starch by flow injection

A tri-enzyme electrode has been developed for determining starch in a flowing stream based on amperometric monitoring of hydrogen peroxide at a potential of +600 mV versus a silver--silver chloride reference electrode. The nylon-based starch-sensing membranes (over a platinum electrode) were prepared from an enzyme cocktail containing various ratios of amyloglucosidase (AMG), mutarotase (MUT) and glucose oxidase (GO). The best starch-sensing membrane (to give the type A electrode) was made from an enzyme cocktail of AMG--MUT--GO (2000 + 100 + 100 U; where 1 U = 16.67 nanokatals), i.e., containing a high level of AMG. In this system, starch samples were first incubated with soluble alpha-amylase (alpha-AMY) (1000 U) for 1 h at room temperature prior to analysis with the tri-enzyme electrode. Attempts were also made to immobilize alpha-AMY on to nylon net, either alone or as a component of a four-membrane starch-enzyme electrode but starch signals were weak compared with those generated by starch pre-treated with soluble alpha-AMY. This system, associated with the type A starch electrode, not only exhibited a calibration of wide linear range (1 x 10(-4)-0.1% m/v starch) but also showed promising operational properties. It has excellent thermal stability over the range 30-70 degrees C.

An economical, upgraded, stabilized and efficient preparation of amyloglucosidase

An economical preparation of amyloglucosidase (AMG) is obtained from a transglucosidase-nonproducing mutant of Aspergillus awamori by qualitative and quantitative optimization of the ingredients of its production medium as well as production parameters. Further, it is upgraded by filtration and 14-fold concentration through an ultrafiltration membrane. Finally, its maximal stabilization during storage at ambient temperature is attained by using 14% (w/v) partial hydrolysate of starch as a stabilizer. While the concentrated preparation without stabilizer showed less than 50% of the original activity after 24 weeks of storage, the concentrated preparation, fortified with 14% (w/v) partial hydrolysate of starch, showed 80% stabilization during the same period. This stabilizer could be substituted by 1 × 10 −3 M thiomersal for comparable stabilization of concentrated amyloglucosidase preparation. This enzyme preparation afforded efficient (96% DX, w/w) saccharification of 35% (w/w) tapioca starch in 48–54 h. Thus, these steps have cumulatively provided an economical, upgraded, stabilized and efficient alternative to commercially available AMG preparations.

ChemInform Abstract: The Substrate Specificity of the Enzyme Amyloglucosidase (AMG). Part 3. Synthesis of Epimers and Mono‐O‐methyl Ethers of Methyl β‐Maltoside.

ChemInform Abstract: The Substrate Specificity of the Enzyme Amyloglucosidase (AMG). Part 3. Synthesis of Epimers and Mono‐O‐methyl Ethers of Methyl β‐Maltoside.

Immobilization of Zymomonas mobilis and amyloglucosidase for ethanol production from sago starch

Immobilization of amyloglucosidase ( AMG) and Zymomonas mobilis cells was studied in order to produce ethanol from sago starch economically. Among various immobilization methods tested, a coimmobilized system using chitin and sodium alginate appeared most promising with respect to ethanol productivity and operational stability. When the system was run in the continuous simultaneous saccharification and fermentation ( SSF) mode, the maximum ethanol productivity was found to be 72.2 g l −1 h −1 at a dilution rate of 3.28 h −1. This system could be run in the stable manner over 40 days with a steady-state ethanol concentration of 44 g l −1 and an ethanol conversion yield of 93%.

PROPERTIES OF A GENETICALLY-ENGINEERED DEXTRIN-FERMENTING STRAIN OF BREWERS' YEAST

A dextrin-fermenting strain of brewer's yeast was obtained by transformation with a recombinant plasmid (pLHCD6) containing a DEX gene. The transformant produced five-fold more extracellular amyloglucosidase (AMG) than the strain of Saccharomyces diastaticus from which DEX was cloned. The growth rate, however, was adversely affected and, consequently, the transformant fermented wort more slowly than the parental (untransformed) strain. A mixed culture was therefore used at pilot-scale, to maintain a rate of fermentation similar to that of the parental strain. The transformant by itself was capable of superattenuating wort (original gravity 1.040) to a specific gravity of 1.0023 (compared with 1.0046 for the parental strain). This represents the expected degree of dextrin breakdown by an AMG with no “debranching” activity which can only hydrolyse alpha-1,4 linkages. A high copy number for the plasmid pLHCD6 was maintained throughout fermentation. The beers produced using the Dex+ transformants were judged to be of sound quality.

ChemInform Abstract: The Substrate Specificity of the Enzyme Amyloglucosidase (AMG). Part 1. Deoxy Derivatives.

AbstractThe various monodeoxy ((Ia)‐(Ic), (IIb), (VIb), (VIII), (IXb)) as well as the bis‐deoxy ((Xa), (Xb)) derivatives of maltose are synthesized.

The Substrate Specificity of the Enzyme Amyloglucosidase (AMG). Part III. Synthesis of Epimers and Mono-O-methyl Ethers of Methyl beta-Maltoside.

The Substrate Specificity of the Enzyme Amyloglucosidase (AMG). Part III. Synthesis of Epimers and Mono-O-methyl Ethers of Methyl beta-Maltoside.

Isolation of a transglucosidase-nonproducing mutant ofAspergillus awamori yielding improved quality and production of amyloglucosidase preparations

The fermentative production of amyloglucosidase (AMG) by differentAspergillus species simultaneously yields transglucosidase (TG), which is undersirable in the conversion of starch to dextrose, as it catalyses the reversion of dextrose and maltose to maltosaccharides, in turn providing disproportionately lower yields of dextrose (DX). To overcome this problem, using UV-irradiation, a novel mutant (ND-1-283) has been isolated fromAspergillus awamori, which has lost the ability to produce TG and which secretes 45% more AMG than its parent strain, giving the mutant a dual operational advantage. The inability of this mutant to produce TG was demonstrated by thin layer chromatography (TLC) of starch hydrolysate; this was substantiated by obtaining 96.0% DX (w/w) at the end of saccharification.

The substrate specificity of the enzyme amyloglucosidase (AMG). Part I. Deoxy derivatives.

The eight possible monodeoxy derivatives of methyl beta-maltoside and two bisdeoxy derivatives have been synthesized. The unprotected glycosides have all been investigated by NMR (1H and 13C) spectroscopy in order to confirm their structures and to obtain supporting information about their preferred solution conformations. The compounds have all been tested as substrates toward the hydrolase, amyloglucosidase (AMG) and it has been demonstrated that three hydroxy groups (3, 4' and 6') are essential for the compounds to act as substrate for the enzyme. The kinetic parameters KM (Michaelis-Menten constant) and VM (maximum rate for the reaction) have been determined using 1H NMR spectroscopy at 500 MHz.

Carbohydrate-protein interactions: substrate specificity of enzymes used in the degradation of oligosaccharides related to starch and cellulose

Treatment of amylopectin with an ct—amylase (Terma— myl) produces a branched pentasaccharide in good yield. The structure of this product has been determined by high field NMR spectroscopy, where particularly homonuclear 2-dimensional methods have been indispensable. A new sensitive and fast 1-dimensional experiment for the correlation of the carbon— and proton chemical shifts of the above mentioned pentasaccharide will be presented. Chemical modifications of maltose and cellobiose have shown that specific hydroxyl groups are required for the binding and subsequently for enzymatic reaction with the enzymes amyloglucosidase (AMG) and cello— biase, whereas removal of other hydroxyl groups enhances the affinity of the modified substrates towards the enzymes in agreement with the polar gate theory proposed by Lemieux. Furthermore, the preferred conformations of the oligosaccha— ride in solution can be correlated with experimental results in terms of hydrophobic and hydrophilic surfaces of the carbohydrate substrates. Thus X—ray data of an amylase inhibitor, acarbose, in the active site of an o—amylase from por— chine pancreas, show that acarbose is bound to the enzyme very near its ground state minimum energy conformation proposed on the basis of experimental NMR data and HSEA calculations. Finally, it will be demonstrated how high field NMR spectro— scopy can be used as a sensitive and powerful tool in the study of dynamic enzymatic reactions with oligosaccharide substrates.

Sago starch saccharification and simultaneous ethanol fermentation by amyloglucosidase and Zymomonas mobilis

AbstractSimultaneous saccharification and ethanol fermentation (SSF) of sago starch was studied using amyloglucosidase (AMG) and Zymomonas mobilis. The optimal concentration of AMG and operating temperature for the SSF process were found to be 0.5% (v/w) and 35°C, respectively. Under these conditions with 150 g dm−3 sago starch as a substrate, the final ethanol concentration obtained was 69.2 g dm−3 and ethanol yield, YP/S, 0.50 g g−1 (97% of theoretical yield). Sago starch in the concentration range of 100–200 g dm−3 was efficiently converted into ethanol. When compared to a two‐step process involving separate saccharification and fermentation stages, the SSF reduced the total process time by half.

Simultaneous saccharification and ethanol fermentation of sago starch using immobilized Zymomonas mobilis

Simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and immobilized Zymomonas mobilis ZM4 on sodium alginate was studied. The immobilized Zymomonas cells were more thermo-stable than free Zymomonas cells in this system. The optimum temperature in the SSF system was 40°C, and 0.5% (v/w) AMG concentration was adopted for the economical operation of the system. The final ethanol concentration obtained was 68.3 g/ l and the ethanol yield, Y p/s, was 0.49 g/g (96% of the theoretical yield). After 6 cycles of reuse at 40°C with 15% sago starch hydrolysate, the immobilized Z. mobilis retained about 50% of its ethanol fermenting ability.

Ethanol production from cassava and sago starch using Zymomonas mobilis

Cassava and sago starch were evaluated for their feasibilities as substrates for ethanol production using Zymomonas mobilis ZM4 strain. Before fermentation, the starch materials were pretreated employing two commercial enzymes, Termamyl (thermostable α-amylase) and AMG (amyloglucosidase). Using 2 μl/g of Termamyl and 4 μl/g of AMG, effective conversion of both cassava and sago starch into glucose was found with substrate concentration up to 30%(w/v) dry substances. Fermentation study performed using these starch hydrolysates as substrates resulted in ethanol yield at an average of 0.48g/g by Z. Mobilis ZM4.

Immobilization of amyloglucosidase on alkylamine derivatives of metal‐link‐activated inorganic supports

AbstractA new process to couple amyloglucosidase (AG) to inorganic supports is described. The technique consists in activating the support with a transition metal salt according to the metal‐link method and subsequent amination and linkage of the alkylamine derivative using glutaraldehyde. The various parameters susceptible of influencing the properties of the immobilized enzyme (IME) preparation are investigated. The best result are obtained when 100 mg of 1000‐Å controlled porous glass (CPG) are treated with 45 mg of TiCl4 and the activated carrier aminated using a 10‐g/L solution of hexamethylenediamine (HMDA) in carbon tetrachloride (10 Ml/100 mg). Preparation obtained according to the process here described show operational stabilities much superior to those of AG immobilized on the same support by the traditional metal‐link method or its variations. The mechanism involved in the preparation of the amino derivative of CPG is proposed.

REGULATION OF AMYLOGLUCOSIDASE PRODUCTION BYSACCHAROMYCES DIASTATICUS

In wort fermentations, production of extracellular amyloglucosidase (AMG) by Saccharomyces diastaticus was greatest, and initiated earliest, when the requirement of yeast for oxygen or unsaturated lipid was incompletely satisfied. During such fermentations, maltotriose disappeared before maltose, glucose, the product of AMG action, accumulated and the wort was inadequately attenuated. In all circumstances examined, commencement of dextrin breakdown coincided with initiation of AMG excretion. Prior to excretion, active enzyme was associated with yeast cells but was located externally to the plasma-membrane. Dextrin or starch was not required to induce AMG production which is initiated, we conclude, when the carbon and energy-source becomes growth-limiting. S. diastaticus strains hydrolysed only a small proportion of wort dextrine and AMG produced in beer was unable to release glucose from pullulan. For production of low carbohydrate beer, strains which (i) synthesise significant amounts of a ‘debranching’ enzyme and (ii) are catabolite-derepressed for AMG production would be advantageous.

Glycogen phosphorylase from human normal and leukemic leucocytes: Activities and interconversions between active and inactive forms

1. 1. Leukemic leucocyte extracts show a lower glycogen phosphorylase and amylo (1,6) glucosidase activities than normal leucocytes. 2. 2. Two forms of phosphorylase, interconvertibles by phosphorylation-dephosphorylation reactions, are present in all types of leucocytes isolated. 3. 3. Total phosphorylase activity in leucocyte extracts can be measured with 1 mM AMP −0.4 M Na 2SO 4. 4. 4. Lymphocyte and leukemic leucocyte phosphorylase is related to the liver type. 5. 5. Glucose, glucose-6-phosphate and ATP appear to be important metabolites in regulating phosphorylase activity.

Electrophoretic characterization of acidic and neutral amylo 1–4-glucosidase (acid maltase) in human tissues and evidence for two electrophoretic variants in acid maltase deficiency

Acidic and neutral amylo 1–4-glucosidase activities were clearly separated by cellulose acetate electrophoresis. An extra isozyme was found in kidney extracts. Two variants, one fast and one slow, were detected in two cases of amylo glucosidase deficient patients.