Production and Characteristics of Raw Starch-Digesting Glucoamylase O from a Protease-Negative, Glycosidase-Negative Aspergillus awamori var. kawachi Mutant

Production of a raw starch-digesting glucoamylase O (GA O) by protease-negative, glycosidase-negative mutant strain HF-15 of Aspergillus awamori var. kawachi was undertaken under submerged culture conditions. The purified GA O was electrophoretically homogeneous and similar to the parent glucoamylase I (GA I) in the hydrolysis curves toward gelatinized potato starch, raw starch, and glycogen and in its thermostability and pH stability, but it was different in molecular weight and carbohydrate content (250,000 and 24.3% for GA O, 90,000 and ca. 7% for GA I, respectively). The chitin-bound GA O hydrolyzed raw starch but the chitin-bound GA I failed to digest raw starch because chitin was adsorbed at the raw starch affinity site of the GA I molecule. The removal of the raw starch affinity site of GA O with subtilisin led to the formation of a modified GA O (molecular weight, 170,000), which hydrolyzed glycogen 100%, similar to GA O and GA I, and was adsorbed onto chitin and fungal cell wall but not onto raw starch, Avicel, or chitosan. The modified GA I (molecular weight, 83,000) derived by treatment with substilisin hydrolyzed glycogen up to only 80% and failed to be adsorbed onto any of the above polysaccharides. The N-bromosuccinimide-oxidized GA O lost its activity toward gelatinized and raw starches, but the abilities to be adsorbed onto raw starch and chitin were preserved. It was thus suggested that both the raw starch affinity site essential for raw starch digestion and the chitin-binding site specific for the binding with chitin in the cell wall could be different from the active site, located in the three respective positions in the GA O molecule.     

Raw Starch-digestive Chitin-immobilized Amylase from a Protease—Glycosidase-less Mutant of Aspergillus awamori var. kawachi

The α-amylase and glucoamylase produced by a protease-, glycosidase-less mutant HF-15 of Aspergillus awamori var. kawachi were found to be adsorbable onto chitin. This adsorption was pH-independent, different from the adsorption onto raw corn starch. The binding between amylases and chitin was so tight that a chitin-immobilized amylase was obtained without the aid of a cross linking agent, glutaraldehyde, and it retained more than 90% of the original activity of the free enzyme. The immobilized amylase digested gelatinized potato starch, glycogen and even raw corn starch to the same high extent as glucose similar to the free enzyme, but it was different from the unbound crude enzyme in the lack of transglucosidase activity, and slightly different in pH- and thermo-stabilities. An experiment using the immobilized amylase for alcohol fermentation demonstrated the possibility of recycling the enzyme for raw starch saccharification.     

Production of raw cassava starch-digestive glucoamylase by a 2-deoxyglucose-resistant mutant of Rhizopus sp.

In order to improve the productivity of raw cassava starch-digestive glucoamylase of Rhizopus sp. MB46 in a liquid culture, a mutant strain, AF-1, which is resistant to 2-deoxyglucose, was derived. The mutant strain produced glucoamylase in the presence of 0.5% glucose though the parent strain did not. With a rice bran liquid medium the productivity was over 2-times that of the wild type strain. A rice bran liquid medium supplemented with β-cyclodextrin was also effective for glucoamylase production. Other maceration enzymes were also produced at a higher level with mutant strain AF-1 than with the wild type strain in a liquid culture as well as in a solid culture. The elution patterns of these enzymes on CM-cellulose column chromatography were principally the same with both strains except for glucoamylase. When 10% of raw cassava starch and cassava waste were digested with the culture filtrate of mutant strain AF-1, glucose was produced in 7% after 60-h incubation and 3.2% after 48-h incubation, respectively.     

Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

High Level Secretion of Calf Chymosin Using a Glucoamylase-prochymosin Fusion Gene in Aspergillus oryzae

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1–603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1–511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.     

Production of raw cassava starch-digestive glucoamylase by Rhizopus sp. in liquid culture

Among about 200 Rhizopus strains isolated in Thailand, Rhizopus sp. MB46 was selected as a producer of raw cassava starch-digestive glucoamylase. Rice bran was effective for the enzyme production in a solid culture as well as wheat bran. Addition of turpentine oil into the rice bran solid culture increased the productivity. Rhizopus sp. MB46 was found to produce glucoamylase in a liquid culture containing 1% rice bran but not in one consisting of 10% raw cassava starch of 2% glucose. The productivity per 1 g solids in the medium in liquid culture was finally improved 6-times by utilization of n-hexane-treated rice bran, supplement of 0.1% meat extract and addition of gauze as a support. The activity was superior to that in turpentine oil-supplemented solid culture.     

Characteristics of raw-starch-digesting glucoamylase from thermophilic Rhizomucor pusillus

A newly isolated thermophilic fungus, NH-139, identified as Rhizumucor pusillus (Lindt) Schipper produced only a single form of raw-starch-absorbable, raw-starch-digesting glucoamylase on solid wheat bran medium at 45°C. The electrophoretically homogenous preparation of glucoamylase, molecular weight 68,000, had its optimal temperature on gelatinized starch at 65°C and on raw corn starch at 50°C. However, this raw-starch-digesting glucoamylase, unlike other glucoamylases, could not completely hydrolyze glycogen but hydrolyzed it to the extent of 80% as glucose, and is classified as type B. The subtilisin-modified glucoamylase of this strain, molecular weight 60,000, still belonged to type B in the hydrolysis curve on glycogen and lost the ability to digest and adsorb onto raw starch.     

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%).     

Production and Characteristics of Raw-Starch-Digesting alpha-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher alpha-amylaseactivity than the parent strain under submerged culture at 30 degrees C for 24 h. About 70% of the total alpha-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable alpha-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable alpha-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal alpha-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Production of a novel raw-starch-digesting glucoamylase by <Emphasis Type="Italic">Penicillium</Emphasis> sp. X-1 under solid state fermentation and its use in direct hydrolysis of raw starch

Penicillium sp. X−1, isolated from decayed raw corn, produced high level of raw-starch-digesting glucoamylase (RSDG) under solid state fermentation (SSF). Maximum enzyme yield of 306.2 U g⁻¹ dry mouldy bran (DMB) was obtained after 36 h of culture upon optimized production. The enzyme could hydrolyse both small and large granule starches but did not adsorb on raw starch. The enzyme exhibited maximum activity at 65°C and pH 6.5, which provided an opportunity of synergism with α-amylase. It significantly hydrolysed 15% (w/v) raw corn starch slurry in synergism with the commercial α-amylase and a degree of hydrolysis of 92.4% was obtained after 2 h of incubation.     

Isolation of Highly Proteolytic Mutants from Aspergillus sojae

Mutational experiments were performed to improve the protease productivity of the mutant strain X-816 previously induced by X-ray irradiation from Asp. sojae K. S. as a high protease producer. The significant correlation was found between the protease production in wheat bran koji and the clear zone around a fungal colony formed on the special medium containing casein. Several mutants of high protease productivity were obtained by the use of the clear zone as a simple criterion for a primary screening test. In this paper, the composition of some other hydrolytic enzymes in wheat bran koji and the genetic stability of these mutants are also discussed.     

Raw starch fermentation to ethanol by an industrial distiller’s yeast strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing glucoamylase and α-amylase genes

Industrial strains of a polyploid, distiller’s Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l⁻¹) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.     

Degradation of raw starch granules by alpha-amylase purified from culture of Aspergillus awamori KT-11

Raw-starch-digesting alpha-amylase (Amyl III) was purified to an electrophoretically pure state from the extract of a koji culture of Aspergillus awamori KT-11 using wheat bran in the medium. The purified Amyl III digested not only soluble starch but also raw corn starch. The major products from the raw starch using Amyl III were maltotriose and maltose, although a small amount of glucose was produced. Amyl III acted on all raw starch granules that it has been tested on. However, it was considered that the action mode of the Amyl III on starch granules was different from that of glucoamylase judging from the observation of granules under a scanning electron microscope before and after enzyme reaction, and also from the reaction products. Glucoamylase (GA I) was also isolated and it was purified to an electrophoretically pure state from the extract. It was found that the electron micrographic features of the granules after treatment with the enzymes were quite different. A synergistic effect of Amyl III and GA I was observed for the digestion of raw starch granules.     

Purification and properties of two forms of glucoamylase fromAspergillus niger

A. niger produced α-glucosidase, α-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5–9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65°C, respectively, and were stable for 1 h at temperatures of up to 60°C. The kinetic parametersK _m andV showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.     

Effect of adsorption of an inhibitory factor on raw starch hydrolysis by glucoamylase

An inhibitory factor (IF) produced byAspergillus niger strain 19, and which inhibits the action of glucoamylase on starch, has the ability to be tightly adsorbed on to various raw starches, though the amount differs from starch to starch. Based on the hydrolysis of the IF-starch complex by glucoamylase, the inhibitions per unit IF adsorbed are similar for some varieties of starch. The effectiveness ratio of IF (% hydrolysis inhibition per % IF adsorbed on raw starch) for corn, sweet potato, waxy rice and waxy corn starches are 1.1, 1.0, 0.85 and 0.96, respectively. These results support the hypothesis that both glucoamylase and IF are adsorbed on to a common binding site on raw starch. However, the effectiveness ratio of IF for cassava and wheat starches are 0.71 and 1.65, respectively, which differ significantly from other varieties of starch.

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Résumé Un inhibiteur (facieur IF) produit par la souche 19 d'Aspergillus niger et qui inhibe l'action de la glucoamylase sur l'amidon a la propriété d'être fortement adsorbé sur dives amidons, bien que la quantité varie d'amidon à amidon. Sur la base de l'hydrolyse du complexe amidon-IF par la glucoamylase, les inhibitions par unité d'IF adsorbé sont sembiables pour quelques variétés d'amidon. Le rapport d'efficience de IF (% d'inhibition de l'hydrolyse par % de IF adsorbé sur l'amidon cru), pour le maïs, la patate douce, et les amidons de riz cireux et de maîs cireux vaut respectivement 1.1, 1.0, 0.85 et 0.96. Ces résultats soutiennent l'hypothèse que tant la glucoamylase que le lacteur IF sont adsorbés sur un site commun de liaison de l'amidon cru. Toutefois, le rapport d'efficience du facteur IF pour les amidons de manioc et de froment valent respectivement 0.71 et 1.65, valeurs significativement différentes de celles pour les aufres variétés d'amidon.

     

Preparation of Koji from corn hulls for alcoholic fermentation without cooking

Corn hulls, the outer peel covering the corn grain, were used for preparation of koji, which was applied to alcoholic fermentation without cooking of raw strachy materials. Corn hull koji had lower saccharifying power, α-amylase, CMCase and xylanase than wheat bran koji, but higher protease and pectinase activities, Its alcoholic fermentations of cassava starch and sweet potato were also superior to those of wheat bran koji: corn hull koji gave 10.3% (v/v) of alcohol with 93% yield from 20 g of cassava starch, while wheat bran koji gave 9.4% (v/v) of alcohol with 90.4% yeild; and corn hull koji gave 9.1% (v/v) of alcohol with 92.6% yielded from 50 g of sweet potato, while wheatbran koji gave 8.1 (v/v) of alcohol with 88.6% yield.     

Production of the raw-starch digesting amylase of Aspergillus sp. K-27

Summary Aspergillus sp. K-27, isolated from soil, produced extracellular glucoamylase and -amylase using wheat starch as a carbon source, and its productivity was doubled by the addition of -methyl-d-glucoside to the medium. The crude enzyme preparation, which was found to be a mixture of 70% glucoamylase and 30% -amylase, well degraded not only cereal starches but also tuber and root starches, and the initial velocity for potato starch was 72% of that for corn starch.     

Production of raw-starch digesting amyloglucosidase by Aspergillussp GP-21 in solid state fermentation

Aspergillus sp GP-21 produced a raw-starch digesting amyloglucosidase which showed optimum activity at 65°C and pH 5.0–5.5. At 50°C the enzyme converted about 40% of raw corn starch to glucose within 48 h. Enzyme production was studied in solid state fermentation using wheat bran. Productivity was affected by the level of moisture, incubation temperature and the presence or absence of supplements. Maximum enzyme production was observed at a moisture level of 75% and at 30°C. Enzyme production was stimulated by supplementing wheat bran with 0.25% proteose peptone, 1% trace mineral solution, 0.01% CaCl₂ and 0.01% MgSO₄. Received 27 October 1998/ Accepted in revised form 15 March 1999     

Construction of a direct starch-fermenting industrial strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> producing glucoamylase, α-amylase and debranching enzyme

To develop a strain of Saccharomyces cerevisiae that produces ethanol directly from starch, two integrative vectors were constructed to allow the simultaneous multiple integration of the Aspergillus awamori glucoamylase gene (GA1) and the Debaryomyces occidentalis α-amylase gene (AMY) and glucoamylase with debranching activity gene (GAM1) into the chromosomes of an industrial strain of S. cerevisiae. The GA1 and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae using the double δ-integration system. The GAM1 gene was constitutively expressed under the corresponding promoter using the double 18S rDNA-integration system. The recombinant industrial strain secreting biologically active α-amylase, glucoamylase and debranching enzyme was able to ferment starch to ethanol in a single step. The new strain produced 8% (v/v) ethanol (62.8 g l⁻¹) from 20% (w/v) soluble starch after 2 days, fermentation.