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.     

Production and Characteristics of Raw-Potato-Starch-Digesting α-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting α-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30°C. The raw-potato-starch-digesting α-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying α-amylase.     

Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of β-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60°C and produced 9 U of β-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.     

Fermentation of Barley by Using Saccharomyces cerevisiae: Examination of Barley as a Feedstock for Bioethanol Production and Value-Added Products

The objective of this study was to examine the ethanol yield potential of three barley varieties (Xena, Bold, and Fibar) in comparison to two benchmarks, corn and wheat. Very high gravity (VHG; 30% solids) fermentations using both conventional and Stargen 001 enzymes for starch hydrolysis were carried out as simultaneous saccharification and fermentation. The grains and their corresponding dried distiller''s grain with solubles (DDGS) were also analyzed for nutritional and value-added characteristics. A VHG traditional fermentation approach utilizing jet-cooking fermentation revealed that both dehulled Bold and Xena barley produced ethanol concentrations higher than that produced by wheat (12.3, 12.2, and 11.9%, respectively) but lower than that produced by corn (13.8%). VHG-modified Stargen-based fermentation of dehulled Bold barley demonstrated comparable performance (14.3% ethanol) relative to that of corn (14.5%) and wheat (13.3%). Several important components were found to survive fermentation and were concentrated in DDGS. The highest yield of phenolics was detected in the DDGS (modified Stargen 001, 20% solids) of Xena (14.6 mg of gallic acid/g) and Bold (15.0 mg of gallic acid/g) when the hull was not removed before fermentation. The highest concentration of sterols in DDGS from barley was found in Xena (3.9 mg/g) when the hull was included. The DDGS recovered from corn had the highest concentration of fatty acids (72.6 and 77.5 mg/g). The DDGS recovered from VHG jet-cooking fermentations of Fibar, dehulled Bold, and corn demonstrated similar levels of tocopherols and tocotrienols. Corn DDGS was highest in crude fat but was lowest in crude protein and in vitro energy digestibility. Wheat DDGS was highest in crude protein content, similar to previous studies. The barley DDGS was the highest in in vitro energy digestibility.The growing need for energy independence and proposed renewable fuels has led recently to a major expansion of fuel ethanol production. In North America, this activity primarily uses corn as a feedstock. The need to find other cost-effective and efficient grains for ethanol production has increased in significance. Cereal grains are high in starch and are currently being utilized for ethanol production (26, 41). To ensure long-term viability of the industry, fermentation strategies that focus on holistic utilization of the feedstock that maximize value addition will increase in importance. The focus of industry is slowly moving from biorefineries that anticipate subsidy and government policy to integrated biorefineries that produce multiple products. Multiple product streams and integrated by-product management are thought to ensure better financial stability and opportunities for diversified income streams.Barley is a potential candidate for industrial ethanol production (10) since its ethanol yield is comparable to that of wheat but below that of American corn, which is currently the preferred industrial feedstock. Barley contains on average 63 to 65% starch, 8 to 13% protein, 2 to 3% fat, 1 to 1.5% soluble gums, 8 to 10% hemicellulose, ca. 2.9% lignin, and 2 to 2.5% ash (15, 27). Barley also contains a hull that could be fermented using cellulolytic enzymes, providing opportunities for integrated biorefineries that utilize more feedstocks than corn. Potential coproducts of ethanol production from barley include protein, fiber, fatty acids, tocopherols, and tocotrienols (40). The nutritional value of barley, based on amino acid content, is greater than that for corn and is not significantly affected by the fermentation process (40). A range of nutraceutical and functional food products, as well as amylase, amylase inhibitors, β-amylase, and oxalate oxidase, are found in barley grains and may have potential for extraction and commercial applications (6, 22, 33). Hull-less barley lines, high in both protein (particularly lysine) and starch, and low in fiber, have recently been developed (11, 14, 32). Since starch recovery and thus ethanol yields are lower for barley than corn, coproduct recovery becomes even more essential for profitability (43).Enzymes used for the pretreatment of grains prior to fermentation have traditionally been α-amylases and glucoamylases. The α-amylase decreases the viscosity of the mash (25) and performs the liquefaction of the pretreatment process. The liquefaction step is typically done at high temperatures of 100 to 120°C (38) with direct steam injection (jet-cooking). The α-amylase action serves to break starch at α-(1,4)-glucosidic bonds, producing smaller dextrin chains. During the saccharification step of the pretreatment, the dextrins produced by α-amylase are then acted on by glucoamylase. This conventional method has a considerable economic drawback, because the mash must undergo a cooking step prior to fermentation. Many industrial ethanol producers use jet-cooking to raise the mash temperature to 100 to 120°C. Because of this temperature requirement, the conventional process uses a large amount of energy to produce ethanol.Recently, a new line of cold starch hydrolyzing enzymes was developed. An example of these enzymes is Stargen 001, which is referred to as a raw starch hydrolyzing enzyme because starch is hydrolyzed to fermentable sugars while the temperature remains at or below a temperature of 48°C (38). Stargen 001 replaces the liquefaction and saccharification steps performed by conventional digestion enzymes (i.e., α-amylase and glucoamylase) and releases free glucose and other fermentable sugars for use by yeast cells. Stargen 001 is a cocktail of modified α-amylase and glucoamylase enzymes that work together to convert starch into dextrins, followed by the hydrolysis of dextrins to fermentable sugars (37, 38). With the absence of a cooking stage in the cold hydrolysis method, the potential exists that the dried distiller''s grain plus solubles (DDGS) produced by fermentation would have less damage so that the proteins contained in the DDGS could be of more value (18).The objectives of the present study were to examine the ethanol yield potentials of three barley varieties (Xena, Bold, and Fibar) and two benchmark grains (Pioneer Hi-Bred corn and CPS wheat) using conventional (jet-cooking) and cold starch hydrolysis with Stargen 001. In addition, dehulling was tested for the potential to increase ethanol yields, because hull does not contain fermentable starch; both hulled and dehulled mashes were studied where possible. The grains and their corresponding DDGS were analyzed for nutritional value and the presence of potential value-added products such as fatty acids, tocopherols, tocotrienols, sterols, and polyphenols.     

Occurrence of an Affinity Site apart from the Active Site on the Raw-Starch-Digesting but Non-Raw-Starch-Adsorbable Bacillus subtilis 65 α-Amylase

α-Cyclodextrin specifically inhibited raw starch digestion by Bacillus subtilis 65 α-amylase. The raw starch digestibility and α-cyclodextrin-Sepharose 6B adsorbability of this α-amylase were simultaneously lost when the specific domain corresponding to the affinity site essential for raw starch digestion was deleted by proteolysis. Occurrence of the affinity site on raw-starch-digesting enzymes was proven also with bacterial amylase.     

Ethanol production from raw starch by simultaneous fermentation using Schizosaccharomyces pombe and a raw starch saccharifying enzyme from Corticium rolfsii

Alcoholic fermentation from raw corn starch using Schizosaccharomyces pombe AHU 3179 and a raw starch saccharifying enzyme (RSSE) from Corticium rolfsii AHU 9627 was investigated. The optimum ethanol production was achieved at pH 3.5, 27°C and under the yeast cell concentration of 2.7 × 10⁹ cells/ml. Addition of RSSE 5 units (as glucoamylase)/g raw corn starch was found sufficient. Under these optimum conditions, 18.5% (v/v, at 15°C) ethanol was obtained from 30% raw corn starch (30.8% as glucose) after incubation for 48 h.     

Development of Rapidly Fermenting Strains of Saccharomyces diastaticus for Direct Conversion of Starch and Dextrins to Ethanol

Alcoholic fermentation, growth, and glucoamylase production by 12 strains of Saccharomyces diastaticus were compared by using starch and dextrins as substrates. Haploid progeny produced from a rapidly fermenting strain, SD2, were used for hybridization with other S. diastaticus and Saccharomyces cerevisiae haploids. Alcoholic fermentation and enzyme production by hybrid diploids and their haploid parents were evaluated. Although the dosage of the STA or DEX (starch or dextrin fermentation) genes may enhance ethanol production, epistatic effects in certain strain combinations caused decreases in starch-fermenting activity. Both the nature of the starch or dextrin used and the fermentation medium pH had substantial effects on alcohol production. Commercial dextrin was not as good a substrate as dextrins prepared by digesting starch with α-amylase. Crude manioc starch digested by α-amylase was fermented directly by selected hybrids with almost 100% conversion efficiency. The manioc preparation contained adequate minerals and growth factors. This procedure should be suitable for direct commercial application in manioc-producing regions in Brazil and elsewhere. A rapidly fermenting haploid strain, SD2-A8, descended from strain SD2, contains two unlinked genes controlling formation of extracellular amylase. A convenient method for detecting these genes (STA genes) in replica plates containing large numbers of meiotic progeny was developed.     

Fermentation Methods for Protein Enrichment of Cassava and Corn with Candida tropicalis

Candida tropicalis grows on soluble starch, corn, and cassava powders without requiring that these substrates be previously hydrolyzed. C. tropicalis possesses the enzyme needed to hydrolyze starch, namely, an α-amylase. That property has been used to develop a fermentation process whereby C. tropicalis can be grown directly on corn or cassava powders so that the resultant mixture of biomass and residual corn or cassava contains about 20% protein, which represents a balanced diet for either animal fodder or human food. The fact that no extra enzymes are required to hydrolyze starch results in a particularly efficient way of improving the nutritional value of amylaceous products, through a single-step fermentation process.     

Enzyme-Synthesized Highly Branched Maltodextrins Have Slow Glucose Generation at the Mucosal α-Glucosidase Level and Are Slowly Digestible In Vivo

For digestion of starch in humans, α-amylase first hydrolyzes starch molecules to produce α-limit dextrins, followed by complete hydrolysis to glucose by the mucosal α-glucosidases in the small intestine. It is known that α-1,6 linkages in starch are hydrolyzed at a lower rate than are α-1,4 linkages. Here, to create designed slowly digestible carbohydrates, the structure of waxy corn starch (WCS) was modified using a known branching enzyme alone (BE) and an in combination with β-amylase (BA) to increase further the α-1,6 branching ratio. The digestibility of the enzymatically synthesized products was investigated using α-amylase and four recombinant mammalian mucosal α-glucosidases. Enzyme-modified products (BE-WCS and BEBA-WCS) had increased percentage of α-1,6 linkages (WCS: 5.3%, BE-WCS: 7.1%, and BEBA-WCS: 12.9%), decreased weight-average molecular weight (WCS: 1.73×10⁸ Da, BE-WCS: 2.76×10⁵ Da, and BEBA-WCS 1.62×10⁵ Da), and changes in linear chain distributions (WCS: 21.6, BE-WCS: 16.9, BEBA-WCS: 12.2 DP_w). Hydrolysis by human pancreatic α-amylase resulted in an increase in the amount of branched α-limit dextrin from 26.8% (WCS) to 56.8% (BEBA-WCS). The α-amylolyzed samples were hydrolyzed by the individual α-glucosidases (100 U) and glucogenesis decreased with all as the branching ratio increased. This is the first report showing that hydrolysis rate of the mammalian mucosal α-glucosidases is limited by the amount of branched α-limit dextrin. When enzyme-treated materials were gavaged to rats, the level of postprandial blood glucose at 60 min from BEBA-WCS was significantly higher than for WCS or BE-WCS. Thus, highly branched glucan structures modified by BE and BA had a comparably slow digesting property both in vitro and in vivo. Such highly branched α-glucans show promise as a food ingredient to control postprandial glucose levels and to attain extended glucose release.     

Degradation of Raw Starch by a Wild Amylolytic Strain of Lactobacillus plantarum

Lactobacillus plantarum A6, isolated from fermented cassava, can break down cassava raw starch that has not been subjected to preliminary physicochemical treatment. When the pH was kept at 6, the microorganism cultured in a bioreactor excreted a high α-amylase activity (60 U/ml). Synthesis of the enzyme occurred during the stationary phase and resulted in full hydrolysis of the cassava starch granules. This gave 41 g of lactic acid from 45 g of raw starch after 3 days of fermentation. Enzymatic attack was evident under scanning electron microscopy in the rougher appearance of the surface of starch granules and in the presence of large cavities in some of them. In contrast, when the pH was not regulated, only a small amount of α-amylase activity was produced (2 U/ml) and no decrease in the starch content of the medium was observed. However, under scanning electron microscopy, some granules displayed a rougher surface, which might have been the result of weak enzymatic attack.     

Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His₆) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.     

Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.     

A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity

Background

Glucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.

Methodology/Principal Findings

A glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H¹-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.

Conclusion/Significance

GLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.     

Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

Production of Thermostable α-Amylase, Pullulanase, and α-Glucosidase in Continuous Culture by a New Clostridium Isolate

The production of α-amylase, pullulanase, and α-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60°C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of α-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.     

Two New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast

Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external β-glucosidase by simultaneous saccharification and fermentation. A β-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new β-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a K_m of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a K_i of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a β-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.     

Comparative Characterization of Complete and Truncated Forms of Lactobacillus amylovorus α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding

Two constructs derived from the α-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyAΔ) forms of α-amylase; AmyAΔ lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyAΔ exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and α-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K_M and V_max values of AmyAΔ were slightly higher than those of AmyA, whereas the thermal stability of AmyAΔ was lower than that of AmyA. In contrast to AmyA, AmyAΔ is unable to bind to β-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyAΔ cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.     

Degradation of Native Starch Granules by Barley alpha-Glucosidases

The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.     

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.     

General Biochemical Characterization of Thermostable Pullulanase and Glucoamylase from Clostridium thermohydrosulfuricum

Cell extracts of Clostridium thermohydrosulfuricum, an anaerobic bacterium which ferments starch into ethanol at 65°C, contained both pullulanase and glucoamylase activities. The general physiochemical and catalytic properties of these enzyme activities were compared. Pullulanase and glucoamylase activities were stable and optimally active at 85 and 75°C, respectively. The pH optima for activity and pH stability ranges were, respectively, 5.5 to 6 and 4.5 to 5.5 for pullulanase and 4 to 6 and 5 to 6 for glucoamylase. The apparent [S]_0.5v and V_max for pullulanase activity on pullulan were 0.33 mg/ml and 2.6 U/mg of protein. The apparent [S]_0.5v and V_max for glucoamylase activity on starch were of 0.41 mg/ml and 0.31 U/mg of protein. These enzymes were active and stable in the presence of air or 10% (vol/vol) ethanol. These enzyme activities allowed the organism to actively degrade raw starch into glucose in the absence of significant α-amylase activity.