Efficient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated α-amylase and glucoamylase genes

The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae, which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3′-half of the α-agglutinin gene as the anchor protein, and the α-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67, 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process.     

Optimization of α-amylase and glucoamylase production by recombinant strains ofSaccharomyces cerevisiae

Summary Replacement of the regulatory sequence of theBacillus amyloliquefaciens α-amylase gene (AMY1) by the yeast alcohol dehydrogenase gene promoter (ADC1 _p) resulted in increased levels of extracellular α-amylase production inSaccharomyces cerevisiae. Negative regulation of glucoamylase synthesis by theSTA10-encoded repressor was alleviated by replacing the nativeSTA2 gene promoter fromS. cerevisiae var.diastaticus withADC1 _p. Enhanced degradation of starch was achieved when the modified versions of theAMY1 andSTA2 genes were introduced jointly intoS. cerevisiae.     

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.     

Comparison of action patterns of gelatin-entrapped and surface-bound glucoamylase on an α-amylase degraded starch substrate: a critical examination of reversion products

The action pattern of gelatin-entrapped and surface-bound glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) on an α-amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) partially hydrolysed starch is reported. Differences have been observed between the action patterns of the two forms of gelatin-immobilized glucoamylase. Both the forward and reverse reactions have been critically examined in depth by sophisticated analysis techniques. The entrapped enzyme favoured the synthesis of (1→6) linked oligosaccharides, mainly isomaltose (9.8%). These reversion products were found in very low concentrations (0.75–1.5%) with gelatin-TiCl₄(liquid) chelate/metal link-coupled enzyme, and no (1→6) linked reversion products were found on gelatin-glutaraldehyde coupled glucoamylase. The level of (1→6) linked reversion products appeared to influence the formal DE value of the d-glucose syrup, being 94.2 and 98.1 for the gelatin-entrapped enzyme and the gelatin-glutaraldehyde surface bound enzyme, respectively. These action patterns and the production of reversion products are discussed in the light of the application of immobilization techniques to the production of high DE d-glucose syrups and the likely failure of systems to achieve 100% conversion.     

Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis

Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an α-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S. cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D. occidentalis. On the other hand, α-amylase activity in the corresponding strain expressing the D. occidentalis AMY gene increased 10-times relative to D. occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and α-amylase for efficient one-step utilization of starch. Growth, substrate utilization and enzyme activity of these strains are described.     

Cytochemical localization and antigenicity of α-amylase in barley aleurone tissue

Summary Gibberellic-acid(GA₃)-induced -amylase has been localised in barley aleurone layers using cytochemical methods and light microscopy. Evidence obtained from the use of a starch substrate film method as well as immunofluorescence indicated that the first amylase to appear in the cell was associated with aleurone grains, apparently with the outer membrane, and also with the peripheral cytoplasm. In GA₃-treated tissue, the amylase distribution was much more diffuse, although patchy, throughout the cytoplasm and it tended to accumulate in the endosperm side of the cell. The possibility that the aleurone grain membrane is the site of gibberellin-induced enzyme synthesis and that it proliferates to become rough endoplasmic reticulum is considered. Immunological information was obtained which supports earlier indications that induced -amylase consists of two different proteins, each with molecular heterogeneity.     

Production of α-amylase by Bacillus amyloliquefaciens in batch and continuous culture using a defined synthetic medium

Summary Using a totally defined synthetic medium the effect of lactose and nitrogen on cell physiology and -amylase production by Bacillus amyloliquefaciens B155 were investigated. Results showed cell growth and -amylase production patterns to be similar regardless of the limiting nutrient and suggested stationary phase gene control of -amylase production as opposed to a direct response to nutrient limitation.     

Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae

A fusion gene containing the Bacillus subtilis -amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both -amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.     

Expression and secretion of barley α-amylase and A.niger glucoamylase in Saccharomyces cerevisiae

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Efficient separation of solids from fermentation broth in bacterial α-amylase production

Various coagulating-flocculating agents were tried in different combinations for the clarification of bacterial broth having high α-amylase enzyme activity. Best separation was achieved using a combination of calcium chloride, sodium hydroxide and sodium alginate.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.     

Enhanced production of extracellular α-amylase and glucoamylase by amylolytic yeasts using β-cyclodextrin as carbon source

Summary Several amylolytic yeasts from the genera Candida, Cryptococcus, Filobasidium, Lipomyces, Saccharomycopsis, Schwanniomyces, and Trichosporon can utilize -cyclodextrin as a sole carbon source. For most species significantly higher yields of both -amylase and glucoamylase are obtained as compared to with starch. This novel inducer of yeast amylases should therefore be useful in the characterization of these amylolytic enzymes and their regulation.     

Final digestion of starch in Musca domestica larvae. Distribution and properties of midgut α-d-glucosidases and glucoamylase

There are low-Mr (72,700) and high-Mr (330,000) soluble α-glucosidase activities in the hind-midgut of Musca domestica that can be isolated by ultracentrifugation. The low-Mr α-glucosidase is less stable and less inhibited by Tris than is the high-Mr α-glucosidase, and occurs mainly in hind-midgut contents, whereas the high-Mr α-glucosidase is found only in hind-midgut cells. Subcellular fractionation of hind-midgut cells showed that the high-Mr α-glucosidase is associated mainly with brush-borders, from where it is set free by freezing and thawing. The low-Mr α-glucosidase is recovered chiefly in the soluble fraction of the cell. The data suggest that the high-Mr and the low-Mr α-glucosidase occur mainly tightly and loosely bound to the cell glycocalyx, respectively. Based on subcellular fractionation, ultracentrifugation, and thermal inactivation data, there is only one molecular species of α-glucosidase and glucoamylase, which are solubilized by Triton X-100 from hind-midgut cell microvillar membranes. The results suggest that starch digestion is accomplished stepwise by luminal amylase, then by membrane-bound glucoamylase, and finally by glycocalyx-associated α-glucosidase and membrane-bound α-glucosidase. Luminal α-glucosidase probably digests ingested oligomaltodextrins and, since it is significantly excreted, it may also be involved in extracorporeal digestion.     

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis–Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.     

The effect of monensin on intracellular transport and secretion of α-amylase isoenzymes in barley aleurone

The effect of monensin on the secretion of -amylase and other enzymes from the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya) was studied by electrophoresis followed by fluorography and by pulse-chase and organelle-isolation experiments. Monensin markedly inhibits the secretion, but not the synthesis, of -amylase, acid phosphatase, and at least four other proteins from the aleurone layer. Monensin treatment causes -amylase to accumulate within the protoplast, but its effect on the different -amylase isoenzymes is not equal. The accumulation of isoenzyme 2 is not influenced by monensin while isoenzymes 1, 3 and 4 are not secreted but rather accumulate in the cell when monensin is included in the incubation medium. The -amylase and acid-phosphatase activities which accumulate within the aleurone cells following treatment with monensin are localized in an organelle having a buoyant density greater than that of endoplasmic reticulum and less than that of mitochondria. In pulse-chase experiments with [³⁵S]methionine, labelled proteins accumulate in this organelle in the presence of monensin and do not appear in the incubation medium. We conclude that monensin inhibits the secretion of proteins from the barley aleurone layer by influencing their intracellular transport.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium dodecyl-sulfate polyacrylamide-gel electrophoresis     

Fast and reliable method for simultaneous zymographic detection of glucoamylase and α-amylase in fungal fermentation

Detection of α-amylase and glucoamylase in crude fermentation extracts using a single native electrophoresis gel and zymogram is described in this article. Proteins were printed on substrate gel and simultaneously onto a membrane in a three-sandwich gel. α-Amylase was detected on the substrate gel with copolymerized β-limit dextrins and iodine reagent. Glucoamylases were detected on the membrane using a coupled assay for glucose detection. Both amylases were detected in native gel using starch and iodine reagent. The described technique can be a helpful tool for monitoring and control of fermentation processes because fungal amylase producers almost always synthesize both amylases.