Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch

An extracellular glucoamylase [exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] of Endomycopsis fibuligera has been purified and some of its properties studied. It had a very high debranching activity (0.63). The enzyme was completely adsorbed onto raw starch at all the pH values tested (pH 2.0–7.6). Amylase inhibitor from Streptomyces sp. did not prevent the adsorption of glucoamylase onto raw starch although the enzyme did not digest raw starch in the presence of amylase inhibitor. Sodium borate (0.1 m) eluted only 35% of the adsorbed enzyme from raw starch. The optimum pH for raw starch digestion was 4.5 whereas that of boiled soluble starch hydrolysis was 5.5. Waxy starches were more easily digested than non-waxy starches, and root starches were slowly digested by this enzyme.     

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.     

Calcium gluconate production by a nonconventional fermentation method

Summary A strain of Aspergillus niger was grown in still (liquid), shake and semi-solid fermentation for calcium gluconate production from glucose, starch, or molasses. The yield from glucose or starch hydrolysate was acceptably high in both shake and semi-solid fermentation indicating that the semi-solid fermentation process offers a promising practical alternative.     

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5–6.0 for E. fibuligera and 5.5–7.0 for S. castellii.     

Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: Synergism and limitations

Summary A mixed culture of Endomycopsis fibuligera NRRL 76 and Zymomonas mobilis ZM4 could directly and more efficiently ferment cassava starch (22.5% w/v) to ethanol (10.5% v/v) than the monocultures. The combination of culture filtrate of E.fibuligera containing amylases and Z.mobilis simultaneously saccharified and fermented the cassava starch to ethanol equally well. Glucoamylase (0.01%) added to the fermenting medium improved ethanol (13.2% v/v) production by the above mixed culture to almost the theoretical level (98%) indicating that this enzyme is a rate-limiting factor in E.fibuligera. Z. mobilis alone converted the enzymehydrolyzed starch only to almost theoretical level (98%).     

Alcoholic fermentation of raw cassava starch by Rhizopus koji without cooking

Using only wheat bran koji from the Rhizopus strain, raw cassava starch and cassava pellets converted reasonably well to alcohol (ethanol) without cooking at 35 degrees C and pH 4.5-5.0. When the initial broth contained 30 g raw cassava starch, 10 g Rhizopus sp. koji, and 100 mL tap water, 12.1 g of alcohol was recovered by final distillation from fermented broth. In this case, 12.1 g alcohol corresponds to an 85.5% conversion rate based on the theoretical values of the starch content. When the initial broth contained 40 g cassava starch, 14.1 g of alcohol was recovered, where 14.1 g corresponds to a 74.5% conversion rate. The alcoholic fermentation process described in the present work is considered more effective and reasonable than the process using raw starch without cooking reported until now, since the new process makes it unnecessary to add yeast cells and glucoamylase preparation.     

Production of ethanol from extruded degermed corn grits by a nonconventional fermentation method

Degermed corn grits (DCG) were extruded at 150°C, with a compression rate of 1 : 2, and the materials were fermented by a nonconventional fermentation method. Yields of ethanol from various concentrations (12–30 g/200 ml) of extruded DCG were 0.37–0.38. It was found that the mold brad enzyme was superior to the glucoamylase preparations for ethanol fermentation, but combinedenzyme systems such as a mixture of glucoamylase, acid protease, and cellulolytic enzymes are as efficient in converting the extruded DCG to ethanol as the mold bran enzyme alone.     

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Glucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC₅₀ value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.     

Tetracycline production with sweet potato residue by solid state fermentation

For saving energy in antibiotic production and reducing the amount of agricultural wastes, solid state fermentation was used in this study to produce tetracycline with sweet potato residue by Streptomyces viridifaciens ATCC 11989. It was found that the optimal media for tetracycline production were sweet potato residue 100 g, organic nitrogen (rice bran, wheat bran, or peanut meal) 20 g, (NH(4))(2)SO(4) 2.4 g, KH(2)PO(4) 0.4 g, CaCO(3) 1.8 g, NaCl 0.6 g, MgCl(2) 0.8 g, soluble starch 10 g, methionine 0.2 g, histidine 0.8 g, and monosodium glutamate 1.6 g with initial moisture content 68-72%, and initial pH 5.8-6.0. Each gram of dry weight substrate was inoculated with 1.0 x 10(8) conidia and incubated at 26 degrees C for 5-7 days, producing 4720 mug of total tetracycline equivalent potency. When incubated at 26 degrees C with the initial moisture content 68%, the conidia in solid media germinated on the second day, mycelia grew abundantly on the third day and reached stationary phase on the sixth day. The antibiotic production was consistent with the morphogenesis of S. viridifaciens: activity could be detected on the third day, had the maximal potency on the sixth day, and decreased slightly on the tenth day. (11-3-88 tly).     

A new method for the preparation of beta-amylase from sweet potato.

A simple method for the preparation of sweet potato beta-amylase by thymol amylose adsorption is described. The method is far more efficient and gives higher recovery of the enzyme. The crystalline enzyme thus obtained is found to be homogeneous by gel chromatography, polyacrylamide gel electrophoresis.     

Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera

Lipomyces kononenkoae and Saccharomycopsis fibuligera possess highly efficient alpha-amylase and/or glucoamylase activities that enable both of these yeasts to utilize raw starch as a carbon source. Eight constructs containing the L. kononenkoae alpha-amylase genes (LKA1 and LKA2), and the S. fibuligera alpha-amylase (SFA1) and glucoamylase (SFG1) genes were prepared. The first set of constructs comprised four single gene cassettes each containing one of the individual amylase coding sequences (LKA1, LKA2, SFA1 or SFG1) under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator, while the second set comprised two single cassettes containing SFA1 and SFG1 linked to their respective native promoters and terminators. The third set of constructs consisted of two double-gene cassettes, one containing LKA1 plus LKA2 under the control of the PGK1 promoter and terminator, and the other SFA1 plus SFG1 controlled by their respective native promoters and terminators. These constructs were transformed into a laboratory strain Saccharomyces cerevisiae (Sigma1278b). Southern-blot analysis confirmed the stable integration of the different gene constructs into the S. cerevisiae genome and plate assays revealed amylolytic activity. The strain expressing LKA1 and LKA2 resulted in the highest levels of alpha-amylase activity in liquid media. This strain was also the most efficient at starch utilization in batch fermentations, utilizing 80% of the available starch and producing 0.61g/100 mL of ethanol after 6 days of fermentation. The strain expressing SFG1 under the control of the PGK1 expression cassette gave the highest levels of glucoamylase activity. It was shown that the co-expression of these heterologous alpha-amylase and glucoamylase genes enhance starch degradation additively in S. cerevisiae. This study has resulted in progress towards laying the foundation for the possible development of efficient starch-degrading S. cerevisiae strains that could eventually be used in consolidated bioprocessing, and in the brewing, whisky, and biofuel industries.     

Protein enrichment of sweet potato residue with amylolytic yeasts by solid-state fermentation

Starchy agricultural wastes were inoculated with amylolytic yeasts for protein enrichment by solid-state fermentation. The moisture content of substrate was 65-69%, and water activity was equivalent to 0.98-0.99. The optimum conditions for protein enrichment were initial moisture content 65%, initial pH 4.5, a 1:1 mixture of ammonium sulfate and urea was incrementally added to the ferment with 1% added at zero time, 1% added at 24 h, and 0.5% added at 48 h, and incubation with amylolytic yeasts (1.0 x 10(10)/100 g substrate) at 30 degrees C for 2-3 days. The final product contained 16.11-20.82% protein.     

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α‐amylase from Aspergillus oryzae as well as α‐amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:338–347, 2014     

Protein enrichment of sweet potato residue with co-culture of amylolytic fungi by solid-state fermentation

Protein enrichment of sweet potato residue with amylolytic moulds by solid-state fermentation was higher than that obtained with amylolytic yeasts. The optimum initial moisture content for protein enrichment was 66% to 75%. Incrementally added nitrogen sources to the culture at zero time and at 24 h considerably improved the final protein content. During the cultivation, the moisture, ash and ATP contents increased, while the pH value decreased. A 1:1 co-culture of amylolytic mycelial fungi yielded a product with 32.4% crude protein after 4 days incubation at 30 degrees C.     

Alcohol fermentation of sweet potato. Membrane reactor in enzymatic hydrolysis

Use of Ultrafiltration membrane systems in stirred cell and in thin-Channel systems for immobilizing enzyme (sweet potato intrinsic and β-amylase) in hydrolysis of sweet potato through a continuous operation mode were studied. Both the filtration rate and reducing sugars, produced as the result of enzymatic hydrolysis, decreased with the filtration time. The immobilized enzymes in the thin-channel system showed a much better performance compared to that in the stirred cell system. Addition of crystalline sweet potato β-amylase to the sweet potato increased both the filtration rate and reducing-sugars content. Alcoholic fermentation of the filtrate resulted in an alcohol content of 4.2%. This represented fermentation of 95% of the sugars with an efficiency of 88%.     

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.