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-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.     

Raw cassava starch-digestive glucoamylase of Aspergillus sp. N-2 isolated from cassava chips

One hundred and eighty strains of black aspergilli isolated from cassava fields and factories in Thailand were screened for the activity of raw cassava starch-digestive glucoamylase. Aspergillus sp. N-2 was selected as the best producer and its extracellular glucoamylase production was investigated. Conditions for the production were optimized for both liquid and solid cultures, and solid culture was found to be approximately three times more efficient than liquid culture. The culture filtrate showed strong glucoamylase activity at low pH (pH 2.0) and high temperature (55°C), and could digest high concentration raw cassava starch. The glucoamylase activity was separated to four fractions (A, B, C and D) by DEAE-Sephacel column chromatography. Fraction C was obtained in a homogeneous state with a molecular weight of 92,000. Each fraction was characterized in terms of the properties of the glucoamylase activity and the efficiency of digestion of cooked and raw cassava starch.     

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.     

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

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting alpha-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 degrees C. The raw-potato-starch-digesting alpha-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 alpha-amylase.     

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.     

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.     

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.     

Cloning and expression of raw-starch-digesting alpha-amylase gene from Bacillus circulans F-2 in Escherichia coli

The raw potato-starch-digesting alpha-amylase gene of Bacillus circulans F-2 was cloned for the first time in Escherichia coli C600, using plasmid pYEJ001. The recombinant plasmid, named pYKA3, has a 5.4 kb insert from a chromosome of the donor bacterium. Subcloning of this amylase gene gave plasmid pHA300 which carried 3.15 kb of the inserted DNA. The transformed bacterium, E. coli C600 (pYKA3), produced the amylase in the periplasmic space, whereas it is secreted outside the cell in the donor bacterium. The cloned raw-starch-digesting alpha-amylase has a molecular weight of 93,000 on SDS-PAGE, and its action pattern was absolutely the same as that of the potent raw-starch-digestible amylase produced by B. circulans F-2. The periplasmic amylase produced by the transformed E. coli (pHA300) could digest raw starch granules such as potato, corn and barley raw starch granules, indicating that the raw-starch-digesting amylase is active in E. coli. Furthermore, this amylase crossreacted with the rabbit antiserum raised against the raw potato-digesting alpha-amylase of B. circulans F-2. From these results it was concluded that the cloned amylase is the same amylase protein as B. circulans F-2 amylase, which has a potent raw-starch digestibility. Thus, this paper is to our knowledge the first describing the molecular cloning of raw-starch-digesting alpha-amylase from Bacillus species and its successful expression in E. coli.     

Thermostable α‐Amylase and Glucoamylase from Thermomyces lanuginosus F1

An α‐amylase and a glucoamylase produced by Thermomyces lanuginosus F₁ were separated by ion‐exchange chromatography on Q‐Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G‐100 and Phenyl‐Sepharose CL‐4B.The molecular weights and isoelectric points of the enzymes were 55,000 Da and pHi 4.0 for α‐amylase and 70,000 Da and pH_i 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60 °C for α‐amylase, and 6.0 and 70 °C for glucoamylase,respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90 °C (1 h), the α‐amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α‐amylase had highest affinity for soluble starch with K_M values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α‐amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α‐amylase and glucoamylase increased in the presence of Mn²⁺, Co²⁺, Ca²⁺, Zn²⁺ and Fe²⁺, but were inhibited by guanidine‐HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.     

Characterization of an organic solvent‐tolerant thermostable glucoamylase from a halophilic isolate,Halolactibacillus sp. SK71 and its application in raw starch hydrolysis for bioethanol production

A halophilic bacterium Halolactibacillus sp. SK71 producing extracellular glucoamylase was isolated from saline soil of Yuncheng Salt Lake, China. Enzyme production was strongly influenced by the salinity of growth medium with maximum in the presence of 5% NaCl. The glucoamylase was purified to homogeneity with a molecular mass of 78.5 kDa. It showed broad substrate specificity and raw starch hydrolyzing activity. Analysis of hydrolysis products from soluble starch by thin‐layer chromatography revealed that glucose was the sole end‐product, indicating the enzyme was a true glucoamylase. Optimal enzyme activity was found to be at 70°C, pH 8.0, and 7.5% NaCl. In addition, it was highly active and stable over broad ranges of temperature (0–100°C), pH (7.0–12.0), and NaCl concentration (0–20%), showing excellent thermostable, alkali stable, and halotolerant properties. Furthermore, it displayed high stability in the presence of hydrophobic organic solvents. The purified glucoamylase was applied for raw corn starch hydrolysis and subsequent bioethanol production using Saccharomyces cerevisiae. The yield in terms of grams of ethanol produced per gram of sugar consumed was 0.365 g/g, with 71.6% of theoretical yield from raw corn starch. This study demonstrated the feasibility of using enzymes from halophiles for further application in bioenergy production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1262–1268, 2014     

Purification and properties of the raw-starch-digesting glucoamylases from Corticium rolfsii

Corticium rolfsii AHU 9627, isolated from a tomato stem, is one of the strongest producers of a raw-starch-digesting amylase. The amylase system secreted by C. rolfsii AHU 9627 consisted of five forms of glucoamylase (G1–G5) and a small amount of α-amylase. Among these amylases, G1, G2 and G3 were able to hydrolyze raw starch. Five forms of glucoamylase were separated from each other and purified to an electrophoretically homogeneous state. The molecular masses were: G1 78 kDa, G2 78 kDa, G3 79 kDa, G4 70 kDa, and G5 69 kDa. The isoelectric points were: G1 3.85, G2 3.90, G3 3.85, G4 4.0, and G5 4.1. These glucoamylases showed nearly identical characteristics except that G4 and G5 were unable to hydrolyze raw starch. Received: 16 December 1997 / Received last revision: 6 May 1998 / Accepted: 1 June 1998     

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.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

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     

Soluble and immobilized enzyme technology in bioconversion of barley starch

Homogeneous and heterogeneous biocatalysis were both investigated as tools for barley starch syrup production. Barley starch was first liquefied by soluble heat-stable Bacillus sp. α-amylase EC 3.2.1.1 (1,4-α-d-glucan glucanohydrolase) Termamyl 60 L at 95°C, pH 6.5, to obtain slurries of varying DE-values up to ≈37. Alternatively, it was extruded with a Creusot-Loire BC 45 twin-screw extruder at 25% moisture, 150°C, for denaturation. After cooling and adjusting the pH to 4.5 or grinding, respectively, the pretreated starch was saccharified either by soluble or by immobilized Aspergillus niger glucoamylase EC 3.2.1.3 (1,4-α-d-glucan glucohydrolase) at 60°C, pH 4.5, to obtain glucose syrup of up to DE 96. The course of hydrolysis was followed by automated Biogel P-2 chromatographic analysis. Glucoamylase was immobilized either on a phenol-formaldehyde resin Duolite S 761 or on silanized Spherosil porous silica beads. Barley glucose syrup obtained was further continuously converted to high fructose syrup by a packed bed reactor of Actinoplanes missouriensis whole cell glucose isomerase (EC 5.3.1.5) Maxazyme entrapped within α-cellulose beads. We could conclude that barley starch may be used as an alternative raw material for biocatalytic starch syrup production.     

Purification and characterization of an extracellular glucoamylase from the yeastCandida tsukubaensis CBS 6389

The starch-degrading yeastCandida tsukubaensis CBS 6389 secreted amylase at high activity when grown in a medium containing soluble starch. The extracellular α-amylase activity was very low. The major amylase component was purified by DEAE-Sephadex A-50 chromatography and Ultrogel AcA 44 gel filtration and characterized as a glucoamylase. The enzyme proved to be a glycoprotein with a molecular weight of 56000. The glucoamylase had a temperature optimum at 55°C and displayed highest activity in a pH range of 2.4–4.8. Acarbose strongly inhibited the purified glucoamylase. Debranching activity was present as demonstrated by the release of glucose from pullulan.     

A thermophilic glucoamylase fromCephalosporium eichhorniae

A novel exocellular glucoamylase produced by a thermophilic fungus,Cephalosporium eichhorniae, was purified by a combination of membrane filtration and Sephadex chromatography. The enzyme was a glycoprotein, 28% carbohydrate by weight. It was composed of a single polypeptide chain with a molecular weight of 26,850. The enzyme was thermostable with optimum activity between 45 and 62°C. It had a substrate preference of amylose>amylopectin. Analysis by thin-layer and gas-liquid chromatography showed the major hydrolytic product of starch was glucose, classifying this enzyme as a thermophilic glucoamylase.     

A thermoactive glucoamylase with biotechnological relevance from the thermoacidophilic Euryarchaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40–90°C) and pH (pH 3.5–7) and was maximal at 75°C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl₂, the half-life increased from 15 min to 2 h at 80°C.     

Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae.

A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.