Amylolysis of raw corn by Aspergillus niger for simultaneous ethanol fermentation

The novelty of this approach was hydrolysis of the raw starch in ground corn to fermentable sugars that are simultaneously fermented to ethanol by yeast in a non-sterile environment. Thus, the conventional cooking step can be eliminated for energy conservation. A koji of Aspergillus niger grown on whole corn for 3 days was the crude enzyme source. A ratio of 0.2 g dry koji/g total solids was found sufficient. Optimum pH was 4.2. Ethanol concentration was 7.7% (w/w) in the aqueous phase with 92% raw starch conversion. Agitation increased rate. Sacharification was the rate-limiting step. The initial ethanol concentration preventing fermentation was estimated to be 8.3% by weight.     

海洋环境来源的淀粉酶AmyP对生玉米 淀粉的降解特性

来自海洋宏基因组文库的 α-淀粉酶（AmyP）属于最新建立的糖苷水解酶亚家族GH13₃7。AmyP 是一个生淀粉降解酶,能有效降解玉米生淀粉。在最适反应条件 pH 7.5和 40 °C 下,生玉米淀粉的比活达到 39.6 ± 1.4 U/mg。酶解反应动力学显示 AmyP 可以非常快速的降解生玉米淀粉。对 1%的生玉米淀粉仅需要 30 min;4%和 8%的生玉米淀粉只需 3 h。DTT 可以显著提高 AmyP 对生玉米淀粉的降解活性,1% DTT 促使活性增加 1倍。根据电镜观察和产物分析,认为 AmyP 是以内腐蚀的模式降解生玉米淀粉颗粒,释放出葡萄糖、麦芽糖和麦芽三糖作为终产物。     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

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.     

Domain C of thermostable α-amylase of Geobacillus thermoleovorans mediates raw starch adsorption

The gene (1,542 bp) encoding thermostable Ca²⁺-independent and raw starch hydrolyzing α-amylase of the extremely thermophilic bacterium Geobacillus thermoleovorans encodes for a protein of 50 kDa (Gt-amyII) with 488 amino acids. The enzyme is optimally active at pH 7.0 and 60 °C with a t _1/2 of 19.4 h at 60 and 4 h at 70 °C. Gt-amyII hydrolyses corn and tapioca raw starches efficiently and therefore finds application in starch saccharification at industrial sub-gelatinisation temperatures. The starch hydrolysis is facilitated following adsorption of the enzyme to starch at the C-terminal domain, as confirmed by the truncation analysis. The adsorption rate constant of Gt-amyII to raw corn starch is 37.6-fold greater than that for the C-terminus truncated enzyme (Gt-amyII-T). Langmuir–Hinshelwood kinetic analysis in terms of equilibrium parameter (K _R) suggested that the adsorption of Gt-amyII to corn starch is more favourable than that of Gt-amyII-T. Thermodynamics of temperature inactivation indicated a decrease in thermostabilisation of Gt-amyII upon truncation of its C-terminus. The addition of raw corn starch increased t _1/2 of Gt-amyII, but it has no such effect on Gt-amyII-T. It can, therefore, be stated that Gt-amyII binds to raw corn starch via C-terminal region that contributes to its thermostability. Phylogenetic analysis confirmed that starch binding region of Gt-amyII is, in fact, the non-catalytic domain C, and not the typical SBD of CBM families. The role of domain C in raw starch binding throws light on the evolutionary path of the known SBDs.     

Production of ethanol from extruded corn starch by a nonconvencional fermentation method

Summary In single-step 48-hour fermentations of extruded, liquefied and raw corn starch, the yields of ethanol from extruded starch were similar to those from liquefied starch, whereas the yields of ethanol from raw starch were lower.     

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 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.     

Alcohol fermentation of corn starch digested by Chalara paradoxa amylase without cooking

Alcohol fermentation of corn starch without cooking was performed by using Chalara paradoxa glucoamylase preparation, which had stronger raw starch digesting activity than those of the conventionally known glucoamylases. A raw corn starch-enzyme-yeast mixture was fermented optimally at pH 5.0 and 30 degrees C for five days and produced ethanol. The yields of ethanol were between 63.5 and 86.8% of the theoretical value by baker's yeast (Saccharomyces cerevisiae), and between 81.1 and 92.1% of the theoretical value by sake yeast (Saccharomyces sake).     

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.     

Exopolysaccharide production from Sclerotium glucanicum NRRL 3006 and Botryosphaeria rhodina DABAC-P82 on raw and hydrolysed starchy materials

AIMS: Evaluation of fermentative usage of raw starchy materials for exopolysaccharide (EPS) production by Sclerotium glucanicum NRRL 3006 and Botryosphaeria rhodina DABAC-P82. METHODS AND RESULTS: Non-hydrolysed corn starch, soft wheat flour, potato flour, cassava flour, sweet and industrial potato flours, and corn starch hydrolysed to different dextrose equivalent (DE) were tested in shaken culture for EPS production. Both fungal strains produced EPS on all tested materials but the production was maximum on hydrolysed corn starch (30.5 and 19.8 g l(-1) by B. rhodina and S. glucanicum on corn starch at 100 and 62 DE, respectively). CONCLUSIONS: Raw starchy materials as such and, in particular, partially or totally hydrolysed corn starch could be used profitably for EPS production by S. glucanicum and B. rhodina. SIGNIFICANCE AND IMPACT OF THE STUDY: The excellent EPS production, productivity and yield of B. rhodina DABAC-P82 when grown on 60 g l(-1) of totally hydrolysed corn starch.     

Purification and characterization of a thermostable raw starch digesting amylase from a Streptomyces sp. isolated in a milling factory

A raw starch utilizing microbe was isolated from mud in a milling factory. The 16S ribosomal DNA (rDNA) sequencing and morphological properties of the strain indicated that it belongs to the genus Streptomyces. A strongly raw starch digesting amylase was purified from the culture supernatant of the strain by chromatographic procedures. The specific activity of the enzyme was 11.7 U/mg, molecular mass 47 kDa, optimum pH 6.0, and optimum temperature 50 to 60 degrees C. The enzyme showed sufficient activity even at 70 degrees C. It was activated by calcium, cobaltous, and magnesium ions, and inhibited by copper, nickel, zinc, and ferrous ions. It formed maltose mainly from raw and gelatinized starch, and glycogen. No products were formed from glucose, maltose, maltotriose, pullulan, or cyclodextrins (CDs). The enzyme digested raw wheat, rice, and waxy rice starch rapidly, and raw corn, waxy corn, sweet potato, tapioca, and potato starch normally.     

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.     

Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.     

Chemistry and properties of starch based desiccants

Desiccants currently used in industry include molecular sieves, lithium chloride, silica gel, and corn grits. Of these, only corn grits (a form of ground corn) are biodegradable and derived from a renewable resource. A major component of the corn grits, starch, is the primary adsorptive material in the corn grits. Other polysaccharides, including cellulose and hemicellulose also have adsorptive properties. The use of alpha-amylase (EC 3.2.1.1) to modify porosity and surface properties of starch resulted in materials with enhanced water sorption properties compared to the native material. This paper reviews the chemical and structural properties of starch, corn grits, and cellulose-based scaffolds on which starch can be affixed, in order to attain structures that might someday find uses in a range of desiccant applications for industrial, commercial, and residential processes.     

Direct fermentative production of lactic acid on cassava and other starch substrates

Lactobacillus amylovorus utilized raw corn, rice and wheat starch medium to produce lactic acid with a productivity of 10.1, 7.9 and 7.8 g lactic acid/L, but had lower productivities of 4.8 g/L and 4.2 g/L on cassava and potato starch in basal medium respectively. When peptone (1%) is added to basal medium with cassava starch as substrate, conversion rate increased from 43% conversion to 70% conversion (7.7 g lactic acid/L). The availability of some components of protein in corn starch is assumed to be the reason for high lactic acid production as compared to that of 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.     

Identification of three proteins up-regulated by raw starch in Cytophaga sp.

Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars. We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI–TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin (cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide an insight into how Cytophaga sp. cells respond to raw starch stimulation.     

Small grains for starch production

Wheat, sorghum, rice, barley, oat and rye grains are actual or potential raw materials for the industrial production of starch, but only the first three are so used. All six contain about 60% to 70% starch, and yield oil and protein as valuable byproducts of starch manufacture. Successful competition of these grains with the present major industrial sources of starch— corn, potatoes and cassava— depends on a number of factors, including comparative costs of the raw materials, efficiency of processing methods, and value of the byproducts.     

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-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 α-amylase by using the C-terminal-half region of α-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.