Academic literature on the topic 'Starch. Hydrolysis. Amylases'

Several natural isolates of Bacillus strains namely 5B, 12B, 16B, 18 and 24B were grown on two different temperatures in submerged fermentation for the raw-starch-digesting a-amylases production. All strains except Bacillus sp. 18 produced more ?-amylase on 37?C. The hydrolysis of raw corn starch followed same pattern. Efficient hydrolysis was obtained with ?-amylases from Bacillus sp. 5B, 12B, 16B and 24B grown on 37?C and Bacillus sp. 18 grown on 50?C. Zymography after isoelectric focusing shown that ?-amylases were produced in multiple forms, from 2 to 6, depending on the strain when they were growing at 37 ?C, while growing at 50?C induced only 1 or 2 isoforms. TLC analysis of hydrolysis products of raw corn and soluble starch by ?-amylases revealed production of various mixtures of oligosaccharides. In most cases G3 was the most dominant product from soluble starch while G2, G3 and G5 were the main products of raw starch hydrolysis. This indicates that obtained a-amylases can be used for starch liquefying or short-chain-oligosaccharide forming, depending on what type of starch (raw or soluble) was used for the hydrolysis.

Analyses of two varieties of Cyperus esculentus (tigernuts) showed that the 100-nutweight of the yellow variety (49.1 g) was higher than the brown variety (14.8 g). The percentage of moisture contents for the yellow and brown varieties were 13.50% and 5.78% respectively. Treatment of soluble starch with tigernut extracts showed that starch hydrolysis occurred. The time for diastatic activity (α- + β- + γ-amylase activities) to completely hydrolyse starch was generally longer than either α- or β-amylase activity at 50℃. Periods and temperatures for complete starch hydrolysis by α-, β- and γ-amylases were virtually the same in the two tigernut extracts. The shortest time for complete starch hydrolyses by diastatic activity occurred at 50℃ and 65℃ for both yellow and brown varieties respectively. Least period for starch hydrolysis by α-amylase activity in both varieties occurred at 50℃, while the least time for β-amylase and γ-amylase activities in both tigernut varieties occurred at 65℃. Quantitative determination of amylolytic enzymes of yellow tigernut extract (TNE) on ‘dry basis’ showed that diastatic activity (183.6º) > α-amylase activity (167.3º) > β-amylase activity (119.8º) > γ-amylase activity (47.5º). Similarly, brown TNE amylolytic enzymes on ‘dry basis’ showed that diastatic activity (175.8º) > α-amylase activity (140.8º) > β-amylase activity (94.9º) > γ-amylase activity (49.6º). The α-amylase activity in yellow tigernut variety was 1.4-fold that of βamylase activity but about 1.5-fold in brown variety. However, α-amylase activity (dry basis) was about 3.5-fold that of γ-amylase in yellow variety but 2.8-fold in the brown variety. Extracts from both tigernut varieties also showed proteolytic and lipolytic activities at about 30℃. Evidently, tigernuts contain various endogenous hydrolytic enzymes and the sweetness of tigernut is invariably due to sugars produced from amylase hydrolysis of innate starch.

Abstract Enzymatic depolymerisation of starch to glucose or maltose is carried out by starch- degrading amylases during a two-stage hydrolysis: liquefaction using bacterial α-amylase followed by saccharification with glucogenic (fungal amylase) or maltogenic (fungal or bacterial) amylases. As a rule, these enzymes are applied separately, following the recommendations concerning their action provided by the enzyme manufacturers. The study presents our attempts to determine the reaction conditions for a simultaneous action of liquefying and saccharifying enzymes on pre-treated potato starch. Hydrolysis was run by Liquozyme Supra, Maltogenase 4000L and San Super 360L enzymes (Novozymes) at different temperatures. During the single-stage method of starch hydrolysate production the most desirable results was obtained for the maltose hydrolysate at 80°C (51.6 DE) and for the glucose hydrolysate at 60°C (96 DE). The analyses indicate that the application of a single-stage hydrolysis of starch to maltose or glucose makes it possible to obtain a degree of starch saccharification comparable with that obtained in the traditional two-stage hydrolysis.

Natural amylase producers, wild type strains of Bacillus sp., were isolated from different regions of Serbia. Strains with the highest amylase activity based on the starch-agar plate test were grown on solid-state fermentation (SSF) on triticale. The influence of the substrate and different cultivation temperature (28 and 37?C) on the production of amylase was examined. The tested strains produced ?-amylases when grown on triticale grains both at 28 and at 37?C, but the activity of amylases and the number and intensity of the produced isoforms were different. Significant hydrolysis of raw cornstarch was obtained with the Bacillus sp. strains 2B, 5B, 18 and 24B. The produced ?-amylases hydrolyzed raw cornstarch at a temperature below the temperature of gelatinization, but the ability for hydrolysis was not directly related to the total enzyme activity, suggesting that only certain isoforms are involved in the hydrolysis.

Abstract α-Amylases have been of interest in diverse fields for many years because of their importance in basic biology, agriculture, and industry. Starch hydrolysis in plants has been studied extensively in germinating cereal seeds. It is generally accepted that α-amylases are secretory enzymes with a pivotal role in the breakdown of starch reserves in the endosperm. Intriguingly, however, recent investigations reveal that some α-amylases degrade starch in the plastids of living cells. The recent solving of the crystal structure of rice AmyI-1 isoform shows that the binding pocket of starch binding site 1 situated outside of the active site cleft interacts with the substances other than oligosaccharides. These findings provided novel insights into structural and cell biological aspects of α-amylase functions in intracellular transport, organelle targeting, and organ-specific actions. Under global warming, abnormal high temperatures during rice grain filling increase grain chalkiness, resulting in yield loss. Intensive “omics” analyses of developing caryopses and mature grains grown under heat stress showed the downregulation of starch synthesis enzymes and the upregulation of α-amylases. Transgenic studies using ectopic overexpression and suppression of α-amylase revealed that α-amylase is a key factor in grain chalkiness. Here we discuss unique new functions of α-amylase in rice cells.

Tigernut tubers (Cyperus esculentus) are used for the production of vegetable milk, commonly known as “Horchata de chufa” in Spain. The presence of starch in the tuber limits the yield of the milk, since this carbohydrate gelatinizes during the pasteurization of the milk and leads to the considerable solidification of this drink. The present work aims to improve the yields and extraction practice of the milk by an in situ hydrolysis of starch, using exogenous amylases of industrial or vegetable origin. The obtained results show that sprouting improves the extraction yields of tigernut milk, which goes from 50% to about 70%. This improvement in milk yield corresponds to a hydrolysis of about 35% of the starch in the tuber. The use of exogenous amylases leads to starch hydrolysis rates of 45% and 70%, respectively, for amylolytic extracts from sprouted tigernut tubers and amylase, with the corollary of a natural increase in the sweetness of milk. This technical approach makes it possible to produce a naturally sweetened tigernut milk which easily lends itself to pasteurization without a significant increase in viscosity.

Background: Amylases are the most widely used biocatalysts in starch saccharification and detergent industries. However, commercially available amylases have few limitations viz. limited activity at low or high pH and Ca2+ dependency. Objective: The quest for exploiting amylase for diverse applications to improve the industrial processes in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence of metal ions as a function of pH. Methods: The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of ions, as a function of substrate concentration in Lineweaver-Burk Plot. Results: Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and 7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5. Conclusion: These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore, a time-saving and cost-effective solution is proposed to overcome the challenges of the existing methodology of starch hydrolysis in starch and detergent industries.

Amylases catalyze the hydrolysis of starch, a vegetable polysaccharide abundant in nature. These enzymes can be utilized in the production of syrups, alcohol, detergent, pharmaceutical products, and animal feed formulations. The aim of this study was to optimize the production of amylases by the filamentous fungus Gongronella butleri by solid-state fermentation and to evaluate the catalytic properties of the obtained enzymatic extract. The highest amylase production, 63.25 U g−1 (or 6.32 U mL−1), was obtained by culturing the fungus in wheat bran with 55% of initial moisture, cultivated for 96 h at 25°C. The enzyme presented optimum activity at pH 5.0 and 55°C. The amylase produced was stable in a wide pH range (3.5–9.5) and maintained its catalytic activity for 1 h at 40°C. Furthermore, the enzymatic extract hydrolyzed starches from different vegetable sources, presenting predominant dextrinizing activity for all substrates evaluated. However, the presence of glucose was observed in a higher concentration during hydrolysis of corn starch, indicating the synergistic action of endo- and exoamylases, which enables the application of this enzymatic extract to produce syrups from different starch sources.

Amylase is one of the leading enzymes used in industry from decades. The preliminary function of this enzyme is the hydrolysis of the starch molecule into glucose units and oligosaccharides. Amylases have spectacular application in broad spectrum of industries such as food, detergent, pharmaceutical and fermentation industries. Among different type of amylases α- amylase is in utmost demand because of its striking features. This particular enzyme is a good substitute over the chemicals catalyst used in industries. α- amylases can be acquired from different sources such as microorganism, animals and plants. Microorganisms are the major source of production of amylase because of the ease of availability, manipulation and operation. The starch converting enzymes is basically generated using submerged fermentation. Some of the prominent characteristics of amylase are its mode of action, substrate specificity and operating condition (temperature and pH). Amylases from different bacterial sources contribute differently to the particular trait of the enzyme. Bacillus amylases have been studied and applied so far because of their robustness in nature and easy accessible pure form of it. Thus this makes it more specific and fit for distinct application in the industry. The purpose of this manuscript was the comparative analysis of the physical and chemical features of α amylases from Bacillus species. It also focuses on the unique characteristics of this enzyme and their industrial applications.Int J Appl Sci Biotechnol, Vol 4(1): 3-14

In vitro activity measurements indicate that storage sweetpotato roots contain high amounts of extractable amylolytic enzymes. These storage roots also have a very high starch content, a characteristic indicating that the in vitro measurements estimate potential amylolytic activity rather than actual physiological activity. We are interested in optimizing the use of endogenous amylases when processing sweetpotato roots and have undertaken a study to identify physiological parameters that control in vivo starch breakdown. Sweetpotato roots were allowed to germinate for 35 days in controlled conditions. Using a combination of in vitro activity measurements and immunochemical detection, the spatial distribution and changes in activity levels for the three major amylolytic enzymes in storage sweetpotato roots—α-amylase, β-amylase, and starch phosphorylase—have been followed. After 6 days, α-amylase protein increased in the outer starchy parenchymatous tissues surrounding the cambium layers, a result suggesting a de novo synthesis of the enzyme in cambium or laticifers layers. β-Amylase was abundant throughout the root at all times, and its high levels did not directly affect starch degradation rates. Starch phosphorylase protein level remained constant, while its extractable activity increased. Starch content decreased during sweetpotato seed root germination. However, the amount of starch that disappeared during germination was low compared with the calculated starch hydrolysis potential estimated by amylolytic activity measurements.

A knowledge of molecular properties and structure of heat-stable enzymes is important for the understanding of basic principles governing thermo stability of proteins and evolution of life at high temperatures. Information on functional characteristics of thermo stable enzymes is necessary also for improving existing biotechnologies and developing new ones. Because of these reasons enzymes from thermophilic organisms are being exploited. In this context, amylolytic enzymes represent a useful choice for investigation from both basic and applied points of view. a-Amylase and glucoamylase hydrolyse starch into oligosaccharides and glucose, respectively. In the present study a thermophilic fungus, Thermomyces lanuginosus, was selected as a source of thermostable starch-degrading enzymes. The main objectives of this research were to understand the physicochemical properties, mechanism of starch utilization by T. lanuginosus and effect of heat, on amylo1ytic enzymes. Purification of amylolytic enzymes A strain of T, lanuginosus, IlSc 91, isolated from a manure heap in our laboratory was found to produce higher levels of extra cellular amylolytic enzymes than strains obtained from culture collection in U.S.A, and Europe. This strain produced 4 units of glucoamylase and 40 units of a-amylase per ml of culture filtrate when grown on 2% starch at 50 C. Culture filtrate was used as the starting material for purification of these enzymes. Glucoamylase and a-amylase were purified by ultrafiltration and a combination of ion exchange and gel-filtration chromatography 93- and 112-fold with 30 and 41% recovery, respectively; Homogeneity of purified enzymes was established by the criteria of native- PAGE, SDS-PAGE, gel-filtration on HPLC and N-terminal amino acid analysis. Some of the physicochernical properties of these enzymes were studied. Physicochemical characteristics Glucoernylase is a monomeric glycoprotien (carbohydrate content 11 %, w/w) and has a molecular weight, of 45 kDa. It produces only glucose from starch. Km and Vmax, for soluble potato starch are 0.04 mg ml-1 and 666 p mole glucose min-1 mg protein-1, respectively. The enzyme is optimally active at 70 C at pH 6.0. Its activation energy is 14.0 kCal mole-1. It has melting temperature of 73 C. Molar extinction coefficient of glucoamylase is 5.5 x 104 mole-1 cm-l. It is stable at 60°C for > 7h. The enzyme is rich in alanine, serine and aspartate/ asparagine. Glucoamylase contains alanine as the N-terminal amino acid. It does not contain cysteine. Purified a-amylase is a homodimeric protein of 40 kDa and contains 5% (w/w) carbohydrate. It liberates oligosaccharides from starch with maltose being the principle product of hydrolysis. The Km for soluble starch is 2.5 mg ml-1. A high Vmax, of 8000 mg starch min-1 mg protein-1 was found. The enzyme is optimally active at 65°C at pH 5.6. The activation energy is 10.9 kCal mole-1. At 50DC, which is the optimal temperature of growth of T. lanuginosus, purified a-amylase is completely stable for over 6 h. Ca2+ increases the melting temperature of a-amylase from 66°C to 73°C. a-Amylase requires Ca2+ for its activity and structural stabi1it.y The molar extinction coefficient of the enzyme is 4.7 x 10' mole-1 cm-1 a- Amylase is rich in aspartate / asparagine, glutamatme /glutamine, alanine, glycine and leucine. It does not contain cysteine. a- Amylase contains alanine as the N-t.ermina1 amino acid. Hydrolysis of starch by a-amylase and glucoamylase Experiments were done to understand the role of a-amylase and glucoamylase in the utilization of starch by T. lanuginosus. Crude and purified amylase preparations hydrolyse raw potato starch slightly more efficiently than soluble potato starch. The extent of starch hydrolysis by a mixture of a-amylase and glucoamylase is equal to that by culture filtrate containing the same amount of enzyme activities, Electrophoresis of crude culture filtrate proteins on native-PAGE and activity staining on gel showed the presence of one species each of a-amylase and glucoamylase. This suggests that in T. lanuginosus hydrolysis of starch is mediated by one species each of extracellular a-amylase and glucoamylase. The hydrolysis of starch by a mixture of a-amylase and glucoamylase is equal to the arithmetic sum of hydrolysis by individual enzyme showing that the enzymes do not act synergistically. a-Amylase is the main starch depolymerizing enzyme. Conversion of starch into glucose by glucoamylase does not require the presence of a-amylase. Starch is hydralysed to a maximum of 72 and 97% by glucoamylase and a-amylase, respectively. Effect of heat on a-amylase The effect, of heat, on a-amylase and glucoamylase was studied with the view to obtain information on the thermal inactivation of these proteins. Five-min heat treatment of the native a-amylase (40 kDa) results in the specific conversion of all protein molecules into partially active (approximately 50% residual activity) and SDS-undissociable dimer of 45 kDa. a-Amylase (45 kDa) after 5-min heat treatment. is partially active and can he rendered completely active by incubation at 37°C for 3 h. This altered form of a-amylase is not due to the formation of disulfide linkage in protein because the enzyme does not contain cysteine and b mercaptoethanol does not prevent heat-induced structural change. Heat, treatment, for 20 min or more results in further structural changes which result in the irreversible inactivation of the enzyme. Prolonged heating (>40 min) probably causes the degradation of protein. Reactivation of 20-min heat-inactivated a-amylase occurs specifically at 37°C or 50°C within 3 h but not at lower temperatures (0°C or 4°C). Native-PAGE analysis of the native and 20-min heated-reactivated a-amylase shows that the reactivated sample is comprised of two protein species of different charge and/or mass. Activity staining shows that only one of these protein band is active and it has electrophoretic mobility identical to that of the native enzyme. Native and the active fraction of 20-min heated-reactivated a-amylase possess similar specific activity. This suggests that it is cat8alytmically and perhaps structurally similar to the native enzyme. The native and the reactivated a-amylase are resist ant to trypsin digestion. However, heat- inactivated a-amylase is degraded into low molecular weight, peptides. These observation suggest that heat-inactivated a-amylase is partially unfolded, Unlike the native, the heat-treated (94"C, 5 min) a-amylase can not be stained with AgN03 while both forms can be stained with Coomassie brilliant blue R and by Schiff's base. On the basis of these observations a tentative model was proposed for the effect of heat on a-amylase (Fig.) Staining by + + Staining by AgNO3, + + Staining by Schiff 's base + + Sensitivity to trypsin + + Figure : Schematic represent ation of heat-induced changes in a-amylase

碩士
靜宜大學
食品營養學系
87
Starch granules of tapioca, taro, sweet potato, lotus rhizome, potato and yam were subjected to enzymatic degradation by α-amylase(0.455 U/mg starch) from Bacillus sp. Effect of α-amylolysis on starch granule size, morphology microstructure, crystalline structure, molecule weight distribution and branch chain length, and distribution was studied. Results indicated thatα-amylolysis susceptibility of starch was found affected by the starch X-ray pattern, granule size, amylose content, and enzymatic degradation pattern. The first-order constant(k1) of hydrolysis of starch was calculated and used as the index of susceptibility of granular starches toα-amylase hydrolysis. Among the starches studied, tapioca starch showed the highest value of k1 (7.66*10-2), and yam starch had the lowest value(0.12*10-2). Theα-amylase susceptibilities of tuber starches studied were tapioca>taro>sweet potato>lotus rhizome>potato >yam. A-type starches(tapioca and taro) were more susceptible to α-amylase than the B-(potato and yam) and C-type(sweet potato and lotus rhizome) starches. The degradation patterns ofα-amylolysis of these starches could be divided into two types. Alpha-amylase hydrolyzed tapioca, sweet potato, lotus rhizome, and potato starches by boring single holes into the granule, and then hydrolyzed the starch granule from the inside out. On the other hand, α-amylase attacked taro and yam starch granules mainly by hydrolyzing the surface of granule to form groove. For starches with the same X-ray pattern and enzymatic degradation pattern, the starch with smaller granule size showed higher susceptibility to α-amylolysis than the starch with larger granule size. The molecular distribution of starch during α-amylolysis was not significantly changed. Chain length of short chain(F3) of amylopectin decreased, and its content increased, duringα-amylolysis. This result indicated that α-amylase hydrolyzed starch granule not only on amorphous region but also on crystalline region. The coefficient of determination(r2) between the k1 value and the content ratio of amylopectin subfraction (F3/F2) is 0.733, and the coefficient for the k1 value and the short chain contents of starch amylopectin is 0.741. This result revealed thatα-amylolysis susceptibilities of starch was significantly correlated with its short chain contents of amylopectin (p<0.05).

碩士
靜宜大學
化粧品科學系碩士班
98
A thermostable raw starch digesting α-amylase gene (tfa) from thermophilic actinomycete Thermobifida fusca NTU22 was expressed in Pichia pastoris X-33 with pGAPZαA and in Yarrowia lipolytica Po1g with pYLSC1. The extracellular amylase activity of Pichia pastoris transformant (pGAPZα-tfa) was 0.51 U/mL after cultivation in 100 mL YPD broth in 500-mL Hinton flasks and shaken (150 rpm) at 28℃ for 72 h. The amylase of P. pastoris transformant (pGAPZα-tfa) was purified 20.21 fold through ultrafiltration concentration and Ni Sepharose™ High Performance Column Chromatography. The overall yield of the purified amylase was 30.47%. The extracellular amylase activity of Y. lipolytica transformant (pYLSC1-tfa) was 0.73 U/mL after cultivation in 100 mL YPD broth in 500-mL Hinton flasks and shaken (200 rpm) at 28℃ for 60 h. The amylase of Y. lipolytica (pYLSC1-tfa) was purified 10.76 fold through ultrafiltration concentration, DEAE-Sepharose CL-6B chromatography and Sepharose CL-6B chromatography. The overall yield of the purified amylase was 21.87 %. Both purified amylases for P. pastoris (pGAPZα-tfa) and Y. lipolytica (pYLSC1-tfa) showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis. The purified amylase was application in raw sago starch hydrolysis. After 72-h treatment, the starch hydrolysis rate was 29%. The DPw (weight-average degree of polymerization) of raw sago starch obviously decreased from 830,945 to 237,092. The surface of starch granules was rough, and some granules displayed deep cavities.

Biotechnology, being the application of biological organisms and their components in pharmaceutical and other industrial processes, has emerged as the basic transformation tool for starch hydrolysis enzyme. Several advantages over chemical catalysts under mild environmental conditions with efficiency and high specificity have been accrued to this fact. Such include ingredient substitution through continuous fermentation, increased products yield and plant capacity, processing aid substitution, more efficient processing, less undesirable products with improved products. This chapter reports on the molecular properties of thermostable enzymes such as alpha-amylases, alpha-glucosidases, glucoamylases pullulanases as relates to pharmaceutical industries; highlights various technology development, continuous solid-state fermentation, metabolic engineering, sol-gel immobilized enzyme arrays often use in enzyme industries. The new modern biotechnology leads to improvement in the effects of various physiological conditions which may allow various industrial processes to carry out lower energy consumption, harmless to the environment, high efficiency, and the product's properties enhancement.

Biotechnology, being the application of biological organisms and their components in pharmaceutical and other industrial processes, has emerged as the basic transformation tool for starch hydrolysis enzyme. Several advantages over chemical catalysts under mild environmental conditions with efficiency and high specificity have been accrued to this fact. Such include ingredient substitution through continuous fermentation, increased products yield and plant capacity, processing aid substitution, more efficient processing, less undesirable products with improved products. This chapter reports on the molecular properties of thermostable enzymes such as alpha-amylases, alpha-glucosidases, glucoamylases pullulanases as relates to pharmaceutical industries; highlights various technology development, continuous solid-state fermentation, metabolic engineering, sol-gel immobilized enzyme arrays often use in enzyme industries. The new modern biotechnology leads to improvement in the effects of various physiological conditions which may allow various industrial processes to carry out lower energy consumption, harmless to the environment, high efficiency, and the product's properties enhancement.

The effect of enzymes with amylolytic activity on the degree of hydrolysis of white corn flour in order to improve the quality of gluten-free bread for special nutrition was studied. Starch was hydrolyzed using mushroom α-amylase and glucoamylase in an amount of 0.005 % and 0.03 % by weight of flour, respectively. As a result, the number of sugars increased to 5.0 % - 5.5 %. The optimal pH value of 4.7 for the action of enzymes was set by adding 0.065 % citric acid. Hydrolysis was subjected to 50 % of white corn flour from the total amount, the humidity of the hydrolyzate was 65%. In addition to mono-and disaccharides, the hydrolysate accumulated 3.5 % on THE basis of dextrins, of which-1.3 % - achro-and maltodextrins, reducing the degree of stale bread.