Amylase heat stable

T rmamyl . [Novo Nordisk] Alpha-amylase heat-stable enzyme for starch liquefaction, for laundry and dishwash detergents, alcohol, brewing, and textile industries. 

Urea Enzymatic Dialysis Method. This method (16) uses 8 M urea [57-13-6] to gelatinize and facUitate removal of starch and promote extraction of the soluble fiber at mild (50°C) temperatures. EoUowing digestion with heat-stable a-amylase and protease, IDE is isolated by filtration or I DE is obtained after ethanol precipitation. Values for I DE are comparable to those obtained by the methods described eadier, and this method is less time-consuming than are the two AO AC-approved methods. Corrections for protein are required as in the AO AC methods. 

A.mylases. Commercial laundry amylases comprise the a-amylase from bacillus amyloliquefaciens and the heat-stable a-amylase from bacillus licheniformis. 

A slurry of the starch is cooked in the presence of a heat-stable bacterial endo-a-amylase. The enzyme hydrolyzes the a-l,4-glycosidic bonds in pregelatinized starch, the viscosity of the gel rapidly decreases, and maltodextrins are produced. The process may be terrninated at this point. The solution is purified and dried, and the maltodextrins are utilized as blandtasting functional ingredients in dry soup mixes, infant foods, sauces and gravy mixes, etc. 

Meanwhile we have shown that the excision activation of ICOR channels is due to disinhibition [72]. The respective inhibitor, operationally named cytosolic inhibitor (Cl), is present in the cytosol of placenta trophoblast cells HT29- and Tg4-colonic carcinoma cells and RE cells of normal and CF patients. The molecule has an apparent molecular weight of 700-1 500 Da it is amphiphilic heat stable and not digested by trypsin, proteases, nucleotidases, lipases or amylase [72]. Burc-khardt, Fromter and their collaborators [114] have confirmed our results and extracted a similar or identical Cl from kidney cortex. 

In this chapter we describe the use of pea seeds to express the bacterial enzyme a-amylase. Bacterial exoenzymes like the heat stable a-amylase from Bacillus licheni-formis are important for starch hydrolysis in the food industry. The enzymatic properties of a-amylase are well understood [13,14], it is one of the most thermostable enzymes in nature and it is the most commonly used enzyme in biotechnological processes. Although fermentation in bacteria allows highly efficient enzyme production, plant-based synthesis allows in situ enzymatic activity to degrade endogenous reserve starch, as shown in experiments with non-crop plants performed under greenhouse conditions [12,15]. Finally, the quantitative and sensitive detection of a-amylase activ-... 

Starch Liquefaction. Starch in its natural state is only degraded slowly by CC-amylases. To make the starch susceptible to enzymatic breakdown, it is necessary to gelatinize and liquefy a slurry with a 30—40% dry matter content. Gelatinization temperature depends on the type of starch (67) com is the most common source of industrial starches followed by wheat, tapioca, and potatoes. Liquefaction is achieved by adding a heat-stable a-amylase to the starch slurry. The equipment used for liquefaction may be stirred tank reactors, continuous stirred tank reactors (CSTR), or a jet cooker. Most starch processing plants liquefy the starch with a single enzyme dose in a process using a jet cooker (Fig. 9). 

Enzymes such as a-amylase and (3-amylase can break the starch chains into maltodextrins and starch sugars such as glucose and maltose. The heat-stable enzymes convert the starch after dissolving the starch by jet-cooking. Enzymatically converted starches are used in the food industry (confectionery, baking products, sweeteners), paper industry (surface sizing) and the fermentation industry. 

A low DE starch hydrolyzate with improved sweetness and browning capacity is prepared by treating starch with a combination of a- and (3-amylase or a-amylase and glucoamylase (amyloglucosidase).229 A further improved process employs a heat-stable a-amylase to convert the starch, with recovery of the products at high temperature.230... 

In a maltodextrin process using enzyme-catalyzed conversion, the starch slurry (30% to 40% dry solids) is first pasted at a temperature of 80-90°C, and is then treated with a heat-stable bacterial alpha-amylase for liquefication. When stabilized with calcium ions, alpha-amylases from B. licheniformis or B. stearothermophilus can withstand temperatures of 90-105°C for at least 30 minutes,10 allowing sufficient process time to split the 1,4 bonds and form maltose and limit dextrins (see Chapter 7). 

Heat-stable amylases are frequently used in both the United States and Japan to produce syrups with a high fructose content from corn starch. 

Fungal amylase has relative low heat stability and its major application is in the baking industry to supplement the variable activity of amylase present in wheat flour. Bacterial amylase is much more heat stable and it is used in brewing, starch degradation, alcohol, textile, and detergent industries. The organisms commonly used for the commercial production of a-amylase include ... 

Enzyme-enzyme conversion employs heat and an enzyme for starch liquefaction in place of acid. This is the most common form of com processing today Subsequent hydrolysis is by enzymes, as above. The choice of hydrolytic system depends upon economics and the kind of endproduct desired. Enzymes are usually inactivated by heating the symp to 75-80°C, with the exception of the heat-stable a-amylases that have come on the market in the last 10 to 15 years. 

Today, there are several heat stable alpha amylase products on the market, many of which were tailored using mutation and recombinant DNA techniques. A major tailoring target has been improvements of yield during enzyme production in order to be more cost effective in the marketplace. All of the heat stable alpha amylases require calcium for stabilization which must be added to the starch slurry and then removed by ion exchange later in the process. The calcium requirements vary widely. For example, the amylase from B. licheniformis requires only 5 ppm calcium while the... 

The major use of glucoamylases in the corn starch industry today is to digest to as near completion as possible the product resulting from the hydrolysis of starch with heat stable alpha amylase. The starting syrup is usually at about 30% dry solids with a DE of about 12 to 15. Two major things must be done to the syrup as it exits the heat stable amylase step. First, it has to be cooled to 60°C because a heat stable glucoamylase has not yet been tailored. 

Second, the pH must be adjusted to about pH 4.5. A major need of the industry is a cost effective heat stable glucoamylase that performs at about pH 6.5. This would eliminate the pH adjustment step as well as to allow both the alpha amylase and the glucoamylase reactions to proceed at the same time. Glucoamylases also require calcium ions for stability but in practice the calcium level is adjusted before the alpha amylase step to stabilize it. 

Procedure (Use two duplicate samples, with a blank for each sample.) Treat each beaker as follows Stir the Sample Preparation magnetically until the sample is totally dispersed. Add 50 p,L of heat-stable a-amylase solution (Sigma Chemical Co. catalog number A 3306, or equivalent) to the beaker. Cover the beaker with aluminum foil, place it on a water bath, and while stirring, incubate at 95° to 100° for 15 min. (Start the timing when the temperature reaches 95°.) Remove the beaker from the bath, and cool to 60°. Uncover the beaker. With a spatula, scrape any ring on the inside wall of the beaker, and disperse any gels formed at the bottom of the beaker. Rinse the beaker walls and spatula with 10 mL of water. 

Most of the purified alpha-amylases lose activity rapidly above 50°, but, in the presence of an excess of calcium ions, the deactivation may be slowed down. Thus, calcium ions are both essential for the activity and important for the stability of alpha-amylase this is particularly true of the cereal a/p/io-amylases, where binding of calcium is very weak. The heat inactivation of the enzymes is minimized when much inert protein is present, and so, heat treatment at 70° may be used in the purification of plant alpha-amylases without high loss of activity. The enzyme from B. stearothermophilus is remarkably heat-stable, and retains 70 % of the original activity after incubation for 20 hours at 85°. 

The enzyme has not yet been examined in detail. The crude enzyme often contains traces of alpha-amylase. The presence of contaminating alpha-amylase can only be accurately tested for by its effect on the viscosity of linear amylose, and application of this test is essential before the enzyme is used for structural determinations. The crude pullulanase appears to be particularly heat-stable, and, on gel chromatography, two molecular forms, of estimated size 150,000 and 50,000, were obtained. The significance of the two forms is not yet known. The crystallized enzyme has been reported " to have a molecular weight of 145,000. 

From heat stable amylase (Sigma 3306) solution, take 2 cm3 and add it to 80 cm3 of cold NDF solution. 

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