Heat-stable a-amylase

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. 

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). 

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... 

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. 

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. 

AOAC enzymatic gravimetric method The samples are dried, with fat extracted if necessary, gelatinized with heat-stable a-amylase and enzymatically digested with protease and amyloglucosidase to remove protein and starch. The soluble dietary fiber is then precipitated using ethanol, the residue filtered and washed, and corrections made for indigestible protein and ash. This method is widely used in the EC and the USA. 

The commercial production of proteins from micro-organisms began in the United States around 1890 when Takamine introduced a traditional Japanese fermentation process for takadiastase. This product, which was derived from Aspergillus niger (cf. section 6.2.2.2) was a mixture of enzymes which catalysed the hydrolysis of starches and proteins. Some years later, in 1913, Boidin and Effront discovered that Bacillus subtilis produces a heat-stable a-amylase. This enzyme also catalyses the hydrolysis of starches, and was used in the textile industry for desizing cloth. 

In the process a starch suspension containing the heat-stable a-amylase is heated briefly to 140 °C so that it forms a gel. This is sufficiently hydrolysed before the a-amylase is destroyed to allow it to be pumped to a vessel where more a-amylase is added, and the hydrolysis continues for about 30 min at 100 °C. At the end of this period about 10% of the a-1,4 links in the starch are hydrolysed and the gel is thin enough to be cooled to 55 °C without setting solid. Amyloglucosidase and pullulanase are stable at this lower temperature, and enough of these two enzymes are added to catalyse the hydrolysis of the starch to glucose over a two- or three-day period. Only some 2 or 3% of the links between the glucose units remain, and this, in fact, represents the equilibrium position of the hydrolysis. 

Gravimetric methods are the methods of choice for the determination of dietary fibers (cf. 15.2.4.2). In the defatted sample, the digestible components (1,4-a-glucans, proteins) are enzymatically hydrolyzed (heat-stable a- amylase, glucoamylase, proteinase). After centrifugation. 

Examples in Table 15.47 illustrate the effects of a-amylse from various sources on baking quality. While malt and fungal amylases show similar effects, the heat-stable a-amylase from Bacillus subtilis, with its prolonged activity even in the oven, may be easily used to excess. Products formed by the activities of a- and P-amylases are also available as reactants for nonenzymatic browning reactions. This favorably affects the aroma and color of the crust. a-Amylases are added to flour not only to standardize the baking properties, but also to delay the aging of the crumb (cf. 15.4.4). 

Liquefaction Heat-stable a-Amylase Addition pH 6.5-6-S and Temp. 85-95°C... 

This method uses the principle of determining residual starch from the insoluble dietary fiber residue. It consists of the enzymatic hydrolysis of starch with heat-stable a-amylase, followed by a proteolytic degradation with a protease and, finally, the hydrolysis of the starch with amyloglucosidase to yield glucose. Glucose is determined in the supernatant with glucose oxidase and peroxidase. 

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