Amylase thermostable

Low Temperature Process. The low temperature process was developed when B. licheniformis and B. stearothermophilus a-amylases became commercially available in the 1970s. These enzymes ate more thermostable, more acidutic, and requite less calcium for stabiUty than the B. subtilis enzyme used in the EHE process. Consequendy, the high temperature EHE heat treatment step was no longer requited to attain efficient Hquefaction. 

Dual-Enzyme Processes. In some cases, especially in symp production in Europe, a Hquefaction process is used that incorporates both a thermostable enzyme and a high temperature heat treatment. This type of process provides better hydrolyzate tilterabiHty than that attained in an acid Hquefaction process (9). Consequendy, dual-enzyme processes were developed that utilized multiple additions of either B. licheniformis or B. stearothermophilus a-amylase and a heat treatment step (see Eig. 1). 

In the alcohol industry, grain or potato raw materials are milled and water added to form a slurry or mash which is heated either batchwise or continuously. Traditionally, the mash is heated to 150°C by the injection of Uve steam. To reduce viscosity, a-amylases are added both during beating to 150°C and during cooling. Thermostable a-amylases from Bacillus licheniformis are the most commonly used enzymes for these processes (68). 

In addition to proteases, other inhibitors reduce the activity of amylase and other digestive enzymes (Ishimoto et al, 1999). Many varieties of beans produce a glycoprotein that complexes with and inhibits a-amylase (Mirkov et al, 1995). The amylase inhibitors are non-competitive and thermostable (Gallaher and Schneeman, 1986) and, unlike protease inhibitors, do not elicit heightened secretion of amylase (Toskes, 1986). Although over-expression... 

In the metagenomic approach, DNA was directly extracted from uncultured samples followed by cloning and expression [3]. For example, by combination of directed evolution with the metagenome approach, an a-amylase mutant with optimal activity at pH 4.5 and optimal thermostability at 105 °C was discovered for starch liquefaction and EtOH production [4]. 

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-... 

In crude aqueous extracts of malted barley the alpha amylase is usually much more thermostable than the beta amylase. This difference in the stability of the two amylases in crude aqueous extracts was recog-... 

The major current industrial use for saccharidases is in the manufacture of high fructose com syrup from starch. The current process requires thermostable enzymes and three processing steps because the enzymes used are not compatible at the same pH and temperature (see Figure 1). Industry is looking to improve the process by developing 1) an improved a-amylase that works at low pH that has a low Ca" ... 

Thermostability of Thermoanaerohacter sp. CGTase. The addition of 40ppm Ca+ + to the CGTase preparation during incubation at high temperatures in the absence or presence of starch substrate provided no enhancement of the thermostability of the enzyme. A comparison of the thermostable CGTase was made to other thermostable enzymes used in starch liquefaction including Termamyl Bacillus licheniformis) and Bacillus stearothermophilus alpha-amylase. 

Figure 4. Thermostability of CGTase relative to Termamyl and Bacillus stearothermophUus alpha-amylase at 80 C, pH 5.0.

The role of the enzymes is three-fold. Firstly there is the use of very thermostable df-amylases to pre-thin the gelatinised starch, reducing its viscosity so that it can be easily handled and further processed. This process is conceptually very similar to many other commercial uses of hydrolases, especially proteases and glycosidases. Pre-thinning takes place at 105°C and the thermostable df-amylase from B. licheniformis actually has a temperature optimum of almost 100°C. 

Thermostable p-amylase Clostridium thermosuifurogenes SV2 (Rama Mohan Reddy et al., 2003)... 

Rama Mohan Reddy, P, Ramesh, B., Mrudula, S., Reddy, G., Seenayya, G. (2003). Production of thermostable (3-amylase by Clostridium thermosulfurogenes SV2 in solid-state fermentation Optimization of nutrient levels using response surfaee methodology. Process Biochem., 39, 267-277. 

Shukla, J., Kar, R. (2006). Potato peel as a solid state substrate for thermostable a-amylase production by thermophilic Bacillus isolates. World J. Microbiol. BiotechnoL, 22, 417 22. 

The kit includes necessary enzymes (thermostable a-amylase and amyloglucosidase), some reagents (buffer concentrate and glucose oxidase/peroxidase [GOPOD] reagent) and standards (glucose solution and com starch) to carry out 100 starch determinations. The other reagents required for this procedure may be obtained from any chemical supplier. 

NOTE Dilute thermostable a-amylase and prepare GOPOD reagent according to Total Starch Assay Kit instructions. Refer to kit instructions for storage conditions. 

Add 3 ml thermostable a-amylase (300 U) diluted in 50 mM MOPS buffer and vortex vigorously. Add 3 ml of 50 mM MOPS buffer instead of enzyme to sample blank. 

Standard flour or starch sample with known starch content Thermostable a-amylase (120 U/g, 1.20 g/ml Termamyl 120 L, Novo Nordisk Biochem or equivalent)... 

Quantitatively transfer sample into a 50-ml beaker using 15 ml distilled water and add 100 (al thermostable a-amylase (14.4 U) into the suspension using a positive-displacement pipettor. 

Thermostable a-amylase is viscous and should be dispensed with a positive-displacement pipettor. 

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