*1.2.3 Treatment with α-amylase of Bacillus subtilis (BAA)*

BAA synthesized using *Bacillus subtilis* is found to have an optimum pH value between 5.3 and 6.4, and an optimum temperature of 50°C [20]. Fogarty and Kelly [21] reported that with starch as substrate BAA produces limit dextrins. BAA enzyme produces limit dextrins that cannot be hydrolyzed using glucoamylase obtained from mold *A. niger* and starch degradation often remains incomplete BAA is unsuitable for SSF process which mainly uses glucoamylase enzyme. The BAA enzyme activity reaches a maximum for a pH between 5.8 and 6.8 and a temperature of 55–60°C, when corn is used a substrate [22, 23].

### *1.2.4 Treatment with α-amylase expressed by Bacillus licheniformis (BAB)*

BAB, a new technical enzyme produced with a genetically engineered strain of *B. licheniformis* (Liquozyme, NOVO Nordisk, Denmark) [24] for its tolerant even at low pH values down to 4.8–5. But BAB is used to liquefy cereal mashes and is very effective. This enzyme express it activity up to 90°C and is used in pretreatment step for liquefying substrate in SSF process.

## *1.2.5 Treatment with fungal α-amylase of Aspergillus oryzae (FAA)*

Fogarty and Kelly [21], reported that FAA contains only a few amino acid residues and is highly stable in acidic pH. The enzyme activity is maximum in a pH between 5.5–5.9 and at a temperature of 40°C. FAA can hydrolyze starch granules at a pH of 7.2 and temperature of 37°C and only 40% of starch was dextrinized in pretreatment step after 60 hour. The optimum pH ranges from 5.0 to 6.0 while corn is used as a substrate. The optimum temperature is reported between 50 and 57°C. FAA reduces the viscosity which is desirable for saccharification and is more effective in producing dextrins.

### *1.2.6 Enzymes for starch saccharification in SSF process*

Glucoamylase (EC 3.2.1.3) enzyme, hydrolyzes α-1,4, α-1,6, and α-1,3 glycosidic linkages of starch molecules. Hydrolysis rate of starch is based upon the size and structure of the molecules [21].
