1.

Brewing Process
The brewing process consists mainly of nine general steps which are, malting, milling,
mashing, lautering, hoping, fermentation, conditioning, filtering, and canning or bottling.
The process starts with malting, where the grains (usually barley or wheat) are dried into
malt in order to convert complex carbohydrates into dextrin and maltose by three
sequential processes which are, steeping (14–18°C), germination (16–20°C), and kilning
(50–110°C) using water, heat, and action of enzymes (e.g. α-amylases and proteases)
[9,10]. After malting, water is added to the malt before grinding in order to give the malt
homogeneity and further hydrolysis to simple sugars by milling and mashing process.
The wort (resultant of mashing process) will then go through the lautering (from the
German word abläutern which means to rinse off), where the grains are filtered from the
sugar solution extracted during mashing in a lauter tank as it contains small filters that
holds the hulls of the grains from the sweet solution. During hoping, the wort is boiled
(100°C) together with hops in order to sterilize the wort from any contamination and
improve beer’s flavor and stability. The wort will be cooled down and left to be aired so
that the yeast will be added for the fermentation process (6–25°C) in order to ferment the
sugar to ethanol and carbon dioxide to produce the beer [9,10]. Later on, the beer is
conditioned by lowering the temperature to (–2–0°C) in order to get rid of the harmful
and unwanted particles that may affect the carbonation, aroma, and taste of the beer.
Later on, the beer is filtered with sheets (pads) or kieselguhr (diatomaceous earth) filters
in order to get rid of the solid suspended particles such as hops, barley grains, yeast, etc.
Finally the beer is packaged in cans, kegs or bottles, one of the most critical steps during
packaging is to remove oxygen from the product in order to avoid spoilage and
deterioration. Through all the steps that were mentioned above in the brewing flow chart,
enzymes are key factors in maintains and improving those steps. Below each enzyme will
be mentioned and their role in brewing will be discussed.
2. Enzyme in fermentation process
2.1. Fungal α-amylase
Amylases are a group of hydrolases which can specifically cleave glycosidic
bonds in starch. There are two important groups of amylases which includes
glucoamylase and α-amylase. Glucoamylase (exo-1,4-α-D-glucan glucanohydrolase,
E.C. 3.2.1.3) that hydrolyze single glucose units from the non-reducing ends of
amylose and amylopectin (Anto et al., 2006). And α- amylases (endo-1,4-α-D-glucan
glucohydrolase, E.C. 3.2.1.1) are extra cellular enzymes that can randomly cleave
1,4-α-D-glucosidic linkages between adjacent glucose units inside the linear
amylose chain (Castro et al., 2010; Anto et al., 2006; Pandey et al., 2005). Microbial
amylases have successfully replaced chemical hydrolysis of starch in starch
processing industries. Amylase has been derived from several fungi, yeasts, bacteria and
actinomycetes, however, enzymes from fungal and bacterial sources have
dominated applications in industrial sectors. Major advantage of using fungi for the
amylase production is the economical bulk production capacity. Many species of
Aspergillus and Rhizopus are used as a fungal source of α-amylase (Pandey et al.,
2005).
Fungal α-Amylase is one kind of food grade α-amylase. It is made from Aspergillus
oryzal var through fermentation and extraction method. It is available as a liquid or as a
powder formulation. This rapid acting hydrolase is active throughout the acidic pH range,
through the neutral pH range and well into the mildly alkaline range. It readily degrades a
wide variety of starch-containing substrates. This product is not extremely heat-resistant
compared with enzymes from bacterial sources but exhibits adequate heat resistance for
many industrial and most agricultural applications where its pH range is often
complementary to alpha amylase from bacterial sources.
STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF α-AMYLASE
The α-amylase belongs to a family of endo-amylases that catalyses the initial hydrolysis
of starch into shorter oligosaccharides through the cleavage of α-D-(1-4) glycosidic
bonds (9, 36, 42, 80). Neither terminal glucose residues nor α-1,6-linkages can be cleaved
by α-amylase (88). The end products of α-amylase action are oligosaccharides with
varying length with an α-configuration and α-limit dextrins (86), which constitute a
mixture of maltose, maltotriose, and branched oligosaccharides of 6-8 glucose units that
contain both α-1,4 and α-1,6 linkages (88). Others amylolytic enzymes participate in the
process of starch breakdown, but the contribution of α-amylase is the mos important for
the initiation of this process (80).
The amylase has a three-dimensional structure capable of binding to substrate and, by the
action of highly specific catalytic groups, promote the breakage of the glycoside links
(36). The human α-amylase is a classical calcium-containing enzyme composed of 512
amino acids in a single oligosaccharide chain with a molecular weight of 57.6 kDa (88).
The protein contains 3 domains: A, B, and C (Figure 1). The A domain is the largest,
presenting a typical barrel shaped (β/α)8 super structure. The B domain is inserted
between the A and C domains and is attached to the A domain by disulphide bond. The C
domain has a β sheet structure linked to the A domain by a simple polypeptide chain and
seems to be an independent domain with unknown function. The active site (substrate-
binding) of the α-amylase is situated in a long cleft located between the carboxyl end of
the A and B domains. The calcium (Ca2+) is situated between the A and B domains and
may act in the stabilization of the three-dimensinoal structure and as allosteric activator.
Binding of substrate analogs suggest that Asp206, Glu230 and Asp297 participate in
catalysis (56). The substrate-binding site contains 5 subsites with the catalytic site
positioned at subsite 3. Substrate can bind to the first glucose residue in subsite 1 or 2,
allowing cleavage to occur between the first and second or second and third glucose
residues.

α-amylase (EC 3.2.1.1) is an extra cellularenzyme, which catalyzes hydrolysis of

internal α-1,4-O-glycosidic bonds in starch and related polysaccharidesliberating α-
anomeric sugars and limit dextrins1. Fungaland bacterial amylases are widely used
for thecommercial applications in food processing industries. Fungal amylases
particularly from Aspergillus species have a high efficiency insaccharification of
starch when compared to bacterial α-amylases. A.oryzae has an efficient system
forsecretion of proteins and is extensively used to produce industrial enzymes.
Alpha amylase, also called dextrinogenic amylase, causes nonselective, random
endohydrolysis of a-(1 - 4) bonds in amylose and amylopectin. That amylase produces
maltose, maltotriose, and higher oligosaccharides from amylose as well as maltose,
glucose, and, additionally, limit dextrins from amylopectin.
PHYSICOCHEMICAL PROPERTIES:
pH optima: 5,0
pH stability: 4,5 – 8,0
Temperature Optima: 50oC
Temperature Stability: < 50oC
STORAGE CONDITIONS:
The enzyme is supplied as an ammonium sulphate suspension in 0.02% sodium azide and
should be stored at 4°C.
APPLICATIONS: Amylases from Aspergillus oryzae are commonly used as baking
additives to prevent staling in the baking industry, clarify haze from fruit juices and
alcoholic beverages, and to produce glucose and maltose syrup products.
Amylases are extensively employed in processed-food industry such as baking, brewing,
preparation of digestive aids, production of cakes, fruit juices and starch syrups (16). The
α-amylases have been widely used in the baking industry. These enzymes can be added to
the dough of bread to degrade the starch in the flour into smaller dextrins, which are
subsequently fermented by the yeast. The addition of α-amylase to the dough results in
enhancing the rate of fermentation and the reduction of the viscosity of dough, resulting
in improvements in the volume and texture of the product. Moreover, it generates
additional sugar in the dough, which improves the taste, crust colour and toasting
qualities of the bread. Besides generating fermentable compounds, α-amylases also have
an anti-staling effect in bread baking, and they improve the softness retention of baked
goods, increasing the shelf life of these products (29, 86). Currently, a thermostable
maltogenic amylase of Bacillus stearothermophilus is used commercially in the bakery
industry (86). Amylases are also used for the clarification of beer or fruit juices, or for the
pretreatment of animal feed to improve the digestibility of fiber.

2.2. α-acetolactate decarboxylase