Amylases

9

REVIEW OF LITERATURE

Starch degrading amylolytic enzymes is of great importance in biotechnological

application ranging from food, fermentation, and textile to paper industries etc. Alpha

amylase is a key enzyme in metabolism of spacious diversity of living organisms which

utilize starch as carbon and energy sources. It can hydrolyze starch, glycogen and

related polysaccharides by randomly cleaving internal α-1,4-glucosidic linkages to

produce different sizes of oligosaccharides. Amylases are enzymes which hydrolyze the

starch molecules in to polymers consists of glucose units. Alpha amylase is ubiquitous

in distribution, with plants, bacteria and fungi being the major sources. Most of the

microbial alpha amylases belong to the family 13 glycosyl hydrolases, and they

contributed numerous common properties. But different reaction specificities have been

observed across the family members. Structurally alpha amylase possesses barrel

structures and is responsible for hydrolysis or formation of glycosidic bonds in the α-

conformation. Stability of alpha amylase has extensively been studied; pH and

temperature have very vital roles to play.

Alpha amylase acts on starch and breaking them up into sugars (hence the term

saccharification). Starch is a carbohydrate source consisting of two molecules amylose

and amylopectine. Amylose is formed from chains of glucose linked α1,4 and

amylopectine is formed from α1,4 linked chains of glucose with 1,6 linked branch

points. The amylases are enzymes that work by hydrolyzing the straight chain bonds

between the individual glucose molecules that make up the starch chain. A single

straight chain starch is called an amylose. A branched starch chain (which can be

10

considered as being built from amylose chains) is called an amylopectin. These

starches are polar molecules and have different ends.

Alpha amylase can be derived from several sources such as plants, animals and

microbes. The microbial enzyme meets the industrial demands a large number of them

are available commercially and have almost replaced chemical hydrolysis of starch

processing industry (Pandey et al., 2000). The major advantage of using

microorganisms for the amylase production is economical bulk production capacity

and microbes are also easy to manipulate to obtain enzymes of desired characteristics

(Lonsane and Ramesh, 1990). Alpha amylase has been derived from several fungi,

yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial

sources have dominated applications in industrial sectors. Fungal sources are mostly

terrestrial isolates such as Aspergillus species. Mode of action, properties and product

of hydrolysis differ, some what and depend on the source of enzyme. Two types of

enzymes have been recognized called as liquefying and saccharifying. The main

difference between them is that the saccharifying enzyme produces a higher yield of

reducing sugar than liquefying enzyme. Many scientists carried out extensive work on

11

alpha amylase production. The enzyme production is dependent on the type of strain,

composition of media and methods of cultivation. Generally fungi secrete alpha

amylase (dextrinizing enzymes) although a few fungi have been known to secreted

alpha amylase and beta amylase (saccharifying enzymes). A. oryzae EI 212 secrete

alpha and beta amylase or both depending upon the composition of media and

fermentation conditions. The nature and amount of extracellular amylase produced by

Aspergillus species determine the efficiency of conversion of starch to

oligosaccharides.

Tokhadze et al. (1975) isolated 86 strains of the Aspergillus producing

maximum acid stable alpha-amylase. Repeated cultivation of the selected strains in

the Minoda agar medium along with sodium nitrate during submerged cultivation

showed a 3-fold increase in the alpha amylase production. Yabuki et al. (1977) studied

rapid induction in the alpha amylase production by A. oryzae using inducer such as

maltose. The mycelia were taken from 20 h old cultures and cultivated on the medium

containing peptone and glycerol. Afterwards these cultures were starved for 5 h; in

this case maltose was added as inducer. During first hour of induction, both extra and

intracellular alpha amylases were produced with the same rate (70-80/µg of cells/h).

After 1.5 h remarkable increase in alpha amylase production takes place and enzyme

production reached at optimum rate. No significant increase was occurred in the

weight of mycelia during 2 h of induction. When the purified samples of these intra

and extracellular enzymes were tested by using diethylaminoethylcellulose column

and techniques of gel filtration, both enzymes were showed similar properties in all

respects. Vallier et al. (1977) observed alpha amylase production after the lysis of

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mycelia. For this purpose, mineral medium was used which consist of starch and

glucose. The lysis of mycelium seems to be due to the action of hydrolyzing enzyme

dextranase and levulanase on the cell wall. The pH of the media has great impact on

the lysis of cell wall and alpha amylase secretion. With increase in the pH of mineral

medium up to 8.8 the secretion of enzyme and lysis of mycelial wall were greatly

increased. This method makes it easy to get 3 times more enzyme production.

Sinha and Chakrabrty (1978) reported Aspergillus wentii hydrolysed the soluble

starch in to maltose. The optimum amylase production by using A. wentii was obtained

when fermentation medium consisted of Tryptophan as nitrogen source along with 1

% starch which was incubated for 72 h at 20°C and pH of medium was adjusted at 6.

The enzyme activity was greatly inhibited with the addition of 1mM sodium

iodoacetate. However, enzyme production was increased 3.51 to 6 mg/ml with the

addition of 10 mM sodium citrate. Varnavskaia et al. (1978) studied the impact of pH

on the protein conformation and alpha amylase activity produced by using Aspergillus

terricola. Dispersion of optical rotation technique showed that macromolecule of

alpha amylase consists of alpha helix and beta structures. The change in the values of

pH resulted in two conformational forms. When decrease in pH occurred from 4-2

alpha helix structure uncoiled and degradation of beta forms occurred with the

increase in the pH from 8-12.

Mahmoud et al. (1978) reported the use of different agricultural by-products and

wastes such as wheat bran, rice bran, cane molasses, corn bran, glucose syrup, corn

starch as a substitute of original carbon source in the fermentation medium for the

synthesis of alpha amylase by Aspergillus niger NRRL-337. The medium containing

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rice bran showed maximum alpha amylase activity. The nitrogen source also

substituted by such type of material that makes the medium economic such as corn

steep liquor, corn steep precipitate, dried yeast and gluten-30 and 50. Corn steep

precipitates give highest alpha amylase production compared to other nitrogen

sources. From these results, it was concluded the medium containing rice bran 7.2 %,

corn steep precipitate 2.5 %, magnesium sulfate 0.1 %, potassium di hydrogen

phosphate 0.1 % and calcium carbonate 0.1 % showed maximum activity. The fungal

amylase was isolated and purified from this medium. The purified enzyme showed

optimal activity at 40°C and pH 4.3. Allen and Thoma (1978) studied alpha amylase

produced from A. oryzae acts on reducing ends, and maltotriose which was uniformly

labeled. The enzyme breaksdown the glycosidic bonds during enzyme substrate

formation.

Augustin et al. (1981) examined the activity and production of alpha amylase

and alpha glucosidase in the some members of ascomycetes, imperfect and mucoral

fungi. The factor of polysaccharide system which was responsible for the consumption

of alpha(1 to 4) glucans was described along with screening of the growth of

organism or fungi on soluble starch. Forty nine strains were tested for the production

of amylolytic activity and only twenty nine strains showed this activity. Kasim (1983)

investigated the biosynthesis of alpha-amylase and amyloglucosidase (EC.3.2.1.3) by

A. oryzae in submerged fermentation. For this purpose different sources of carbon and

nitrogen were tested. The medium which shows maximum production of alpha

amylase and glucoamylase was not very costly and consists of following components

in (%) corn steep liquor 3, magnesium sulfate 0.1, potassium dihydrogen phosphate

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0.1, defatted rice bran 8 and calcium chloride 0.1. The pH of medium was adjusted at

5. The optimum conditions for enzymes production were incubation at 28°C for 96 h

and the inoculum consists of 0.5 % mycelial suspension. Erratt et al. (1984) reported

that starch was used as inducer for alpha amylase production from the A. oryzae.

When glucose was used as carbon source the production of both intra and extracellular

amylase was very low. While starch was used as carbon source increase in the activity

of alpha amylase was noticed. In glucose grown cultures intracellular activity of alpha

amylase increased 6.5 fold; however, 20 fold increase was observed in extracellular

activity. Regardless of type of carbon source used, the active protein react only those

antibodies which showed specificity only for alpha amylase and active protein have

molecular weight 52 500 +/- 1800.

Ustiuzhanina et al. (1985) studied the regularities in the biosynthesis of protease

and alpha amylase by using washed cells of selected strain of A. oryzae. The results

enabled us to compare the constitutive characters of protease and alpha amylase by

selected strain of A. oryzae. Carbon, nitrogen and sulfur play very important role in the

regulation of protease synthesis. However, in alpha amylase synthesis, merely carbon

source played an important role. Phosphorous was vital for the synthesis of both alpha

amylase and protease. Removal of phosphorous from the medium adversely affects the

production of both enzymes. The alpha amylase and protease production was

stimulated by the addition of celatin.

Hayashida and Teramoto (1986) reported that a protease negative mutant M33 of A.

ficum was obtained by treating A. ficum with MNNG. This strain showed highest alpha

amylase activity compared to parent strain in submerged fermentation at optimal

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condition i.e. 30°C for 24 h. The molecular weight of purified enzyme was 54, 0000.

MacGregor (1988) studied two computerized methods which explain the sequence of

amino acid in the secondary structure of protein in alpha amylase which was produced

by A. oryzae. Alpha-amylase produced by A. oryzae, showed three dimensional

structures. The computerized methodology explained the position of amino acid and

gave the predictions about the structure of alpha amylase from different sources. It

was noticed all alpha amylase having known amino acid sequence possess same basic

structure, these alpha amylase possess barrel shape structure which was surrounded by

eight helices. The strong resemblance were found in those part of protein which take

part in binding the Ca+2 ions and active site of enzyme which play important role in

catalyzing the substrates hydrolysis. The active site was composed of amino acids

which were specifically found in the loop joining the adjacent helix. The changes in

the length and sequence of amino acid created the differences in binding the substrate

and produced modifications in the action pattern of alpha amylase from different

origins.

Ali and Abdel-Moneim (1989) reported that the best temperature for the

preservation of A. flavus var. columnaris alpha-amylase was -5°C followed by 5°C.

CaCl2 at 0.005 M had no effect on the activity in both temperatures. Repeated freezing

(-5°C) and thawing followed by freezing (-5° C) had no effect on stability of alpha-

amylase. On the other hand, 25°C was the lowest preservation temperature without

any effect on the stability of alpha-amylase. 0.005 M CaCl2 decreased the activity of

alpha amylase and reached a 100 % inhibition at 35th day. The fungal alpha amylase

had an optimum temperature of 55°C at pH 4.6, but had 60°C in buffer containing

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0.005 M CaCl2 and 50°C in buffer containing 0.005 M Na2-EDTA. The addition of

0.01 M CaCl2 greatly increased the thermostability of alpha amylase at 40, 45, 50, 55

and 60°C for 30 min. Optimum pH for alpha amylase was 5, but in the presence of

0.01 M CaCl2 or Na2-EDTA 5.6. The enzyme was only stable for 4 h at 25°C.

Whereas, addition of 0.01 M CaCl2 showed a loss of 4 % compared to a 22 % loss in

the presence of 0.01 M Na2-EDTA after 4 h at 25°C and 65 % loss in the presence of

0.01 M CaCl2 together with 0.01 M Na2-EDTA in the beginning and a 100 % loss

after 4 h at 25°C. The optimum temperature for the activity of alpha-amylase at pH 5

was 50°C for the enzyme only but 55°C in the presence of 0.01 M CaCl2. However, at

pH 6 and 7 optimum temperature was 55°C for the activity of the enzyme only or with

0.01 M CaCl2. The presence of 0.01 M CaCl2 at pH 5, 6 and 7 resulted in increase of

enzyme activity at the temperatures above 50, 40 and 25°C, respectively. However,

0.01 M CaCl2 at pH 5 and 6 resulted in decreasing enzyme activity at temperatures

below 55 and 45°C, respectively.

Rousset and Schlich (1989) screened different species of A. niger for the

synthesis of amylolytic enzymes i.e., alpha amylase and glucoamylase by using the

submerged fermentation. Statistical analysis was used to explain the behaviour of

culture instead of explaining optimization of fermentation conditions. Principal

component analysis (PCA) was used to explain the affect of three agitation rates on

amylase production and the formation of many other factors which affect the growth

in indirect way. The result of Principal component analysis (PCA) describes the

transfer of oxygen at different agitation rate influences enzyme production and carbon

dioxide. The production of carbon dioxide was indirect growth measurement.

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Maximum alpha amylase production was obtained at lower agitation speed while in

case of glucoamylase intermediate agitation speed gave maximum alpha amylase

production. Shah et al. (1991) optimized the conditions for the synthesis and recovery

of alpha amylase from A. oryzae. A. oryzae alpha amylase retained 100 %, 61 % and

58 % respectively when preserved for 12 months at 4°C, ambient temperature and

37°C. Harway (1991) has isolated thermophilic bacteria from the soil which was

preliminary enriched with 0.6 % starch broth at 55°C. These bacteria had ability to

hydrolyze the starch. Of the entire isolated cultures one was Bacillus coagulans, which

was best producer of alpha amylase. The maximum production was obtained in

optimal condition which consists of incubation temperature 55°C, 200 rpm agitation

speed, 48 h incubation period and broth extract starch agar medium.

Tsekova et al. (1993) studied the ability of Aspergillus genus for alpha amylase

production. When 3 % soluble starch was used in Czapek-Dox agar and in liquid

Czapek-Dox media maximum alpha amylase production was obtained. Sudo et al.

(1993) studied the fermentation medium containing all the components which were

necessary for the production of acid stable alpha amylase (asAA) by A. kawachii using

submerged fermentation. One hundred and thirty milligram of acid stable alpha

amylase per liter of medium was produced after 5 days of inoculation at 30°C in

submerged fermentation. Glycogen was present as stored polysaccharide. When the

amount of stored glycogen (CSG) decreased and inducer such as dextrin was present

synthesis of acid stable alpha amylase started. Maximum production of as AA was

obtained when amount of CGS reaches at zero. When the amount of CGS increased

production of acid stable alpha amylase tend to be decreased. The amount of glucose

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in the medium and growth of mycelia was strongly influence by the concentration of

CGS. The quantity of acid stable alpha amylase was directly proportional to the

production of mycelia. Soccol et al. (1994) tested two species of Rhizopus for protein

enrichment of both cooked and raw cassava and also for the synthesis of

amyloglucosidase and alpha amylase in solid and submerged fermentation. The

protein enrichment and maximum enzyme synthesis were obtained in solid state

fermentation. Cooked cassava showed optimum production in solid state fermentation;

however, maximum synthesis of amyloglucosidase by R. oryzae was obtained when

raw cassava was used. Khoo et al. (1994) achieved fifty units per milliliter amylolytic

activity by using A. flavus in liquid medium containing topica starch. The culture

filtrate was subjected to electrophoretic analysis. This analysis showed filtrate contains

only one type of amylolytic enzyme named alpha amylase. The following factor

support the identification of alpha amylase (i) iodine stained starch quickly become

colourless (ii) starch digestion resulted in the formation of a mixture of glucose,

maltose, maltotriose and maltotetrose. Purification of enzyme was involve the use of

ammonium sulfate precipitation, ion exchange chromatography and gel

electrophoresis. The purified enzyme showed 52.5 ± 2.5 kDa molar mass with an

isoelectric point at pH 3.5. Characterization of enzyme showed the maximum activity

of purified enzyme was noticed at pH 6 and 55°C.

Omori et al. (1994) isolated acid labile alpha amylase (A-3) from A. kawachii in

barley koji. The enzyme was purified by using the different techniques such as ion

exchange chromatography and gel filtration. The changes in new alpha amylase

production was compared with two known alpha amylase represented as A-1 and A-2.

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Sodium dodecyl sulfate poly acrylamide gel eletrophoresis showed A-3 has molecular

weight 56,000. The enzyme showed constant production at 40°C approximately for 54

h. However, in traditional method formation of A-3 was not detected after 36 h. In the

presence of 2 % citric acid in barley A3 was formed upto 36 h. The results indicated

production of A3 was influenced both by temperature and initial concentration of

citric acid. Chang et al. (1995) reported that alpha amylase produced by A. oryzae

was purified by passing through the different steps in a specific sequence such as

amylopectin affinity adsorption, DEAE-Sepharose ion exchange chromatography and

sephacryl S-200 HR gel filtration. After passing through these steps the enzyme

showed 16 fold increase in the purity and 45 % of enzyme was recovered. The

optimum conditions for purified enzyme was pH range 4-5, temperature 50°C and km

value for starch hydrolysis was 0.22 %. Incubation for 30 min at 50°C result 80 % lose

in enzyme activity. The heat denaturation constant and molecular weight by gel

filtration was 0.024/m and 52 kDa, respectively. The enzyme activity was inhibited by

using Mercuric ion (0.3m M), DNFB# (6mM), NBSI (6mM) and NAI (6mM). The

hydrolysis of maltoheptaose by the enzyme resulted in the formation of maltotriose

and maltotetraose.

Donmez and Melike (1996) isolated bacteria showing amylolytic activity from

different samples and grouped them on the basis of showing amylolytic activity in the

solid and liquid fermentation media. Of all the isolated strains Bacillus subtilis

produce 24 U/ml alpha amylase. Different carbon sources were added to the

fermentation media to check effect of these carbon sources on alpha amylase

production. The maximum activity of alpha amylase 360 U/ml was obtained in the

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presence of dextrin and optimum temperature was 50°C for enzyme production.

However, if enzyme was incubated for 2 h at 100°C 23 % of this activity was lost.

Carlsen et al. (1996) tested the stability of alpha amylase produced by A. oryzae at

different pH. The enzyme showed highest stability at neutral pH (5-8); however,

beyond this pH range a great loss in the activity of enzyme was noticed. On line FIA

system was used and a rate constant was obtained by the empirical expression k = 1.19

× 107 [H+]1.99 (h−1) explained the inactivation of enzyme was greatly influenced by pH

values. The inactivated enzyme again obtained some of its activity at pH 6 and this

reactivation steps also obey the first order kinetics rules. The contamination of

protease in the protein sample was not result to the irreversible loss of activity.

Abou Zeid (1997) isolated filamentous fungi from cereals and screened to test

the alpha amylase producing potential. The strain which showed highest ability for

alpha amylase production was identified as A. flavus. The enzyme was purified by

using starch adsorption methodology. The polyacrylamide gel electrophoresis (PAGE)

indicated the molecular weight of A. flavus was 75, 000 ± 3,000. The optimum

temperature for purified enzyme was 7 and 30°C, respectively. The use of potassium

ions increased the activity of alpha amylase. However, magnesium ions did not

extremely influence the enzyme activity. The activity of alpha amylase was greatly

inhibited in the presence of manganese, zinc, copper and ferric ions. The hydrolysis of

native starch by A. flavus resulted in the formation of glucose and some other

oligosaccharides

Kajiwara et al. (1997) studied the production of acid stable alpha amylase from

A. kawachii during production of shochu-koji. From barley shochu-koji two types of

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the acid stable alpha amylase (as AA) represented as as A-1 and as A-2 were puified.

The asA-1 and asA-2 showed different adsorption characteristics on raw starch. The

activity of as A-1 slowly increased during the process of shochu-koji production but

dropped after incubation of 24 h. Contrary to as A -1 the activity of as A-2 was

increased with increase in the incubation time. Temperature affect ratio of as A1 to

total as AA activity. When acid protease and as A-1 were incubated along with each

other and this sample was analyzed by SDS-PAGE. A known band was appeared in

the place of as A-1 band after 12 h of incubation. The unknown protein showed all the

characteristics which were present in as A-2. The result showed acid stable alpha

amylase was found in different form just like the glucoamylase produced by A.

awamori during the production of shochu-koji. Spohr et al. (1997) examined alpha

amylase producer strain of A. oryzae for the production of recombinant protein and

affect of growth on the production of protein. The comparison of these strains for

morphology and impact of morphology on the protein indicated the mutant strain

having denser mycelium, produce more alpha amylase compared to other strains.

Arnesen et al. (1998) cultivated thermophilic fungus in the presence of dextran

(having low molecular weight) along with Tween 80 or Triton X-100. The

fermentation was carried out in shake flasks for more than 120 h. The 2.7 fold increase

in the activity of alpha amylase was observed in medium containing Tween 80

compared to the medium with out Tween 80. The medium containing Tween 80

showed increase in the alpha amylase production after 48 h; while general protein

secretion was stimulated after 24 h of inoculation. The Tweeen 80 also influences the

production of biomass. The production of biomass increased gradually with the

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increase in the concentration of Tween 80. Contrary to this Triton X-100 produce

reverse effect. It was noticed increase in the amount of Tween 80 resulted greater than

3 fold increase in the total extracelluar protein. The Tween 80 had no effect both on

the hyphal length and diameter. Glycosylation degree was also not effected by the

Tween 80. Anidyawati et al. (1998) purified three forms of alpha amylase to

homogeneous state by using the methodology of column chromatography. These

forms of alpha amylase were produced from A. awamori. These forms were

designated as Amyl1, Amyl 11, and Amyl 111. The SDS PAGE indicated these three

forms possess 49,000, 63,000 and 97,000 molecular weight, respectively. The

optimum pH for Amyl 11 and Amyl 111 was 5.5 while in the case of Amyl 1 the pH

was 4. Maltose and maltosetriose were formed by the hydrolyzing action of Amyl 1

on malto-tetraose-pentose,-hexaose,-heptose and β and γ-cyclodextrin. However Amyl

1 produces no hydrolyzing effects on raw corn starch, maltose, maltotriose,

isomaltotriose, isomaltosse, and α-cyclodextrin. Unlike Amyl 1 both Amyl 11, and

Amyl 111 have ability to hydrolyze maltotriose, raw corn starch and alpha, beta,

gamma cyclodextrin resulting in the formation of maltose along with minor products

of glucose and maltose. The range of soluble starch hydrolysis through Amyl 1, Amyl

II and Amyl III was 33, 35 and 38 %, respectively.

Jin et al. (1998) used A. oryzae for alpha amylase production and microbial

biomass protein (MBP) from starch processing waste water (SPW) in air lift

bioreactors. The production of MBP and fungal alpha amylase was carried out under

the optimized conditions i.e., pH 5 and 35°C. Bioproduct yield obtained from 12h

batch culture was 6.1 g/l. This yield consists of 55 EU/ml of alpha amylase and 38 %

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protein. The enzyme showed stability at pH 5-9 and 25-35°C. Spohr et al. (1998)

tested three different strain of A. oryzae having the ability to form recombinant protein

with respect to growth and alpha amylase production. One was wild strain and the

second strain was a transfomant strain which consists of additional copies of alpha

amylase gene while third strain was morphological mutant. It was observed the

production and growth of organism were correlated. Comparison of production and

morphology of these strains indicated the variations in the morphology had direct

impact on enzyme production in submerged fermentation.

Moreira et al. (1999) isolated a fungal strain from the soil having the ability to

produce amylolytic enzymes. This strain was identified as A. tamari. A. tamari formed

both alpha amylase and glucoamylase in the mineral medium concomitant with carbon

source i.e., 1 % starch or maltose. The formation of alpha amylase and glucoamylase

indicated tolerance to wide range of initial fermentation medium pH (4-10) and

temperature (25 - 42°C). Ion exchange chromatography was used for the separation of

alpha amylase and glucoamylase. Partially purified alpha amylase and glucoamylase

showed maximum activities at pH 4.5 and 6 and stability at pH 4-7. The temperatures

at which enzymes showed highest activities was between 50 - 60°C.

Pedersen and Nielsen (2000) reported the effect of organic and inorganic

nitrogen sources on alpha amylase production by A. oryzae in continuous cultivations.

Both nitrogen sources were tested along with glucose. In case of inorganic nitrogen

source ammonia was better than nitrate. The comparison between organic and

inorganic nitrogen sources indicated organic nitrogen for example yeast extract or

casine hydrolysate was superior to ammonia. In the presence of 0.05 g/l casine

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hydrolysate 35 % increase in alpha amylase production was found. The transcription

of the alpha amylase genes were not involved in the increase production of alpha

amylase the basic reason was the grater secretion of alpha amylase from the biomass.

Nguyen et al. (2000) optimized the composition of fermentation media for increasing

amylases production through Thermomyces lanuginosus by using the different ways.

The influence of different carbon and nitrogen sources was tested. The carbon and

nitrogen sources, which proved to be good substrates for the growth of T. lanuginosus

and exbhited maximal alpha amylase (92-125 U/ml) and glucoamylase (6-13 U/ml)

activites included starch, maltodextrin, dextrin, maltose, amylopectin, glucose dextran

and L-asparagine. L-asparagine at the level of 6.5 % was good for alpha amylase

production and 2 % L-asparagine was optimum for glucoamylase production. The pH

of medium was adjusted by using hundred millimolar citrate buffer for amylases

production. Response surface method (RSM) was used to find out the suitable

concentration of medium component for the synthesis of amylolytic enzymes. A

second order polynomial model was used at significance level 95 % (p<0.05) for alpha

amylase and glucoamylase. The selected composition of media was tested with respect

to synthesis of amylolytic enzymes.

Mariani et al. (2000) studied impact of Amaranth seed meal and the aeration on

the productiviy of alpha amylase by A. niger NRRL 3112. The assays for the selection

of fermentation media was carried out by using the rotary shaker at 250 rpm and 2.5

cm stroke. The selection of aeration conditions were carried out in New Brunswick

mechanically stirrer fermenter A fermentation medium containing 5.0g/l Amaranthus

cruentus seed meal produce 2750 U.Dun/ml alpha amylase with dry weight of 8.0 g/l

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after 120 h, of inoculation. The optimum condition for alpha amylase production in

fermenter were fermentation period of 120 h, agitation rate 300 rpm and an air flow of

11/l/ min in limited concentration of dissolved oxygen. Although increase in agitation

speed increases the dissolve oxygen but it was not suitable for the formation of alpha

amylase. Morphology of A. niger such as long and branched hyphae was very

important to obtain the maximum alpha amylase production. Petrova et al. (2000)

reported the purification of wild and mutant strains of Thermomyces lanuginosus

ATCC 34626 a thermophilic fungus. The purification was carried out to homogeneity

by using the different techniques in a sequence such as precipitation with ice cold

propanol, anion exchange and molecular sieve chromatographic methods. The SDS-

PAGE results indicated purified alpha amylases (both with PI values of 3.0) have

molecular mass 58 kDa. The optimum pH for the activity of wild and mutant strains

was 5 and 4.5, respectively. 1 – Cyclohexyl - 3 - (2-morpholinyl – 4 - ethyl) -

carbodiimide (40 – 100 mM) and N- bromo succinimide (0.1 – 1mM) produce

inhibitory effect on the enzymes activity due to the presence of carboxylic groups and

tryptophan residues in the catalytic process.

Madihah et al. (2000) isolated and partially purified alpha amylase from the

fermentation of sago starch to solvent by C. acetobuylicum P262. The characterization

of partially purified enzyme showed the following optimal conditions. The highest

activity of alpha amylase was observed at pH 5.3 while enzyme showed stability from

pH 3-9. The highest activity of alpha amylase was found at 40°C; however, if alpha

amylase was placed at 60°C for 60 min merely 50 % of its original activity was

retained. The Km and Vmax values of alpha amylase for soluble starch were 0.31 g/l and

26

10.03 U/ml, respectively. Viswanathan and Surlikar (2001) designed the medium by

the use of fractional factorial method and Plackett-Burman design to study the

influence of component of Amaranthus paniculatas (Rajgeera) medium on alpha

amylase production by A. flavus. Fifteen components were used in developing the

medium. Out of these components only four i.e., CSL, NaCl, CaCl2 and NH4HPO4

were choosed on the basis of contrast coefficient values and selected as independent

variables for the Box-Behnken design. By using SPSS/PC +(version 7.5) statistical

analysis a polynomial multiple regression model was prepared. CSL, NaCl, CaCl2 and

NH4HPO4 increased the yield up to 81.3 % however, NaCl, CaCl2 influence the

product to the tune of 68.3 %. The comparison of control and optimized medium

exhibited 8 fold increase in production of enzyme in the optimized medium.

Carlsen and Nielsen (2001) tested the effect of different carbon sources such as

fructose, galactose, mannitol, glucose, glycerol, sucrose, and acetate on alpha amylase

production by A. oryzae in carbon limited chemostat cultures. A. oryzae was not able

to grow on such a medium which contain galactose as only carbon sources; however, a

combination of glucose and galactose allow the fungal strain to grow and produce

alpha amylase. Medium containing maltose and maltodextin indicated more alpha

amylase production during growth of A. oryzae compared to medium containing

glucose concentration less than10 mg/l. Sucrose, glycerol and mannitol showed low

alpha amylase production. Acetate alone did not show any production of enzyme but

acetate along with little quantity of glucose exhibited alpha amylase production. It was

observed alpha methyl-D-glucoside was acted as an inducer for alpha amylase

production but it was not as good as glucose.

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Agger et al. (2001) evaluated the influential impact of formation of biomass on

the synthesis of alpha amylase by using the wild strain of A. oryzae and recombinant

strain of A. nidulans in submerged fermentation. It was noticed specific rate of alpha

amylase production was inversely proportional to the concentration of biomass

formation. When the concentration of biomass was increases 2-12 g dry weight/kg the

specific rate of enzyme production was decreased. However in case of recombinant

strain of A. nidulans in which gene creA was removed (which cause carbon catabolite

repression) no marked decrease in the specific rate of enzyme formation was observed.

The results indicated less alpha amylase production at high biomass formation was

due to slow mixing rate of vital components in viscous culture medium.

Ray (2001) isolated Penicillium sp possessing the ability to form alpha amylase

and xylanase in the presence of starch and xylan respectively, in fermentation. It was

noticed the optimum amylolytic activity and xylanolytic activity was obtained on 4th

and 6th day of fermentation respectively. The quality of alkalophilic strain of

Penicillium sp to hydrolyze starchy and hemicellulosic wastes made them a potent

strain for the large scale economic production of both enzymes using the cheap

substrates. Bogar et al. (2002) tested different strains of A. oryzae on spent brewing

grain (SBG) and corn fiber for alpha amylase production. A Plackett-Burman

experimental design was practiced to develop optimized media for alpha amylase

production using best producer strain. A. oryzae NRRL 1808 strain produced 4519 U

of alpha amylase/g of dry matter substrate in stationary 500 ml Erlenmeyer flask

culture after 72 h. The crude enzyme, in situ enzyme produced in solid substrate

fermentation material was economic biocatalytic product for animal feed and for the

28

production of bio alcohol from starchy substrate. Francis et al. (2002) investigated the

effect of spent brewing grains on alpha amylase production by A. oryzae NRRL 6270

when spent brewing grains utilized as sole carbon source. Maximum alpha amylase

production was obtained at 25°C. At 30°C almost similar results were obtained.

Optimum alpha amylase production [6870U/g dry substrate (gds)] was obtained in

solid state fermentation at 30°C after 96 h by the use of suspension containing 1×107

spores/ ml. Addition of any external carbon source in the spent brewing grain resulted

decreased in alpha amylase production.

Arnesen et al. (2002) used thermophilic fungus T. lanuginosus for alpha

amylase production in shake flasks. The fermentation medium contained carbon

source in the form of low molecular dextran. The fermentation was carried out up to

120 h. The results showed maximum alpha amylase activity after 96 h of inoculation

during stationary phase while the production of maximal biomass takes place after 48

h of fermentation. A same pattern was observed in the case of total extra cellular

protein. It was found many unidentified proteins and alpha amylase were de novo

synthesis by using pluse labeling techniques of proteins. The sequencing of alpha

amylase from T. lanuginosus using specific primmer and RT-PCR technique indicated

that transcription of alpha amylase was not start before the late growth phase and

reached at its highest value more than 24 h after maximum biomass was produced.

Gigras et al. (2002) used the central composite design along with 3 variables

i.e., starch, yeast extract, and di potassium hydrogen phosphate for alpha amylase

production by A. oryzae in shake flasks and bioreactor. The alpha amylase production

was 133U/ml in shake flasks while in case of bioreactor production was 161 U/ml.

29

However, there was great difference in the fermentation period of shake flasks and

bioreactor. The fermentation period for the maximum alpha amylase production by A.

oryzae in shake flasks was 120 h but in case of bioreactor this time period was reduced

to only 48 h. A high concentration of phosphate in the fermentation medium and use

of low inoculum size was essential to prevent the unnecessary foaming in bioreactor;

but managing the pO2 level and agitation rate was not compulsory for alpha amylase

production. The enzyme production increases with the increase in the pH of medium

and reached at its peak at pH above than 7.5. Thus in present study pH act as sign of

commencement or ending of the enzyme production.

Huang et al. (2003) developed a segregated model to explore the intrinsic

associations between growth, substrate consumption, cell differentiation and enzyme

formation by Bacillus subtilis in bioreactor. The segregated model represented three

different states of cell and the change from vegetative stage to sporangium and lastly

to mature spore. An age-based population balance model was used to explain the

maturity of sporangium in the direction of the formation of spores. Parameters in the

model were found out by placing the experimental data in the model. The model has

ability to describe the temporary behavior of B. subtilis in both batch and fed-batch

cultures.

Francis et al. (2003) optimized incubation temperature, initial moisture contents

and inoculum size by application of Box–Behnken design under the response surface

methodology for the highest production of alpha amylase by A. oryzae NRRL 6270.

The experimental data was added into a polynomial model to find out alpha amylase

production. A Plackett–Burman design was used to test the influence of nineteen

30

nutrient components on alpha amylase production by the A. oryzae. Soybean meal,

CaCl2 and Mg SO4 were chosen on the basis of their positive affect on alpha amylase

production. A Box–Behnken design was used to select the best condition for alpha

amylase production. Incubation temperature 30°C, initial moisture contents 70 % and

an inoculum size of 1×107 spores/g dry substrate were the optimum conditions for the

alpha amylase formation by A. oryzae NRRL 6270 on SBG. Under selected conditions

of solid state fermentation SSF, about 20 % enhancement in enzyme production was

found. Kariya et al. (2003) purified alpha amylase from culture broth of A. oryzae

MIB 316. The enzyme had ability to efficiently hydrolyzed amylopectin, amylose and

starch and break down maltopentose to produce a maltotriose and maltose. However,

maltose did not produce glucose. The N-terminal sequence of first 10 residues and

many other molecular characteristics were similar to Taka-amylase.

Kusuda et al., (2003) isolated alpha amylase from an immobile culture filtrate of

Tricholoma matsutak. The enzyme was purified to homogeneity by sequential steps of

Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The alpha

amylase showed 3580 fold purity and 10.5 % recovery. SDS-PAGE analysis resulted

in a single protein band. The characterization of purified alpha amylase showed that it

was most active at pH 5–6 and having stability between wide range pH i.e., 4–10. The

experimental results also indicated the alpha amylase was somewhat thermostable and

showed thermostability at 50°C while the optimal temperature was 60°C. The size-

exclusion chromatography and SDS-PAGE showed that purified alpha amylase had

molecular mass 34 kDa and 46 kDa, respectively. The mercuric ion did not inhibit the

activity of enzyme. Measurement of viscosity, TLC and HPLC analysis indicated

31

amylases from T. matsutake was of endo type. The specificity of alpha amylase was

tested by using amylose along with various polysaccharides. This alpha amylase

rapidly hydrolyzed the α-1,4 glucoside linkage in soluble starch and amylose A

(MW,2900), but was not able to hydrolyze the α-1,6 linkage and cyclic

polysaccharides e.g α- and β-cyclodextrin.

Kanwal et al. (2004) extracted alpha amylase from Malus pumila (apple) by

homogenizing the apple in buffer for alpha amylase. After extraction, the enzyme was

purified by passing sequential steps of purification. The crude extract exhibited 3.09

U/ml alpha amylase activity was subjected to ammonium sulfate precipitation. This

partially purified enzyme produces 4.76 U/ml and showed 5.01 U/mg specific activity.

The enzyme was further purified by gel filtration chromatography (Sephadex G-150).

After gel filtration chromatograph it produces of 5.025 U/ml and specific activity

38.95 U/ml along with 20-fold purification. SDS-PAGE of enzyme removed the

undesirable proteins and single band of enzyme was appeared. Molecular weight of

alpha amylase was 51,180 D which was finding out by Sephadex G-150 column.

Amylase exhibited optimal pH 6.8, incubation temperature 37°C, Km value 2.0x10-3

g/ml, λmax 540nm and incubation time for enzyme assay was ten min.

Apar and Ozbek (2004) studied the effects of temperature on the enzymatic

hydrolysis of starch from different sources such as corn, rice and wheat. Three

commercial alpha amylases produced from Bacillus sp. A oryzae and B. licheniformis

were employed for hydrolysis of starch. In every starch hydrolysis process, the

concentration of residual starch and the residual activity of alpha amylase in

percentage were determined at 50 and 60°C temperature based upon the processing

32

time in a stirred batch reactor. Mathematical models were developed for using

experimental data of residual starch concentration from each source. Some

inactivation models were used to understand the relation between temperature and

stability of enzyme during hydrolysis of starch from enzymes having different origins.

El-Safey and Ammar (2004) reported that the amylolytic family has great

importance due to its wide spectrum of application. Alpha amylase produced from

Aspegillus flavus var.columinaris was isolated and characterized. The enzyme was

purified by using ammonium sulfate precipitation and Sephadex G200 filtration

method. The purified enzyme showed 9.97 fold purification and 6471.6 (units/mg

port/ml) specific activity. The alpha amylase activity amplified with the enhancement

of enzyme concentration. The optimum condition for the production of alpha amylase

was 0.2% (w/v) starch, while the optimal temperature was 35°C. The purified alpha

amylase showed maximum activity at pH 6.2 after 30 h of incubation. Pimpa (2004)

reported that the highest alpha amylase production by Aspergillus sp. was obtained

after 24 h. Addition of suitable nitrogen sources and inorganic salts to the medium

appreciably increased the enzyme production. The maximum enzyme yield 36.5 U/ml

was obtained in the media containing wastewater, defatted soyabean 10 g/l, potassium

di hydrogen phosphate 10g/l, magnesium sulfate 5 g/l, zinc chloride 0.1 g/l. The alpha

amylase produced by Aspergillus sp. showed catabolic repression. The enzyme was

partially purified by subjecting into 60 % ammonium sulfate. The optimal pH and

temperature of partially purified enzyme was 5 and 50°C, respectively.

Chavez et al. (2004) screened different carbon sources namely sorghum, soluble

potato, corn and cassava starches as well as maltose for the concurrent cultivation and

33

production of both alpha amylase and glucoamylase by a novel Trichoderma sp. even

though maltose gave better results compared to other carbon sources with respect to

activity of alpha amylase (about 28,000 U/l) and alpha amylase production (about 390

U/l/h), cassava and corn starches showed maximum glucoamylase activities (17,000-

18,000 U/l) and production of enzyme was almost similar to those obtained with

maltose (about 100 U/l/h). Because of its capability to produce both alpha amylase and

glucoamylase, the Trichoderma sp used in this study proved to be beneficial in a direct

process of raw starch saccharification with no preliminary gelatinization.

Konsula and Kyriakides (2004) isolated a somewhat thermophilic Bacillus

subtilis strain, from fresh milk of sheep possess the ability to produce extracellular

thermostable alpha amylase. The medium containing low starch concentration showed

maximum alpha amylase production at 40°C. The enzyme exhibited highest activity at

135°C and pH 6.5. The thermostability of alpha amylase increased in the presence of

calcium or starch. This thermostable alpha amylase was employed for the hydrolysis

of different starches. Ammonium sulfate crude enzyme preparations as well as the

cell-free supernatant actively break down the starches. The use of the clear supernatant

as enzyme source was highly advantageous mainly because it decreases the cost of the

hydrolysis. When the reaction temperature increased up to 70°C, all of the substrate

showed higher rates of hydrolysis. Potato starch upon hydrolysis produced higher

concentration of reducing sugars compared to other starches at all tested temperatures.

Soluble and rice starch came at second and third position respectively, with respect to

reducing sugar liberating ability. However, in case of corn and oat starch alpha

amylase showed somewhat less affinity.

34

Ramachandran et al. (2004) investigated alpha amylase production by A. oryzae

in solid state fermentation (SSF).The substrate used was coconut oil cake (COC). Raw

COC was a good substrate and 1372 U/gds alpha amylase was produced in 24 h. As a

result of optimization of media component alpha amylase production was increased

(1827 U/gds) when solid state fermentation was carried out at 30°C for 72 h along

with media contained 68 % moisture contents. Addition of glucose and 0.5 % starch

further increased the alpha amylase production (1911 U/gds). However, maltose

repressed the alpha amylase production. Alpha amylase production increased upon

adding the organic and inorganic nitrogen sources. When peptone at the level of 1 %

was added in the fermentation media 1.7-fold increase in enzyme activity (3388

U/gds) was observed. The enzyme production and growth were correlated. The

activity became maximal when the fungal biomass was at its peak at 72 h.

Kunamneni et al. (2005) employed the response surface methodology to study

the collective impact of the nutritional parameters and to increase extracellular alpha-

amylase production in solid-state fermentation by T. lanuginosus. These nutritional

parameters consist of carbon source (soluble starch), nitrogen source (peptone) and a

concentrated mineral medium. A twenty three factorial central composite design using

response surface methodology was used to optimize the above three variables. The

best calculated values of these variables for optimal amylase production were soluble

starch 71.10 g/Kg, peptone 91.97 g/Kg and mineral salts solution 175.05 ml/Kg with

an estimated alpha amylase activity of 5.085 ´ 105 U/Kg of wheat bran. These

parameters were checked in the laboratory and ultimate alpha amylase activity

obtained, 4.946 ´ 105 U/Kg of wheat bran, was very near to the calculated value.

35

Kiran et al. (2005) isolated the thermophilic Bacillus sp. K-12 from soil samples

having the ability to produced amylolytic enzyme. Effects of different carbon sources

and chemicals on production of alpha amylase were checked in the laboratory.

Medium consist of 1 % starch showed maximum alpha amylase activity after 60 h of

fermentation. However manganese sulfate, zinc sulfate and EDTA showed inhibitory

effect on alpha amylase production by Bacillus sp. Haq et al., (2005a) reported the

choice of the appropriate surfactant for alpha amylase production by Bacillus subtilis

GCBM-25 during shake flasks. Various surfactants (laundry soap, detergent powder,

sulphonic acid, acyle benzene sulphonic acid, liquid soap, Tween 80, sodium silicate,

bath soap, sodium tripolyphosphate, sodium lauryl ether sulphate or sodium lauryl

sulphate) at rate 2.0 % (w/v) were screened for synthesis of enzyme. Among all the

surfactants, tested laundry soap proved to be superior with respect to alpha amylase

production (605 U/ml/min) after 44 h of fermentation using 4.0 % inoculum. The

enzyme production was enhanced (857 U/ml/min) with the addition of Millon soap at

rate 3.2 % (w/v) in the medium. However, addition of surfactants in the medium

reduced the thermostability from 70 to 50°C.

Haq et al. (2005) reported the use of agricultural starchy substrate for alpha

amylase production by Bacillus licheniformis. The use of agriculture by products

made the medium economic. Soluble starch, hordium, pearl millet, rice, corn, gram

and wheat starch were screened for the alpha amylase production by parental and its

mutant derivative. The mutant strain B. licheniformis GCUCM-30 exhibited 10 fold

more enzyme production compared to parental strain when1.5 % pear millet and 0.25

% of nutrient broth was added to fermentation medium.

36

Anto et al. (2006) reported the alpha amylase production by B. cereus MTCC

1305 in solid state fermentation using wheat bran and rice flake manufacturing waste

as substrates. Maximum alpha amylase activity (94±2) U/g was obtained when wheat

bran was used as a substrate. Optimal conditions for alpha amylase production were

inoculum size 10 % substrate moisture ratio 1:1 and glucose, (0.04 g/g). Addition of

different nitrogen sources (0.02 g/g) showed decrease in enzyme production. Optimal

alpha amylase activity was observed at 55°C and pH 5. Swain et al. (2006) reported

the alpha amylase production by B. subtilis isolated earlier from cow dung microflora.

The optimum temperature, pH and incubation period for amylase production were 50-

70°C, 5-9 and 36 h, respectively. Enzyme secretion was very similar in the presence of

any of the carbon sources tested (soluble starch, potato starch, cassava starch, wheat

flour, glucose, fructose, etc.). Yeast extract and ammonium acetate (1 %) as nitrogen

sources gave higher yield compared to other nitrogen sources (peptone, malt extract,

casein, asparagine, glycine, beef extract) whereas ammonium chloride, ammonium

sulfate and urea inhibited the enzyme activity. Addition of Ca+2 (10-40 mM) to the

culture medium did not result in further improvement of enzyme production, whereas

the addition of surfactants (Tween 20, Tween 40, Tween 80, and sodium lauryl

sulphate) at 0.02 % resulted in 2-15 % increase in enzyme production. There were no

significant variations in enzyme yield between shake flask and laboratory fermenter

cultures. The purified enzyme was in two forms with molecular mass of 18.0 ± 1 and

43.0 ± 1 kDa in native SDS-PAGE.

Kathireasan and Manivannan (2006) isolated Penicillium fellutanum from

coastal mangrove soil and screened out the sound effects of different variables such

37

as pH, temperature, incubation time, salinity, carbon and nitrogen sources in shake

flasks fermentation for alpha amylase production. The fermentation medium with no

addition of seawater and supplemented with maltose and peptone as carbon and

nitrogen source was incubated for 96 h, at pH 6.5 and temperature 30°C, gave

maximum alpha amylase production by P. fellutanum. Djekrif-Dakhmouche et al.

(2006) studied the alpha amylase production and optimization from A. niger ATCC

16404 .The statistical analysis has revealed that variation in agitation from 150 rpm -

200 rpm has no effect on the alpha amylase production but it increased biomass. As

far as variation in pH from 5 to 6 has positive effect on alpha amylase production

while its effect on the biomass was negative. The addition of starch at 10 g/l to the

fermentation medium (an inductive substrate and carbon source) stimulated the alpha

amylase production, while it has no effect on biomass production. Calcium chloride at

1 g/l (a structural and stabilizing element for the alpha amylase) solely affect the

enzyme production. The use of other salts (manganese, iron sulfates as well as

magnesium chloride) seemed to be increased alpha amylase production but did not

effect either the production of protein or biomass.

Prakasham et al. (2007) reported fractional factorial design of Taguchi

methodology for the optimization of medium along with eight variables soluble

starch, corn steep liquor, casein, potassium dihydrogen phosphate, magnesium sulfate,

initial pH, incubation temperature and inoculum size for the amylase production in

submerged fermentation by A. awamori. Considerable enhancement approximately

48% in acid amylase synthesis was observed. The optimized fermentation medium

included in (%) soluble starch 3, CSL 0.5, KH2PO4 0.125, casein 1.5 at pH 4 and

38

31°C. Shoji et al. (2007) reported a new submerged culture system of A. kawachii

NBRC4308 with the help of barley whose surface was wholly or partially covered

with husk. Both glucoamylase activity 150.8 U/ml and acid-stable alpha amylase

activity 7.7 U/ml were found in the supernatant in the presence of low concentration of

glucose.

Rao et al. (2007) investigated the formation of spores from B.

amyloliquefaciens B128 in shake flask cultivation. Fermentation media were

optimized by applying two steps approach. A rapid identification of an appropriate

carbon and nitrogen source was obtained by screening experimentation, and use of

response surface methodology (RSM). A five-level four-factor central composite

design was used to find out the highest spore yield at optimal level for lactose, tapioca,

ammonium sulfate and peptone. A noteworthy linear major effect was observed in the

case of topica and peptone, while lactose and ammonium sulfate produced no

important linear effect. Lactose-ammonium sulfate and lactose-peptone extensively

affected spore production. Optimum conditions for the alpha amylase production were

(g/l): lactose 12.7, tapioca 16.7, ammonium sulfate 1.8 and peptone 8. The predicted

spore production was 5.93 × 108 (no/ml). The real experimental results were in

concurrence with the prediction.

Suganuma et al. (2007) reported that highly humid climate of Japan facilitate the

growth of various molds. Among these molds A. oryzae was the most important and

popular in Japan, and has been used as yellow-koji in producing many traditional

fermented beverages and foods, such as Japanese sake, and soy sauce. The koji molds

black-koji and white-koji produce two types of alpha amylase, namely, acid-stable

39

(AA) and common neutral (NA).The latter enzyme was enzymatically genetically

similar to Taka amylase. In this review they investigated AA from three molds, A.

niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the

distinguishable properties between AA and NA. AA has many advantages in industrial

applications, such as its acid-stability, thermostability, and raw-starch digesting

properties. Bhanja et al. (2007) used Growtek bioreactor as modified solid state

fermenter to circumvent many of the problems associated with the conventional tray

reactors for solid state fermentation (SSF). A. oryzae IFO-30103 produced very high

levels of alpha amylase by modified solid state fermentation (mSSF) compared to SSF

carried out in enamel coated metallic trays utilizing wheat bran as substrate. High

alpha amylase yield of 15,833 U/ g dry solid in mSSF were obtained when the fungus

were cultivated at an initial pH of 6 at 32°C for 54 h whereas alpha amylase

production in SSF reached its maximal (12,899 U g–1 dry solid) at 30°C after 66 h of

incubation. With the supplementation of 1 % NaNO3, the maximum activity obtained

was 19,665 U g–1 dry solid (24% higher than control) in mSSF, whereas, in SSF

maximum activity was 15,480 U/ g dry solid in presence of 0.1 % Triton X-100 (20 %

higher than the control).

Tayeb et al. (2007) conducted the alpha amylase production using amplified

variants of B. subtilis (strain SCH) and of B. amyloliquefaciens (strain 267CH) in a

bioreactor with multiprotein-mineral media. The time course of fermentation in a

bioreactor revealed that the highest yield (about 8 x 104 U/ml within 6 h) by strain

SCH was obtained by applying: 3.5 % initial starch, 2 % additional starch after 19 h, 3

vvm aeration and 300 rpm agitation. The highest yield (about 19 x 104 U/ml within

40

100 h) by strain 267CH was obtained by applying: 2.5 % initial starch, 2 % additional

starch after 24 h, 3 vvm aeration, and 300 rpm agitation with the productivity after 60

h reaching only about 14 x 104 U/ml. Production occurred in both the logarithmic and

post logarithmic phases of growth. Maximum consumption of starch and protein

occurred during the first day of incubation. The optical density peak coincided with

enzyme production peak in case of strain SCH and preceded that of enzyme

production in case of strain 267CH. The alpha amylase produced by the two strains

was shown to be the liquefying and not both enzymes liquefied starch to a dextrose

equivalent of about 15-17 at 95°C hence they are classified among thermostable alpha

amylases. They exhibited broad pH and temperature activity profiles. The optimum

pH for activity was 4-7 for alpha amylase produced by strain SCH and 4-8 for alpha

amylase produced by strain 267CH while the optimum temperatures for their activities

were in the range of 37 -75°C at 0.5 % starch and in the range of 85 - 95°C at 35 %

starch.

Poornima et al. (2008) isolated different strains of actinomycetes and tested

these strain for their ability to synthesize the alpha amylase. Among all the strains, the

strain AE-19 showed best alpha amylase production. This strain was identified as

Streptomyces aureofasciculus and selected for subsequent studies. The highest alpha

amylase production was obtained in the presence of mannose and L-histidine as

carbon and nitrogen source, 0.05 % sodium chloride at temperature 45°C and pH 9.

These results indicated that strain can be successfully used for commercial alpha

amylase production after testing strain competence in large scale fermentations. Gupta

et al. (2008) studied the nutritional requirements of A. niger and the factors such as

41

incubation temperature, pH of medium, carbon and nitrogen sources, fermentaion

period, surfactants and concentration of metal ions. The experimental result showed

ideal carbon and nitrogen source for alpha amylase production was 0.5 % starch and

0.3 % peptone. The optimal pH, temperature and fermentation period were 5, 30°C

and 5th day, respectively. Different surfactant at varying level such as Tween-80,

Triton X-100 and Sodium dodecyl sulphate at 0.02, 0.002 and 0.0002 % concentration,

respectively exhibited enhanced alpha amylase productivity. The major purpose of the

present study was to employ an appropriate fungal strain for extracellular alpha

amylase production and find out the fermentation period for the synthesis of alpha

amylase and to determine the effects of external substances that might increased the

synthesis of alpha amylase.

Esfahanibolandbalaie et al. (2008) reported the effect of many chemical and

physical factors on alpha amylase production by A. oryzae in shake flasks

fermentation via an Adlof-Kuhner orbital shaker. The impact of varying pH of

medium ranging from 4-7 was studied. The maximum alpha amylase production was

obtained at pH 6.2. Carbon and nitrogen source has discernible effect on the enzyme

production. The corn starch at level of 15 g/l proved to be best carbon source for alpha

amylase synthesis while glucose represses the alpha amylase production. The medium

consist of corn starch, sodium nitrate as inorganic nitrogen resulted in significant

enzyme production. Among the organic nitrogen sources yeast extract at the level of

2.5g /l was excellent nitrogen source. The impact of different temperatures and

agitation speed from 20 to 40°C and 50 to 200 rpm, respectively was observed. The

maximum activity was obtained at 35°C and 180 rpm. Planchot and Colonna (2008)

42

purified A. fumigatus (Aspergillus sp. K-27) extracellular alpha amylase to

homogeneity by using anion-exchange DEAE-cellulose and affinity α-cyclodextrin-

Sepharose chromatography. The purified enzyme was glycoprotein in nature which

was found to contain 15 % carbohydrate. The purified alpha amylase exhibited an

isoelectric point of 3.7, and SDS PAGE estimated that purified enzyme possess a

molecular weight of 65,000. A large number of neutral hydrophobic residues were

present in an amino acid. The optimum enzyme activity was obtained at pH 5.5, and

the enzyme showed stability at 40°C. It hydrolyzed amylose and amylopectin, with

respective Km of 0.42 and 7.7 mg mL- 1 and kcat/K m of 3.4 and 2.5 mL mg -1 min-1.

The main end-products of maltohexaose, hydrolysis were glucose and maltose. While

intermediate products were maltotriose, maltotetraose, and maltopentaose having an α-

anomeric configuration. Although its capability to gradually degrade some α1-6

linkages, purified enzyme ought to be classified as an alpha-amylase.

Leman et al. (2009) reported alpha amylase from A. oryzae had only very little

effect on the side chain segments of the amylopectin molecules and the reason might

be enzyme hydrolysis the segments of internal chain. Singh et al., (2009) investigated

the effect of various agricultural by products as a substrate such as wheat bran, wheat

straw, rye, straw on the alpha amylase production by Humicola lanuginose in solid

state fermentation. Wheat bran proved to be good substrates for starch degrading

enzymes because highest alpha amylase production was observed when wheat bran

was used as a substrate. Various variables such as moisture content, incubation time

inoculum size and carbon source has marked effect on the enzyme production. It was

noted the optimum condition for the alpha amylase production by Humicola

43

lanuginose in SSF was incubation period 144 h, initial moisture content 90 %, initial

pH of medium 6, incubation temperature 50ºC , size of inoculum 20 % and soluble

starch as best carbon source.

Shafique et al. (2009) tested the five strains of fungi belonging to two

filamentous fungi A. niger and A. flavus for their ability to produce alpha-amylase.

The different chosen strains were cultivated on two different typed of media i.e.,

potato dextrose agar (PDA) and enzyme production medium (EPM), the pH of

medium was fixed at 3 level i.e., 4.5, 5.5 and 6.5. The efficiency or ability of strains

was estimated on the basis of the formation of hydrolysis zone. EPM medium at pH

4.5 was best for the highest activity of alpha amylase. Strain 74 and strain 198 of A.

niger and strain 209 and strain 231 of A. flavus gave best result on solid media; so

these strains were selected for the alpha amylase production in submerged

fermentation. All the selected strains showed highest activity of alpha amylase after 48

h in shake flasks.

44

Uses of alpha amylase

Starch is a major storage product of many economically important crops such as

wheat, rice, maize, tapioca, and potato. A large-scale starch processing industry has

emerged in the last century. In the past decades, we have seen a shift from the acid

hydrolysis of starch to the use of starch-converting enzymes in the production of

maltodextrin, modified starches, or glucose and fructose syrups. Currently, these

enzymes comprise about 30 % of the world's enzyme production. Besides the use in

starch hydrolysis, starch-converting enzymes are also used in a number of other

industrial applications, such as laundry and porcelain detergents or as anti-staling

agents in baking. A number of these starch-converting enzymes belong to a single

family: the alpha amylase family or family13 glycosyl hydrolases. This group of

enzymes share a number of common characteristics such as a (β/α)8 barrel structure,

the hydrolysis or formation of glycosidic bonds in the α conformation, and a number

of conserved amino acid residues in the active site. As many as 21 different reaction

and product specificities are found in this family.

Bread and chapatti industry

The quantities, taste, aroma and porosity of the bread are improved by using the enzyme

in the flour. More than 70 % bread in U.S.A, Russia and European countries contain

alpha amylase. Amylases play important role in bakery products (Goodwin and Mercer,

1972). For decades, enzymes such as malt and fungal alpha-amylases have been used in

bread-making. The significance of enzymes is likely to raise as consumers insist more

natural products free of chemical additives. For example, enzymes can be employed to

45

replace potassium bromate, a chemical additive that has been prohibited in a number of

countries. The dough for bread, rolls, buns and similar products consists of flour, water,

yeast, salt and possibly other ingredients such as sugar and fat. Flour consists of gluten,

starch, non-starch polysaccharides, lipids and trace amounts of minerals. As soon as the

dough is made, the yeast starts to work on the fermentable sugars, transforming them

into alcohol and carbon dioxide, which makes the dough rise. The major component of

wheat flour is starch. Amylases can degrade starch and produce small dextrins for the

yeast to act upon. The alpha-amylases degrade the damaged starch in wheat flour into

small dextrins, which allows yeast to work continuously during dough fermentation,

proofing and the early stage of baking. The result is improved bread volume and crumb

texture. In addition, the small oligosaccharides and sugars such as glucose and maltose

produced by these enzymes enhance the Maillard reactions responsible for the browning

of the crust and the development of an attractive baked flavour (Lundkvist et al., 2007).

Textile industry

Textile industries are extensively using alpha amylases to hydrolyze and solubilize the

starch, which then wash out of the cloth for increasing the stiffness of the finished

products. Fabrics are sized with starch. Alpha amylase is used as desizing agent for

removing starch from the grey cloth before its further processing in bleaching and

dyeing. Many garments especially the ubiquitions‛ Jean ’ are desized after mashing.

The desired fabrics are finally laundered and rinsed (Iqbal et al., 1997; Allan et al.,

1997).

46

Sugar and Glucose industries

Alpha amylase plays a very important role in the production of starch

conversion products of low fermentability. The presence of starch and other

polysaccharides in sugar cane creates problem throughout the sugar manufacturing

which is minimized or eliminated by the action of alpha amylase. The high quality

products depends upon the efficiency of the enzyme which lead to low production,

costs for the starch processor has increased (De-cordt et al., 1994; Ensari et al., 1996;

Hamilton et al., 1998). Many industries used alpha amylases for the production of

glucose. Enzyme hydrolyzed the starch and converted it into glucose. They hydrolyze

α-1,4 glucosidic linkage in the starch polymer in a random manner to yield glucose

and maltose (Akiba et al., 1998). Therefore, alpha amylase is extensively used in

many industries for the production of glucose (Shetty and Crab, 1990). This process

also gives the water-soluble dextrin.

Alcohol Industry

Alpha amylases convert starch in to fermentable sugars. Starches such as grain;

potatoes etc. are used as a raw material that helps to manufacture ethyl alcohol. In the

presence of amylases, the starch is first converted in to fermentable sugars. The use of

bacterial enzyme partly replaces malt in brewing industry, thus making the process

more economically significant. Alpha amylase can also carries out the reactions of

alcoholysis by using methanol as a substrate (Santamaria et al., 1999).

47

Paper industry

Starch paste when used as a mounting adhesive modified with additives such as

protein glue or alum, frequently, causes damage to paper as a result of its

embrittlement. Starch digesting enzymes, e.g. alpha amylase, in immersion or as a gel

poultice are applied to facilitate its removal. Alpha amylase hydrolyzed the raw starch

that is used for sizing and coating the paper instead of expensive chemically modified

starches. So, starch is extensively used for some paper size press publications (Okolo

et al., 1996).

Detergent, Building product and Feed industries

In detergent industries, the enzyme alpha amylase plays a vital role. It is widely used

for improvement of detergency of laundry bleach composition and bleaching with out

color darkening (Borchet et al., 1995; Atsushi and Eiichi, 1998). The addition of

enzyme stabilizes the bleach agent and preserves effectiveness of the bleach in laundry

detergent bar composition (Onzales, 1997; Mirasol et al., 1997) Modified starch is

used in the manufacture of gypsum board for dry wall construction. Enzyme modified

the starch for the industry use. Many starches or barely material are present in the

feed. So, the nutritional value of the feed can be improved by the addition of alpha

amylase.

Chocolate industry

Amylases are treated with cocoa slurries to produce chocolate syrup, in which

chocolate starch is dextrinizing and thus syrup does not become thick. Cocoa flavored

syrups having a high cocoa content and excellent stability and flow properties at room

48

temperature may be produced by using an amylolytic enzyme and a sufficient

proportion of Dutch process cocoa to provide a syrup pH of 5.5 to 7.5. The syrup is

made by alternate addition of cocoa and sweetener to sufficient water to achieve a

solids content of about 58 to 65 weight percent, adding an amylolytic enzyme, heating

to a temperature of about 175 -185°F for at least 10 to 15 min, raising the temperature

to about 200° F. and cooling. The stabilized cocoa flavored syrups may be added at

room temperature to conventional non-acid confection mixes for use in the production

of quiescently frozen chocolate flavored confections (Ismail et al., 1992)