BioEvolution

Vol 2 (1), Feb 2015, ISBN [978-93-83006-04-5], pg 19-25

www.giapjournals.org/bioevolution.html 19

MICROBIAL ENZYMES WITH SPECIAL REFERENCE TO α-AMYLASE

Tejasvi Kaur Sahni1 and Dr. Arti Goel

2*

1B. Tech. Student, Amity Institute of Biotechnology, Amity University, Sector-125, Noida, 201301, (U.P.), India

2Assistant Professor, Amity Institute of Microbial Biotechnology, Amity University, Sector-125, Noida, 201301, (U.P.), India

Corresponding Author E- mail: agoel2@amity.edu, peena_agrawal@yahoo.co.in

ABSTRACT

The microbial enzymes particularly the bacterial enzymes have gained more popularity in the industrial sector as they are

more stable than the enzymes prepared from plants and animal resources. Modern molecular techniques, such as

metagenomics modified by different strategies are used for analysis of microbial enzymes. Bacteria and fungi produce the

majority of industrial microbial enzymes. Production of enzyme is carries out using submerged fementation and also solid

state fermentation in some cases. α-Amylase (1,4- α -D-glucanohydrolase; EC3.2.1.1) is responsible for hydrolysis of the α-

1,4-glucosidic linkage in starch and other similar molecules. It is one of the many enzymes involved in starch degradation.

The production of α-amylase is essential for the coversion of starch to oligiosaccharide. Enzymes like amylases- starch

converting enzymes, are important in production of glucose and fructose syrups, modified starches. For isolation and

optimization of a-amylase from bacillus species, an α-amylase producing bacillus species is isolated from a soil sample and

the soluble starch amylase was characterized. Then, the optimum pH activity,temperature activity and substrated

conectration for bacterial growth is observed. The purification of the enzyme is then carried out and the enzyme growth is

then recorded.

Keywords- Microbial enzymes, α-Amylase, Starch, α – 1, 4 – glycosidic bonds,

INTRODUCTION

Enzymes are proteins that act as catalysts which can speed up chemical reactions that occur in the cells. An enzyme can add

atoms to a molecule, move atoms from a molecule, split a large molecule into two smaller molecules, or join together smaller

molecules to form a large molecule. Enzymes have unique shapes and reactive sites that allow them to bind with specific

molecules namely substrate molecules.

Digestive enzymes turn the food we eat into energy and unlock this energy for the use in the body. Our bodies naturally

produce both digestive and metabolic enzymes. It is believed that taking enzymes to supplement cooked food lack of enzymes

will promote better digestion and preserve your own limited enzyme production. Once enzyme production ceases in the body,

life ends. Enzymes are present in each living cell thus also in all microorganisms.

Hydrolytic enzymes also called as hydrolases are the enzymes that carry out the hydrolysis of a chemical bond. Hydrolysis is

the biochemical reaction that involves the addition of water molecule and the cleavage of the covalent bond holding the two

monomers together.

A-B + H2O A-OH + B-H

Amylases, proteases, lipases are some examples of hydrolytic enzymes.

MICROBIAL ENZYMES

Microbial enzymes term represent the enzymes that are present in the various species of bacteria, fungi and yeast. These

enzymes play a crucial role in industrial biochemical processes. Each single strain of these organisms produces a large number

of enzymes, hydrolyzing, oxidizing or reducing, and metabolic in nature. But the absolute and relative amounts of the various

individual enzymes produced vary markedly between species and even between strains of the same species. Hence, for the

BioEvolution

Vol 2 (1), Feb 2015, ISBN [978-93-83006-04-5], pg 19-25

www.giapjournals.org/bioevolution.html 20

commercial production of specific enzymes the strain of microbes which have the capacity for producing highest amounts of

the particular enzymes is desired.

BACTERIAL ENZYMES

The enzymes obtained and isolated from bacteria are the numerous and have vast variety of applications. The organisms like

Bacillus subtilis, Bacillus liceniformis, Bacillus amylofaciens and other species of Halobacillus are the main produces of

bacterial enzymes. Also various acidophilic and thermo acidophilic bacteria produce enzymes like amylases. Thermostable

enzymes have increased use in industries as the are able to function even in harsh extreme temperature conditions. Enzymes

produced by some halophilic microorganisms have optimal activity at high salinities and thus acn be used in difficult industrial

environments,where the concentrated salt solutions used would interfere with enzyme activity.In addition, most halobacterial

enzymes are considerably thermotolerant and remain stable at room temperature over long periods. Halophilic amylases have

been characterized from halophilic bacteria such as Chromohalobacter sp., and Bacillus dipsosauri.

FUNGAL ENZYMES

Species of Aspergillus like Aspergillus niger, Aspergillus oryzae, Aspergillus flavus are the major fungal species that are

capable of producing industrially functional enzymes. Also these filamentous fungi, produce large quantities of amylases

which can then be used commercially. Aspergillus oryzae has been largely used in the production of food such as soy sauce,

organic acid such as citric and acetic acids and enzymes including α-amylase. Aspergillus niger has significant hydrolytic

capacities in the α-amylase production. Also, due to its tolerance of acidity (pH < 3), it allows the avoidance of bacterial

contamination.

AMYLASES

Amylases are enzymes that hydrolyse starch molecules to give diverse products including dextrins and progressively smaller

polymer composed of glucose units. Amylases are enzymes which hydrolyze starch molecules to give diverse products

including dextrins and progressively smaller polymers composed of glucose units. Today a large number of microbial amylases

are available commercially and they have almost completely replaced chemical hydrolysis of starch in starch processing

industry.

Amylases can be divided into two types on the basis of the manner of catalysis, endoamylases and exoamylases. Endoamylases

catalyze hydrolysis in a random manner in the interior of the starch molecule. This action causes the formation of linear and

branched oligosaccharides of different chain lengths. Exoamylases hydrolyze from the non-reducing end, successively

resulting in short end products. A large number of enzymes are known which hydrolyze starch molecule into different products

and a combined action of various enzymes is required to hydrolyze starch completely.

STRUCTURE

The structure of this enzyme is simple. There are different variations of the amylase molecule, called alpha (α) amylase and

beta (β) amylase. The human body has two types of α-amylase, one in saliva and one in the pancreas. Both molecules are

important in digestion of carbohydrates, since the body cannot digest complex sugars and starches without breaking them down

first. Β-amylase is not present in the human body, and is usually found in plants and seeds.

Even though the basic structure of amylase is not quite complex, there are some variants that are significantly more

complicated than the others. Most α-amylases are very similar in length, but there are some that fold into more than three

common domains. Many food crops have this type of α-amylase, including barley, soybeans, and sweet potatoes, but the size

of the α-amylase molecule does not enhance or detract from the plant's nutritional properties.

BioEvolution

Vol 2 (1), Feb 2015, ISBN [978-93-83006-04-5], pg 19-25

www.giapjournals.org/bioevolution.html 21

SUBSTRATE HYDROLYSIS

The substrate for the amylase enzyme is starch. It is often found in seeds and tubers (e.g., potatoes). It is made up of two kinds

of polymers of glucose. The simpler kind of polymer is called amylose, and it makes up about 20% of starch. It is basically a

chain of glucose units linked by α – 1, 4 – glycosidic bonds. The other 80% of starch is a water insoluble molecule called

amylopectin. It is a branched chain polysaccharide with α – 1, 4 – glycosidic links. This branching of glucose units occurs at

every 25 glucose units. When treated with acid or enzymes, the components of starch are hydrolyzed to dextrins (mixture of

low melting polysaccharides, made up of 3 – 8 glucose units), maltose and finally D-glucose.

Figure 1- Starch (amylose)

Starch is one of the major nutrients in the human diet. Its presence can be detected by the blue-black color produced when

iodine solution is added to a sample of the material ,like food, to be tested.

Starch + I2 blue-black color

TYPES OF AMYLASE

α-AMYLASE (E.C.3.2.1.1) –

α-amylase are enzymes that catalyses the hydrolysis of internal α-1,4-glycosidic linkages in starch in low molecular weight

products, such glucose, maltose and maltotriose units. A-Amylase can be derived from several fungi, yeasts and bacteria.

However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors.

β-AMYLASE (EC 3.2.1.2) -

β -amylase is another form of amylase synthesized by bacteria, fungi, and plants. β -amylase catalyzes the hydrolysis of the

second α-1,4-glycosidic bond. It works from the non-reducing end, cleaving off two glucose units at a time. During the

ripening of fruit, β -amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit. Animal tissues do not have β -

amylase.

γ- AMYLASE (EC 3.2.1.3) -

γ -amylase cleaves α-1,6-glycosidic linkages, in addition to cleaving the last α-1,4-glycosidic linkages at the non-reducing end

of amylose and amylopectin, yielding glucose.

α –AMYLASE

STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF α -AMYLASE

The α-amylase belongs to the family of endoamylases which catalyse the initial hydrolysis of starch into shorter

oligosaccharides through the cleavage of α-D-(1-4) glycosidic bond. The terminal glucose residues and α-1, 6-linkages cannot

be cleared by α-amylase. The end products of α-amylase enzyme action are oligosaccharides 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..

The amylase has a three-dimensional structure capable of binding to substrate and, by the action of highly specific catalytic

groups this enzyme promotes the breakage of the glycoside bonds. The human 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. The protein

contains 3 domains: A, B, and C.

BioEvolution

Vol 2 (1), Feb 2015, ISBN [978-93-83006-04-5], pg 19-25

www.giapjournals.org/bioevolution.html 22

Figure 2 - The domains of α-amylase

Domain A includes the red region; Domain B is the yellow region whereas the purple region is the Domain C

INDUSTRIAL APPLICATION OF α -AMYLASE

STARCH PROCESSING INDUSTRY

The most wide applications of α-amylases are in the starch industry, which are used for starch hydrolysis in the starch

liquefaction process that converts starch into fructose and glucose syrups. The enzymatic conversion of all starch includes:

gelatinization, which includes the dissolution of starch granules, thus forming a suspension. The next process includes

liquefaction, which involves partial hydrolysis and loss in viscosity; and the third is saccharification, involving the production

of glucose and maltose via further hydrolysis.

Initially, the α-amylase of Bacillus amyloliquefaciens was used in starch processing but it has been replaced by the α-amylase

of Bacillus stearothermophilus or Bacillus licheniformis. The enzymes from the Bacillus species hold special interest for large-

scale biotechnological processes, the reason being their marked thermostability and efficient expression systems are available

for these enzymes.

DETERGENT INDUSTRY

The primary consumption of enzymes is in detergent industries. Amylases are second in rank of enzymes that can be used in

the formulation of enzymatic detergent. Amylase is used in detergents for laundry and automatic dishwashing. This leads to

conversion of the residues of starchy foods such as potatoes, gravies, custard, chocolate, etc. to dextrins and other smaller

oligosaccharides. Since, amylases have optimum activity at lower temperatures and at alkaline pH. Maintaining the necessary

stability under washing conditions and the oxidative stability of amylases is a important criterion for use of amylase in

detergents as the washing environment is very oxidizing. Examples of amylases used in the detergent industry are derived from

Bacillus or Aspergillus.

FUEL ALCOHOL INDUSTRY

Ethanol is the most utilized liquid biofuel. For the ethanol production, starch is the most abundant substrate due to its low price

and easy availability as a raw material. During this production, starch is solubilized and then subjected to two enzymatic steps

in order to obtain fermentable sugars. The bioconversion of starch into ethanol involves liquefaction and saccharification,

where starch is converted into sugar using an amylolytic enzyme such as α-amylase, followed by fermentation, where sugar is

converted into ethanol using an ethanol fermenting microorganism such as yeast, for example Saccharomyces cerevisia.

Among bacteria, α-amylase obtained from thermoresistant bacteria like Bacillus licheniformis or from engineered strains of

Escherichia coli or Bacillus subtilis is used during the first step of hydrolysis of starch suspensions.

FOOD INDUSTRY

Amylases are extensively employed in processed-food industry such as brewing, preparation of digestive aids or supplements,

production of cakes, fruit juices and starch syrups. The α-amylases are widely used in the baking industry. These enzymes can

BioEvolution

Vol 2 (1), Feb 2015, ISBN [978-93-83006-04-5], pg 19-25

www.giapjournals.org/bioevolution.html 23

be added to the dough of bread, this degrades the starch in the flour into smaller dextrins, which are then fermented by the

yeast. The addition of α-amylase also lead to enhancing of the rate of fermentation and the reduction of the viscosity of dough,

resulting in improvements in the volume and texture of the product formed. Also, it generates additional sugar in the dough,

which improves the taste, crust color and toasting qualities of the bread.

TEXTILE INDUSTRY

Amylases are used in textile industry for desizing process. Sizing agents like starch are applied to yarn before fabric is

produced to ensure a fast and secure weaving process. Starch is a very attractive sizing agent, because it is cheap, easily

available in many regions of the world. Also it can be quite easily removed. Starch is then removed from the woven fabric in

the textile finishing industry.

Desizing involves the removal of starch from the fabric. The starch acts as a strengthening agent to prevent breaking of the

thread during the weaving process. The α-amylase remove selectively the size that is the starch and do not attack the fibers.

Amylase from Bacillus stain was employed in textile industries for quite a long time.

PAPER INDUSTRY

The use of α-amylases in the pulp and paper industry is for the modification of starch of coated paper, i.e. for the production of

low-viscosity, high molecular weight starch. The coating treatment serves to make the surface of paper sufficiently smooth and

strong, to improve the writing quality of the paper. In this application, the viscosity of the natural starch is too high for paper

sizing and this can be altered by partially degrading the polymer with α-amylases in a batch or continuous processes. Starch is

a good sizing agent for the finishing of paper, improving the quality and erasebility, besides being a good coating for the paper.

The size enhances the stiffness and strength in paper.

Examples of amylases obtained from microorganisms used in paper industry includes Amizyme® (PMP Fermentation

Products, Peoria, USA), Termamyl®, Fungamyl, BAN® (Novozymes, Denmark) and -amylase G9995® (Enzyme Biosystems,

USA).

CONCLUSION

Microbes are able to produce enzymes which will be commercial in nature and obtained from strains of molds, bacteria, and

yeasts. Also, Microorganisms that can survive under extreme pH values could be good sources of thermoalkaliphilic enzymes,

like proteases and lipases, particularly useful for applications as additives in laundry and dishwashing detergents. The reason

for this increasing use of microbial enzyme in industry is the ease with which these enzymes can be obtained and with which

enzyme concentrations can be increased by changes in environmental and genetic manipulation.

Amylases are one of the most widely used enzymes required for the preparation of fermented foods. Apart from food and

starch industries, in which demand for them is increasing continuously, they are also used in various other industries such as

paper and pulp, textile, etc. Knowing the promising applications of α-Amylase as industrial enzyme isolation, screening,

characterisation and production of α-Amylase from Bacillus sp can be carried out.

Research is focused on developing thermotolerant and pH tolerant a-amylase from microbes, modifying them genetically. Also

to acquire desired properties in the enzyme by applying the concept of site-directed mutagenesis. Production of α-amylase is

generally obtained by submerged fermentation, but solid state fermentation can also be seen as a potential productive option.

Using agroindustrial residues as substrate the production of bacterial amylase can be carried out.

REFERENCES

1. Gupta, R.; Gigras, P.; Mohapatra, H.; Goswami, V .K.; Chauhan, B. (2003). Microbial a-amylases: a biotechnological

perspective. Process Biochem 38, 1599 - 1616.

BioEvolution

Vol 2 (1), Feb 2015, ISBN [978-93-83006-04-5], pg 19-25

www.giapjournals.org/bioevolution.html 24

2. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. Advances in microbial amylases. Biotechnol Appl

Biochem 2000; 31:135 -/52.

3. Paula Monteiro de Souza; Pérola de Oliveira e Magalhães. Application of microbial a-amylase in industry – A review.

Brazilian Journal of Microbiology (2010) 41: 850-861

4. Brayer, G.D.; Luo, Y.; Withers, S.G. (1995). The structure of human pancreatic alpha-amylase at 1.8 A resolution and

comparisons with related enzymes. Protein Sci 4, 1730-1742.

5. Baysal, Z.; Uyar, F.; Aytekin, C. (2003). Solid-state fermentation for production of A-amylase by a thermotolerant

Bacillus subtilis from hot- spring water. Process Biochemistry 38, 1665-1668.

6. Tangphatsornruang, S.; Naconsie, M.; Thammarongtham, C.; Narangajavana, J. (2005). Isolation and

characterization of an alpha- amylase gene in cassava (Manihot esculenta). Plant Physiol Biochem 43, 821-827.

7. Iulek, J.; Franco, O.L.; Silva, M.; Slivinski, C.T.; Bloch, C., Jr.; Rigden, D.J.; Grossi de Sa, M.F. (2000). Purification,

biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye).

Int J Biochem Cell Biol 32, 1195- 1204.

8. Couto, S.R.; Sanromán, M.A. (2006). Application of solid-state fermentation to food industry- A review. Journal of

Food Engineering 76, 291-302.

9. Gangadharan, D.; Sivaramakrishnan, S.; Nampoothiri, K.M.; Sukumaran, R.K.; Pandey, A. (2008). Response surface

methodology for the optimization of alpha amylase production by Bacillus amyloliquefaciens. Bioresour Technol 99,

4597-4602.

10. Singhania, R.R.; Patel, A.K.; Soccol, C.R.; Pandey, A. (2009). Recent advances in solid-state fermentation. Biochem.

Eng. J. 44, 13–18.

11. Sajedi, R.H.; Naderi-Manesh, H.; Khajeh, K.; Ahmadvand, R.; Ranjbar, B.; Asoodeh, A.; Moradian, F. (2005). A Ca-

independent a-amylase that is active and stable at low pH from the Bacillus sp. KR-8104. Enzyme Microb. Technol.

36, 666–671.

12. Nielsen, J.E.; Borchert, T.V. (2000). Protein engineering of bacterial alpha-amylases. Biochim Biophys Acta 1543,

253-274.

13. Prakash, O.; Jaiswal, N. (2009). Alpha-Amylase: An Ideal Representative of Thermostable Enzymes. Appl Biochem

Biotechnol.

14. Teodoro, C.E.S.; Martins, M.L.L. (2000). Culture conditions for the production of thermostable amylase by Bacillus

sp. Braz. J. Microbiol. 31, 298-302.

15. Saxena, R.K.; Dutt, K.; Agarwal, L.; Nayyar, P. (2007). A highly thermostable and alkaline amylase from a Bacillus

sp. PN5. Bioresour Technol 98, 260-265.

16. Hmidet, N.; El-Hadj Ali, N.; Haddar, A.; Kanoun, S.; Alya, S.; Nasri, M. (2009). Alkaline proteases and thermostable

a-amylase co-produced by Bacillus licheniformis NH1: Characterization and potential application as detergent

additive. Biochemical Engineering Journal 47, 71–79.

17. Chi, Z.; Chi, Z.; Liu, G.; Wang, F.; Ju, L.; Zhang, T. (2009). Saccharomycopsis fibuligera and its applications in

biotechnology. Biotechnol Adv 27, 423-431.

18. Moller, K.; Sharif, M.Z.; Olsson, L. (2004). Production of fungal alpha- amylase by Saccharomyces kluyveri in

glucose-limited cultivations. J Biotechnol 111, 311-318.

19. Van der Maarel, M.J.; Van der Veen, B.; Uitdehaag, J.C.; Leemhuis, H.; Dijkhuizen, L. (2002). Properties and

applications of starch-converting enzymes of the alpha-amylase family. J Biotechnol 94, 137-155.

BioEvolution

Vol 2 (1), Feb 2015, ISBN [978-93-83006-04-5], pg 19-25

www.giapjournals.org/bioevolution.html 25

20. Feitkenhauer, H. (2003). Anaerobic digestion of desizing wastewater: influence of pretreatment and anionic surfactant

on degradation and intermediate accumulation. Enzyme Microb. Technol. 33, 250–258.

21. Bruinenberg, P.M.; Hulst, A.C.; Faber, A.; Voogd, R.H. (1996) A process for surface sizing or coating of paper. In:

European Patent Application.

22. L.A.Underkofler, R.R.Barton, and S.S.Rennert (1957) Production of Microbial Enzymes and Their Application:

Microbial process report.215-220

23. Jose L. Adrio and Arnold L. Demain (2014) Microbial Enzymes: Tools for Biotechnological Processes; Biomolecules

2014, 4, 117-139.