0115 966 7955 Today's Opening Times 10:00 - 20:00 (BST)

Bacterial α Amylase Production

Published: Last Edited:

Disclaimer: This essay has been submitted by a student. This is not an example of the work written by our professional essay writers. You can view samples of our professional work here.

Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UK Essays.

Introduction

Starch is the primary repository of carbohydrates in evolved plants [1]. It is not only the prime form of a dietary sugar storage molecule but has extensive industrial applications in the manufacturing of packaging substances to the synthesis of alcohol [1]. The enzyme alpha amylase has the ability to degrade starch to simpler sugars with reducing ends [2]. They are termed as endohydrolases as they bring about the schism of -1, 4 D- glycoside linkage within the chain randomly [3].

This catalysing activity makes it a salient feature particularly in the food based and textile industries which have starch as an indispensable raw material that demands hydrolysis as a part of the production [4, 5]. Traditionally, amylases were introduced into the baking mixture or other places requiring the dextrinizing enzyme in the form of barley flour or malted wheat [6]. Alpha amylase contributes towards gas generation and gas retention in bread and give proper colour to the crust [6].

Fuelzyme is an industrially developed, genetically engineered alpha amylase obtained from a combination of three enzymes due to DNA shuffling of the parental enzymes [3]. It is beneficial due to its lower viscosity, activity maintained over a wide pH range and better thermostability [3]. It is of use in biodiesel or ethanol based industry and not suitable for use in food industries due to restrictions in downstream processing [3].

The potential enzyme manufacturers

The bacteria known to produce alpha amylase are Geobacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquefacians [3]. The alpha amylase produced by Bacillus stearothermophillushas more ascendancy than that produced by Bacillus licheniformis due to greater specific activity and lesser pH required for optimum growth [7]. Bacillus subtilis strains are widely exploited owing to their biological features such as easy, evident growth in simple culture medium, lack of secretion of toxic substances and no appreciable pathogenicity [4]. They also possess the highly demanded ability of secreting the formed amylase in the growth medium [3].

Fungi of genus Aspergillus and Rhizopus have been known to synthesize this industrially important sugar producing enzyme [8].Amylases which are fungal in origin can replace malt amylases imparting undesirable colour to the food products but get fairly inactivated by heat making them less applicable in the bakery based industry [9].

Alpha amylases are also derived from anaerobes like Clostridium thermoamylolyticum which are known to be heat stable [10]. Clostidium thermohydrosulfuricum synthesized alpha amylase has exceeding starch hydrolysing characteristics at a pH of around 4 [10]. Amylase is also derived from the heat loving bacterium Bacillus diastaticus. However, its recovery using ammonium sulphate cannot be ensured without contamination [2].

A novel amylase enzyme has been discovered featuring in the bacterium Alicyclobacillus pohilae which increases the shelf life of bread [6]. Yoneda et al discovered a strain of B. subtilis which on genetic modification increased the amylase yield 170 times [4]. The Bacillus subtilis strain AB101 is a recombinant strain known to be the most superior amongst the organisms of the same species [4].

Inside the fermenter

The producers of the biocatalyst, that is, the micro-organisms in action are subjected to aerobic or anaerobic conditions depending upon the need[11].Clostridium spp are cultured under anaerobic conditions whereas Bacilli are grown in submerged media with proper aeration or on the surface of shallow medium[2, 11]. Fungi are cultured on solid media composed of wheat bran, rice bran or straw [8].

The solid medium may contain starch in crude form, that is, in the form of beans, potatoes, grains, corn, arrowroot or other starch rich plants [2, 8]. Starch in the liquid medium can be in the form of uncooked rice flour [8]. Starch in soluble form or maltodextrin is also added to the medium [10,11].The source of starch is not a critical consideration as the enzyme is synthesized using the proteins in the medium and starch is just a stimulator which can be added in the range of 0.2% to 10%[2].

The protein content of the medium should be high [2]. 4% protein based substances in the medium have found to be suitable for the formation of amylase and its further precipitation [2].The media contains yeast extract as supplement of minerals and vitamins [10, 11]. Calcium hydroxide is particularly added to the medium to prevent the inactivation of amylase and stabilize it [11]. Maltose aggravates the production whereas glucose acts as an inhibitor [10].

The most favourable pH of the medium of fermentation for alpha amylase production is 6.5 to 7.0 [10].Temperature is not allowed to exceed 90-C in case of strains of B. subtilis and B. amyloliquefaciens while in case of B. licheniformis, it is allowed to reach 110-C [12]. The most fitting temperature range for most organisms is between 50-C to 70-C [2]. Hence, a fermentation temperature of 65-C is maintained [2]. The time for which a cycle of fermentation is run is from 12hrs to 24hrs though amylase activity is observed within 4hrs from the start of fermentation [2]. Maximum yield is obtained after 12hrs [2].

Downstream processing, purification, confirmation and assays

  • The enzyme is released into the medium [10]. The efforts and cost involved in disrupting the cells to get the protein product is eliminated [10].
  • The cells are removed by filtration and the extraneous media components are precipitated out by calcium chloride [10].Centrifugation can also be carried out for removal of solids [2].
  • Dialysis is a preferred step to be carried out prior to concentration and precipitation as this process ensures increased purity of the final product [2].
  • The solution containing the enzyme thus formed is concentrated and put forth for refinement on starch that is granular in nature [10]. Potency is increased by concentrating the solution [2].
  • Precipitation is carried out using organic solvents, miscible in water such as dioxane, acetone, ethyl methyl ketone, ethanol, methanol, n-propanol, isopropyl alcohol and tertiary butyl alcohol [2]. One or two volumes of the solvent serve the purpose [2].
  • Filtration and centrifugation is carried out after precipitation to remove the precipitated amylase [2]. The final precipitated product is dried [2]. Drying can be brought about at room temperature or at 65-C under vacuum on calcium chloride without affecting the stability of amylase obtained [2].
  • Chromatography techniques are also employed to bring about purification post refinement and removal of starch [10].
  • SDS polyacrylamide gel electrophoresis carried out of the purified sample helps determine the molecular weight with a protein marker run along [10].

Assays to determine amylase activity

Two assays are conducted:

The first assay is a modification of Fuwa method wherein starch is stained with iodine [13]. The decrease in intensity of blue colour per ml of enzyme is determined [13]. The temperature and pH of the system are altered to determine the optimum activity [13].

The second assay is based on the principle of colorimetry [13]. The protocol followed is that of Nelson reducing-sugar assay wherein 100μl of relevantly diluted amylase is incubated with 1.9 ml of starch for 10 minutes at a fixed temperature followed by addition of Nelson Reagent D to give the colour [13]. Spectrophotometer is used to determine the colour intensity [13].

Modifying the enzyme and the way of producing it for better yields and economics

  1. A Bacillus strain has been subjected to manipulation following the principles of genetic engineering and plasmid expression vectors such as coliphage 105 for enhanced alpha amylase production [4]. The gene coding for the protein of interest is interpolated under the transcriptional influence of a promoter of phage origin [4]. Addition of antibiotic is essential to selectively promote the proliferation of the plasmid bearers [4]. It is also important for retention of the genetically recombinant cells over extended periods of time in a continuous culture overcoming vulnerability of plasmid to curing [4]. The cloned genes get over expressed in association with the pro-phage based vectors with high stability, apt location on the chromosome and conducive regulation by the phage system [4].
  2. To make the baking process more efficient, alpha amylase obtained from Bacillus subtilis and Bacillus stearothermophillusis modified to reduce its thermostability as its application in the food industry requires[9].The modification brought about is acylation employing acylating agents such as the monocarboxylic acid anhydrides, ethanoic, propanoic, butanoic, valeric and even benzoic anhydrides [9]. Dicarboxylic and cyclic anhydrides do not serve the purpose [9]. The quantity of acylating agent incorporated to bring about the reaction is not crucial but the amount has to be enough to acylate at least fifty percent of the free amino acids of the amylase molecule [9]. The original enzyme is introduced to acylation in association with starch to prevent its degeneration during the process [9].
  3. Modification is also carried out with respect to the process of deriving the enzyme [14]. An organosilicon polymer is introduced in the fermentation medium at the beginning itself which is known to be water insoluble at the temperature to which fermentation is subjected and soluble in water at a lower temperature [14]. The organosilicon is a copolymer composed of dimethyl silicon and propylene oxide [14]. The approved concentration of the copolymer is one part of it for every 4000 parts of the fermentation medium [14]. The copolymer addition is greatly applauded due to the enhanced yield and reduction in cost due to lack of requirement of special antifoam agents [14]. Better downstream processing and recovery is ensured as no oily residues otherwise created by the antifoam agents are produced [14].

Conclusion

Amylases yielded from the different organisms differ significantly with respect to features such as molecular weight, optimum temperature and pH requirement, enzyme activity and affinity towards calcium ions [13]. This broadens the scope of their appositeness. For e.g. amylase produced by a novel strain of Bacillus stearothermophilus has high affinity for calcium ions which makes its inclusion in detergents and use in presence of chelating agents and soft water apropos [13].

Mixed culture system with varying proportion of various species and strains of genus Aspergillus and Rhizobium have shown to give better yields than pure cultures of the same [7]. The enzyme once purified can be immobilized using befitting carriers like ceramics, organic polymers and glass [13]. Immobilization can be brought about by crosslinking, entrapment, adsorption and bonding [13].

Research is carried out to modify the amino acid sequence in the enzyme active site of amylase for enhanced activity [12]. For e.g. Bacillus licheniformis mutant G475R has the amino acids methionine, tryptophan and aspargine replaced by valine, histidine, alanine and serine [12].

Use of antibiotics as a selective pressure for harnessing recombinant techniques increases the expense of downstream processing since presence of antibiotics ordinarily in food products is not appreciable [4].Hence alternative way of selective isolation need to be worked out.

Properties such as specificity towards the substrate, its binding to it, the cleavage of the substrate, activity and stability profile with respect to pH, stability at higher temperature, towards oxidation and low calcium ion concentration and its activity alone and in combination can be altered [3].

References

[1] Myers Alan M. and James Martha Graham. Isolation of Su1, a starch debranching enzyme, the product of the maize gene Sugary 1. Februaury 7, 2006. US 6,995,300 B2.

[2] Tetrault Philip A., Lafayette West and Stark Egon. Process for preparing alpha amylase. November 30, 1954. US 2,695,863.

[3] Nedwin Glenn F., Sharma Vivek and Shetty Jayarama K. Alpha amylase blend for starch processing and method of use thereof. October 1, 2013. US 8,545,907 B2.

[4] Leung Yun Chung, Lo Wai Hung and Errington Jeffery. Method for production of alpha-amylase in recombinant Bacillus. June 23, 2009. US 7,550,281 B2.

[5] Leung Yun Chung, Lo Wai Hung and Errington Jeffery. Method for production of alpha-amylase in recombinant Bacillus. December 12, 2002. US 0187541 A1.

[6] Lucie Parenicova. Alpha Amylase. July 31, 2013. EP 2620,496 A1.

[7] Carrol et al. Alpha-Amylase mixtures for starch liquefaction. July 8, 1987. EPO 252730 A2.

[8] Kitakyushu Foundation. 2012. JP20100293378. EPO.

[9] Brumm Phillip J. Reduced-stability alpha-amylase and process for its production. December 10, 1987. EPO 273268 A2.

[10] Zeman Nancy W. Novel thermostable, aciduric alpha-amylase and method for its production. September 23, 1986. US 4613570.

[11] Katkocin et al. Novel thermostable, aciduric alpha-amylase and method for its production. November 25, 1987. EPO131253 B1.

[12] Robert M. Caldwell, Colin Mitchinson and Traci H Ropp. Mutant α amylase. 2001. US 6211134 B1.

[13] Kindle et al. Thermostable alpha amylase having a low requirement for m calcium ions, derived frombacillus microorganism. July 15, 1986. US 4600693 A.

[14] Wynes Robert A., Llyod Norman E., Linton and Iowa. Process for the preparation of bacterial alpha-amylase. December 3, 1968. US 3414479.

Reference table

Sr. No.

Inventor

Company name

Year

Title

Number

1

Myers Alan M. and James Martha Graham

Iowa State Research Foundation, Inc ., Ames US

2006

Isolation of Su1, a starch debranching enzyme, the product of the maize gene Sugary 1

US 6,995,300 B2

2

Tetrault Philip A., Lafayette West and Stark Egon

Purdue Research Foundation, Lafayette, Indiana

1954

Process for preparing alpha amylase.

US 2,695,863

3

Nedwin Glenn F., Sharma Vivek and Shetty Jayarama K

Danisco US Inc., Palo Alto, CA (US)

2013

Alpha amylase blend for starch processing and method of use thereof.

US 8,545,907 B2

4

Leung Yun Chung, Lo Wai Hung and Errington Jeffery

The Hongkong Polytechnique University, Kowloon (HK)

2009

Method for production of alpha-amylase in recombinant Bacillus.

US 7,550,281 B2

5

Leung Yun Chung, Lo Wai Hung and Errington Jeffery

The Hongkong Polytechnique University, Kowloon (HK)

2002

Method for production of alpha-amylase in recombinant Bacillus.

US 0187541 A1

6

Lucie Parenicova

DSM IP Assets B.V.

2013

Alpha Amylase.

EPO 2,620,496 A1

7

Carrol et al.

NOVO Industries

1987

Alpha-Amylase mixtures for starch liquefaction.

EPO 252730 A2

8

 

Kitakyushu Foundation

2012

. Production method of amylase by using mixed bacterial culture of Aspergillus bacterium and Rhizopus bacterium.

JP20100293378

9

Brumm Phillip J

Enzyme Bio-systems Ltd.

1987

Reduced-stability alpha-amylase and process for its production.

EPO 273268 A2

10

Zeman Nancy W

CPC International Inc., Englewood Cliffs, NJ.

1986

Novel thermostable, aciduric alpha-amylase and method for its production.

US 4613570

11

Katkocin et al.

CPC International Inc., Englewood Cliffs, NJ.

1987

Novel thermostable, aciduric alpha-amylase and method for its production.

EPO131253 B1

12

Robert M. Caldwell, Colin Mitchinson and Traci H Ropp

Genecor International, Inc.

2001

Mutant α amylase.

US 6211134 B1

13

Kindle et al

Corning Glass Works, Corning, N.Y.

1986

Thermostable alpha amylase having a low requirement for m calcium ions, derived from bacillus microorganism.

US 4600693 A

14

Wynes Robert A., Llyod Norman E., Linton and Iowa

Standard Brands Incorporated, New York

1968

Process for the preparation of bacterial alpha-amylase.

US 3414479


To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Request Removal

If you are the original writer of this essay and no longer wish to have the essay published on the UK Essays website then please click on the link below to request removal:


More from UK Essays

We can help with your essay
Find out more
Build Time: 0.0026 Seconds