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In biological systems, an indispensable element in any biochemical pathway is an enzyme. Without enzymes, many biological phenomena would have remained unaccomplished; our very existence and persistence, as well as that of any other organism on Earth would have been at stake! Owing to their exceptional catalytic properties and applications, along with biological systems, enzymes also dominate the domain of biotechnological industries. One such industrially invaluable enzyme is alpha amylase (or α-amylase). Alpha amylase belongs to the category of hydrolase enzymes with the ability to break down polysaccharides such as starch and glycogen into simpler reducing sugars.
Alpha amylases have tremendous industrial applications such as in food industry, brewery, fermentation, textiles, paper, pharmaceuticals and detergents, as well as in research and development oriented sectors. A significant proportion of the commercial enzyme market in the world constitutes of alpha amylases [1.1]. Alpha amylases can be universally isolated from plant, animal as well as microbial sources. The variable attributes of alpha amylases from above mentioned sources are stability and the temperature of inactivation. Microbial counterparts have proven to be more stable and therefore exhibit higher efficacy and allow ease of manipulations for enhancing the activity; making them superior candidates for industrial processes [1.2].
This article gives a brief description of bacterial alpha amylases, its production and industrial applications referring to patents filed in the U.S and European Patent Offices.
Brief Account on Alpha Amylase
Eighteenth century marked the discovery of amylases; first observed by Kirchoff then identified by French chemists Anselme Payen and Jean-François Persoz and further named by Kuhn [1.2]. Alpha amylase (E.C. 220.127.116.11) is a metalloenzyme, requiring Ca++ for its activity. It hydrolyzes polysaccharides containing glucose units linked by α-1, 4- glycosidic bond. Particularly starch and glycogen are endo-hyrolyzed randomly by alpha amylases by catalytically cleaving the α-1, 4-glycosidic bonds present at internal positions [1.3]. As mentioned earlier, alpha amylases have been isolated from sources covering all kingdoms; however microbial sources have gained attention of investigators in the industrial sector [1.4]. Bacterial alpha amylases being exclusively thermo-stable are of paramount importance in the starch industry. Their desirable features have also made them instrumental in paper, textile, biofuel, alcohol and detergent industries. Bacterial sources of alpha amylase majorly include Bacillus species such as Bacillus subtilis, Bacillus licheniformis, Bacillus amyloloquefaciens, Bacillus stearothermophilus, etc [1.3, 1.4].
Production Methodologies Summarizing Patents
Various patents have been filed in the U.S. and European Patent Offices providing protocols for industrial production of alpha amylases. Traditionally alpha amylases were extracted from plant and fungal sources. In the early nineteenth century, malt extract was found to be a good source [2.1]. Thereafter, fungal strains belonging to Aspergillus genera have been popular sources for industrial production. However heat inactivation and difficulty of manipulations and scale-up defamed these conventional sources, whereas popularized bacterial sources, particularly Bacillus species for industrial production.
Bacterial alpha amylases have been produced by submerged fermentation by many investigators [1.5]. Previously mesophilic bacteria were used as sources. However these enzymes were found to be unstable when incorporated for alcohol production and other industrial applications, as fermentation is carried out at temperatures above 60°C. Thereafter protocols were redesigned to use thermophilic bacteria wherein the enzymes could withstand up to 100°C. However recovery of the enzyme from the production medium posed inherent problems.
US2695863: This patent provides an improvised protocol devised by a group of investigators, wherein cell-free alpha amylase was isolated from Bacillus stearothermophilus. The medium was then filtered to remove cells and enzymes were subjected to ammonium sulphate precipitation followed by dialysis, extraction in organic solvent and drying. By performing a volumetric assay (iodometry), high enzyme activity was found at 25°C- 95°C [2.2].
US3414479: Alpha amylase produced from Bacillus subtilis posed problem of excessive foaming in the medium and subsequently hampering the production. This patent describes an economical and peculiar organosilicon copolymer, acting as an efficient antifoaming agent. It significantly increased the yield, when incorporated into the medium at the start of fermentation [2.3].
US3642582: In this patent, the property of adsorption was exploited to specifically purify alpha amylase from the protein extract obtained from the production medium of Bacillus species. The extract was first treated with calcium chloride to eradicate any polyvalent anions present in the extract and further confer stability to the enzyme, followed by passing the extract through a column of adsorbent alumina fibrils [2.4].
US3808102: This invention provides an efficient method for obtaining a high yield of alpha amylase from Bacillus cereus strains by incorporating foul excrement, wheat chaff and glycine in the nutirient medium and further purifying the recovered enzyme by ion exchange chromatography with DEAE-cellulose columns [2.5].
Bacterial cell wall components are potential inhibitors of alpha amylases and have high allergenicity. Enzymes recovered from fermentation if contaminated with these, cannot be further utilized for any industrial application. Enzyme extracts thus need to be highly purified.
US5281526: The inventors in this patent have developed a novel method to get rid of bacterial cell wall components from recovered enzyme by adding a metal halide; particularly sodium chloride and/or 4-hydroxybenzoic acid and its derivatives (specifically methyl esters) for precipitation [2.6].
In many cases it was observed that the activity of the enzyme was gradually lost after few days of extraction. Precipitation and adsorption are partial purification methods; contaminants can lead to inactivation of the enzyme.
WO200141794: This inventor described a method to retain the activity of alpha amylase produced by Bacillus amyloliquefaciens by addition of calcium chloride in the purified enzyme extract, at specific concentration and amount. The activity was not only retained but also significantly increased even after several weeks of extraction [2.7].
Genetic engineering has revolutionized the industrial production of enzymes in the past few decades. Only the gene encoding the specific alpha amylase is incorporated into suitable host bacteria via a vector and over-expressed. This reduces the down streaming steps and production cost.
EP0034470: This patent demonstrated how strains of Bacillus megatarium, Bacillus cereus, Bacillus coagulans and Klebsiella pneumonia have been genetically engineered to over-produce alpha amylase. The gene encoding the enzyme is either incorporated into bacteriophage lambda vector or a plasmids vector such as pBR322 or pACYC184 and then transfected or transformed into suitable host- an Escherichia coli strain. When lambda vector is used, the lysogenic host cells are cultivated till maximum cell density is obtained and then lytic cycle is induced by temperature shock to release the enzyme. Plaque assays are used to screen for the recombinant cells. Further to check activity of the enzyme, starch is incorporated in the medium and iodine is added; enzyme activity is indicated by zone of clearance around the enzyme releasing cells. For plasmid vectors, antibiotic resistance markers are used for identification. After transformed cells are cultivated till maximum density, chloramphenicol is added to inhibit protein synthesis, but plasmid replication continues; thereby amplifying the enzyme encoding gene. The amplified genes are then expressed by re-cultivating the cells in fresh medium and further sub-cloning. The cells are then subjected to osmotic shock to release the enzyme which is further purified [2.8].
EP0124076: The scope of this patent is to produce thermo-stable alpha amylase by cloning the alpha-amylase gene from thermophilic bacteria such as Bacillus stearothermophilus into an alpha amylase non-producing thermophilic bacteria via a compatible plasmid vector- pTB90. The recovered enzyme was claimed to have high activity [2.9].
EP0772684: This patent demonstrated how alpha amylase from Bacillus lincheniformis was subjected to a series of site directed mutagenesis, to develop an alpha amylase with 70- 90% identity and having better thermo-stability and oxidation stability. The mutant gene fragments were placed under the transcriptional control of tac promoter, facilitating induction by IPTG and were transformed into suitable Escherichia coli strain via plasmid vector pBHATLAT [2.10].
In most of the industrial processes inclusive of alpha amylases as ingredients, the initial starch degradation step is carried out at very high temperatures above 100°C. Thus, inventors have put constant efforts to improvise the stability and yield of the enzyme.
WO91/00353: By employing chemical and enzymatic site directed mutagenesis, these inventors have modified the alpha amylase from Bacillus licheniformis to produce an enzyme with better thermo-stability and stability over a wide range of pH. Bisulphite method for chemical mutagenesis and nucleotide misincorporation method for enzymatic mutagenesis was used to replace specific amino acids with proline and incorporating branched amino acids to restrict the conformations, decrease entropy and thereby increase stability [2.11].
US5763385: This patent provided a method wherein alpha amylase from Bacillus licheniformis was modified chemically and genetically to alter its calcium binding domains and further transformed into amylase negative, protease deleted host Bacillus strains, so as to enhance the overall stability [2.12].
US7550281: These inventors placed the alpha amylase encoding gene from Bacillus amyloliquefaciens under the transcriptional control of a temperature inducible strong phage promoter and transformed into competent Bacillus subtilis strain to overproduce the enzyme [2.13].
As the demand for alpha amylase in the commercial market has increased, novel strains are being discovered by researchers for isolation.
WO2013/077723: The inventors here have isolated a novel alpha amylase encoding gene from Geobbacillusthermoleovorans and amplified it by PCR, followed by cloning into pCR4Blunt- TOPO vector and transformation into Escherichia coli. The recovered enzyme had efficient activity in a wide range of temperature [2.14].
The hunt for novel sources and techniques that elevate the productivity continue to be explored by researchers.
Applications: An Industrial Perspective
It is clearly evident that bacterial alpha amylases have a wide range of applications in food and bakery industries, textiles, biofuel production, detergents, etc. due to their inherent starch hydrolyzing function and profitable stability. Many patents have been registered providing novel methods for incorporating bacterial alpha amylases into the manufacture protocol of mentioned products [2.15].
WO1992019744: The inventors here have developed a method to express recombinant alpha amylase in Lactobacillus species which can be use in food and fodder preparations containing high amounts of starch so as to facilitate ease of digestion [2.16].
EP0252730: This patent demonstrated how alpha amylase mixtures obtained from Bacillus licheniformis and Bacillus stearothermophilus are used for manufacturing high-fructose syrup which acts as a substitute for sugar in cold drinks and other food products [2.17].
US5209938: The inventors here have developed a technique to incorporate bacterial thermo-stable alpha amylases from Bacillus megatarium and Bacillus subtilis into bakery product recipes to prevent spoilage [2.18].
WO2008092919: This patent showed how recombinant alpha amylases can be used for starch liquefaction, manufacturing maltose syrup and dishwashing and laundry detergents [2.19].
US8097444: This patent provides a description of formulations incorporating recombinant alpha amylase originated from Bacillus licheniformis which has potential application in the detergent industry. [2.20].
Desirable functional characteristics and stability over wide range of temperature and pH has led to the supremacy of bacterial alpha amylases and their recombinant variants in the commercial market. Their extensive applications in a vast array of industries are remarkable and have signified their beneficial future prospects. As the scope of bacterial alpha amylases in industries is broadening, demands have increased several folds. Over the years, investigators have been on a constant quest to isolate alpha amylase from novel bacterial sources and improvise their strategies and formulations to enhance stability and productivity. Genetic engineering has superseded conventional methods and aided to accomplish the goal of researchers to an impressive extent. Site-directed mutagenesis accompanied by recombinant DNA technology has been successfully employed to produce alpha amylases with greater efficacy. Investigators continue to customize their methodologies, as per the requirements of the individual industrial process.