Team:Utah State/Results/Amylase

From 2014.igem.org

Revision as of 01:42, 18 October 2014 by Ryanputman (Talk | contribs)


USU 2014iGem2014;

Mechanism

The enzyme α-amylase is a hydrolase that is responsible for breaking down starch and other related sugars. To break down starch, α-amylase hydrolyzes the α-1,4 glycosidic linkages between each glucose monomer [1]. Because α-amylase is an enzyme, it has many operating conditions that need to be met in order to work at its maximum capacity. The optimum temperature is anywhere between 30 and 37°C, and the optimum pH is between 7.0 and 8.0. With that said, these enzymes can still be very active at lower temperatures and higher pH, which makes them useful in the detergent industry [2] For a bacterial culture of α-amylase producing bacteria, it is crucial that starch or maltose is included to provide a substrate for the α-amylase [3]. The active site of α-amylase consists of a trio of acidic residues: glutamate 233, aspartate 197, and aspartate 300 (see Figure 1) [4].


USU 2014iGem2014;

Figure 1. Active site of α-amylase (PDB 1PPI). The active site is shown in red and white, with a short starch chain shown in yellow with its cleavage site in pink. A chloride ion (green) and a calcium ion (gray) are necessary cofactors for the enzymatic cleaving and overall structure of the enzyme [5].

Results

Protein generator part for α-amylase. Part BBa_K523001 was obtained from the parts registry and is the malS coding sequence encoding E.coli α-amylase. In order to be compatible for fusion protein applications, primers were designed to make BBa_K523001 RCF 23 compatible. These primers were also designed to remove the native ribosome binding site and the predicted cleavage signal peptide . The product of this PCR reaction was cloned into pSB1C3 and is designated as BBa_K1418020. The double stop codons were then removed via PCR. The PCR product was then cloned into pSB1C3 and this construct is designated as BBa_K1418021. Part BBa_K1418021 was then cloned behind a lac inducible promoter and a ribosome binding site (K208010 contains both R0010 and B0034). To aid in protein purification, a 10x histidine tag with two transcriptional terminators (K844000) was cloned in frame on the 3' end to generate the final composite construct, BBa_K1418022.


USU 2014iGem2014;

To test for protein production from the new BBa_K1418012 construct, protein purification using a nickel column will be performed. After protein purification, enzyme activity analyses will also be performed.

Future Applications

α-amylase is found in many different environments including the human body, plants and fungus, bacteria, and has had many applications in industry, especially in the laundry detergent industry [6, 7]. Hydrolytic enzymes, like α-amylase, are “100% biodegradable and enzymatic detergents can achieve effective cleaning with lukewarm water” [7]. Even though α-amylase is popular among the detergent and food industries, future uses of α-amylase could include genetically engineering it to produce biofuels or other energy sources, to help degrade packaging and other wasted materials, or to be used as a supplement for those who suffer from digestion issues.

References

[1] Davies, Gideon, and Bernard Henrissat. “Structures and Mechanisms of Glycosyl Hydrolases.” Structure 3.9 (1995): 853–859.

[2] Monteiro de Souza, P., and de Oliveira e Magalhaes, P. “Application of Microbial α-Amylase in Industry-A Review”. Brazilian Journal of Microbiology 41 (2010): 850-861.

[3] Gupta, Rani et al. “Microbial α-Amylases: a Biotechnological Perspective.” Process Biochemistry 38.11 (2003): 1599–1616. Web. 18 July 2014.

[4] Qian, M., Haser, R., Buisson, G., Duee, E., Payan, F. “The active center of a mammalian alpha-amylase. Structure of the complex of a pancreatic alpha-amylase with a carbohydrate inhibitor refined to 2.2-A resolution.” Journal of Biochemistry 33 (1994): 6284-6294.

[5] Image of 1PPI. Goodsell, D., RCSB PDB Molecule of the Month “Alpha-amylase”. doi: 10.2210/rcsb_pdb/mom_2006_2

[6] Oudjeriouat, Naïma et al. “On the Mechanism of α-Amylase. Acarbose and Cyclodextrin Inhibition of Barley Amylase Isozymes.” European Journal of Biochemistry 270 (2003): 3871–3879.

[7] Mitidieri, Sydnei et al. “Enzymatic Detergent Formulation Containing Amylase from Aspergillus Niger: a Comparative Study with Commercial Detergent Formulations.” Bioresource technology 97.10 (2006): 1217–24.