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Team:Utah State/Results/IBP
From 2014.igem.org
Functionalized Bioplastic
Biosynthetic Pathway
Three genes are needed to produce PHB from acetyl-CoA in E. coli. These genes are phaA, phaB, and phaC. The pathway for PHB production is shown in the figure below, where 2 acetyl-CoAs are converted to acetoacetyl-CoA then 3-Hydoxybutyryl-CoA is made via phaB enzyme. Finally 3-Hydoxybutyryl-CoA is polymerized to make PHB (Agnew and Pfleger 2013). The PhaC synthase remains covalently attached to PHB after polymerization (Rehm 2009).
Figure 1. Figure shows the biosynthesis of PHB from acetyl-CoA adapted from Rehm 2009.
Granule Formation
As mentioned earlier, the PhaC synthase remains covalently bound to the PHB polymer and during granule formation (Tomizawa, Hyakutake et al. 2011). Other proteins, such as phasin, can also bind to the PHB granule and help granule structure and control granule size (York, Junker et al. 2001; York, Stubbe et al. 2001).
Purification and Quantification
Traditional Purification
There are a variety of PHB purification strategies ranging from mechanical, chemical, and biological treatments to liberate PHB from cells. The different strategies are outlines in Jacquel et al. 2008. The most common method of PHB purification is to use a chloroform/bleach extraction. The purpose of bleach is to lyse the cells and chloroform helps agglomerate the PHB together. The chloroform/bleach method has been reported to give extremely high purities with minimal cell contamination (Jacquel et al. 2008). There are, however, several aqueous-based isolation procedures for recovering bacterially produced PHB.
NMR-GC quantification of PHB
E. coli containing the BBa_K1418050 part was grown in M9 media supplemented with glucose (see protocols page for details). The BBa_K1418050 part was modified from the BBa_K934001 BioBrick to include the lactose inducible promoter system BBa_K208010. After growth and purification, PHB concentrations were determined from a NMR-GC correlation as mentioned in the protocols page. The concentration found was approximately 48.5% PHB of the dry cell weight. The figure below shows an NMR spectra of PHB produced from XL1 Blue E. coli harboring the BBa_K1418050 part, confirming the presence of PHB.
Figure 2. Figure shows NMR spectra for PHB produced using BBa_K1418050 in XL1 Blue E. coli .
Immobilized Bioplastics
Enzymes are produced by an organism or cell culture to catalyze metabolic reactions, defensive deactivation of compounds, and other chemical conversions (Nisha et al., 2012). Enzymes can be expensive to produce, and have low stability and are sensitive to process conditions (Cao et al., 2005). To resolve these problems, immobilization techniques were developed as a means of stabilizing enzymes for prolonged activity. Berna & Batista (2006) classified the methods of enzyme mobilization into two categories. The first type is reversible enzyme immobilization, which include adsorption, ionic binding, affinity binding and metal binding. The second is the irreversible enzyme immobilization methods which include covalent bonding and entrapment processes.
Enzyme Immobilization Methods
Covalent binding is an irreversible process, which involves the direct attachment of the enzyme to the material through covalent linkage (Wong et al., 2008). Enzymes are attached to the support material such as polyacrylamide, porous glass, agarose and silica. In this process, enzymes are kept on the support material after the reaction process.
Another irreversible process is called entrapment. The process use natural or synthetic polymeric networks which act as a permeable membrane, allowing the substrate to pass through while trapping the enzymes in its network. Entrapment is a fast and inexpensive process which requires controlled, mild conditions (Bernfield & Wan, 1963). Different methods of entrapment include gel entrapping, fiber entrapping and microencapsulation.
Adsorption is a simple, reversible immobilization process using adsorbents such as activated carbon, alumina, and ion exchange resin. While inexpensive, the bonding force between the enzyme and the adsorbent is weak compared with other methods (Brady & Jordan, 2009). Chemical bonding present are normally ionic and Van der Waals forces due to partial charge of the substrate and the carrier. It is the oldest and easiest enzyme immobilization technique.
Affinity binding generally involves one of two methods. Support material can be activated which contains the coupled affinity ligand followed by the addition of the enzymes. This procedure allows the enzyme protection from exposure to harsh conditions. In the second method, the enzyme’s structure is changed in order to promote enzyme affinity to the matrix (Nisha et al., 2012).
Immobilized enzymes are applied in numerous industries including biomedical use, food industry, biodiesel production and wastewater treatment.
Immobilization methods for Bioplastics
The immobilization of enzymes on plastics has been a practice utilized several times to create a more stable, efficient enzymatic reactions. Enzymes immobilized on plastics can perform operate multiple times before disassociating which improves their overall operational stability. Therefore, an immobilized enzyme is a reusable enzyme.
The most common forms of enzyme attachment are currently through covalent coupling. This requires treatment of the plastic with a nitrating acid, or something similar, to create cleaved chain polymer ends projecting from the surface. Most procedures then use a glutaraldehyde solution to create a binding location for the subsequent enzymes. This method has shown relative success allowing enzymes to withstand multiple uses and retain activity for longer.
Although the enzymes primarily put to use in study have been protease, lipase, and amylase (major enzymes in laundry detergent,) other enzymes are being tested for various applications such as: cleaning pollutants out of ground water, and medical testing/diagnosis/treatment.
Biological Functionalization of Bioplastics
Using a synthetic biological approach, we aimed to functionalize the bioplastic, polyhydroxybutyrate (PHB), with the enzymes described earlier (chlorophyllase, cellulase, and amylase). Functionalizing the bioplastics with these enzymes could allow for various applications of the enzymes, including reusable use with laundry detergents.
As mentioned previously, PHB can be produced via a three enzymatic pathway converting acetyl-CoA to PHB in E. coli. Three genes code for beta-ketothiolase (phaA), acetoacetyl-CoA Reductase (phaB), and PHA polymerase (phaC, or PHA synthase) (Agnew et al. 2013). These genes are currently in the Biobrick Registry of Standard Biological Parts and were available in the 2013 distribution kit( BBa_K934001). An interesting characteristic of PhaC is that as the PHB polymer is being synthesized the PhaC remains covalently attached to the PHB granule. PhaC helps in formation of a amphipathic molecule with hydrophilic (polar) and hydrophobic (non-polar) ends (Rehm 2009). Since the PHA synthase remains attached to the bioplastic granule, creating fusions of PhaC with chlorophyllase, cellulase, and amylase will allow for the creation of functionalized bioplastic
Figure 3. Typical PHB granule and attached proteins. PhaC and Phasin can be found on the surface of a PHB granule (adapted from: Rehm 2010).
To test the feasibility of the system, we used a green fluorescent protein (GFP) as a marker. Using a synthetic biological approach, we wanted to determine if by genetically fusing GFP to PhaC we could generate bioplastics containing GFP activity (and thus potentially activity of our detergent enzymes). This fusion construct is designated as BBa_K1418061. The schematic for this genetic design and general concept are shown in Figure 4.
Figure 4. Schematic for functionalization of PHB granules. PhaC remains covalently bound to the PHB granule after polymerization and GFP can be fused to the N-terminal of PhaC, thus functionalizing the PHB granule. Direct production of PHB with the phaABC operon and expression of the GFP-PhaC fusion protein are both under lactose control.
To test for GFP functionalized bioplastic granules, E. coli cells containing BBa_K1418061 were grown for 24 hrs in the presence of IPTG for induction of the Lac promoter. Cells were then visualized with a fluorescent microscope for GFP expression and with Nile Blue staining for PHB production. Results from these experiments are shown in Figure 5. NMR was also used to confirm PHB production of BBa_K1418061 (using methods outlined above). PHB peaks were observed in BBa_K1418061 similar to that seen in BBa_K1418050, this demonstrates that by fusing GFP to PhaC PHB can still be produced by E. coli. Additionally, these results demonstrated that by using Biobrick part BBa_K1418061, PHB granules can be produced with GFP activity. This finding suggests that by using a similar procedure, we should be able to functionalize PHB with chlorophyllase, cellulase, and amylase.
Figure 5. A.) Nile Blue staining and B.) GFP expression of E. coli cells containing BBa_K1418061. Microscope images of GFP bound PHB granules in E. coli after 24 h of growth with IPTG induction. White bar represents 5 µm.
References
Berna, B. M. & Batista, F. 2006. Enzyme immobilization literature survey methods in Biotechnology Immobilization of Enzymes and Cells, 2nd (ed.). 15-30.
Bernfield, P. & Wan, J. 1963. Antigens and enzymes made insoluble by entrapping them into the lattices of synthetic polymers. Polymer Science, 142. 678-679.
Brady, D. & Jordan, A. 2009. Advances in online immobilization. Biotechnol. Lett. 31. 1639-1650.
Cao, L. 2005. Carrier-bound immobilized enzymes: principles, applications and design (first edition). Wiley – VCH, Weinheim.
Nisha, S., Arun Karthick, S. and Gobi, N. 2012. A Review on Methods, Application and Properties of Immobilized Enzyme. Che Sci Rev Lett 1(3), 148-155.
Wong, L. S., Thirlway, J., and Micklefield, J. 2008. J. Am. Chem. Soc. 130(37). 12456-12464.
Agnew DE, Pfleger BF:Synthetic biology strategies for synthesizing polyhydroxyalkanoates from unrelated carbon sources. Chem Eng Sci 2013:58-67.
Rehm BHA: Polyester synthases: Natural catalysts for plastics. Biochemical Journal 2003,376(1):15-33.
Immobilization of porcine pancreas lipase onto bristles of plastic brush: Kinetic properties Vandana Panwar*1, Umesh Kumar Shandilya*1, Dinesh Dahiya2, Chander Shekhar Pundir3 Journal of Chemical and Pharmaceutical Research, 2012, 4(1):91-95
Pseudomonas aeruginosa Biofilm Growth Inhibition on Medical Plastic Materials by Immobilized Esterases and Acylase. Kisch JM1, Utpatel C, Hilterhaus L, Streit WR, Liese A. Chembiochem. 2014 Sep 5;15(13):1911-9. doi: 10.1002/cbic.201400023. Epub 2014 Jul 15.
Coimmobilization of Detergent Enzymes onto a Plastic Bucket and Brush for Their Application in Cloth Washing C. S. Pundir* and Nidhi Chauhan Department of Biochemistry, M. D. University, Rohtak-124 001, Haryana, India
