Team:Hong Kong UCCKE/Basic Part
Basic Parts
BBa_K3077101
This part, BBa_K3077101, is a cytoplasmic esterase encoded as lip8 in Pseudomonas aeruginosa.The genomic sequence is referenced from [1].
Lip8 is a cytoplasmic esterase that catalyzes both the hydrolysis and synthesis of ester bonds, especially that of water-soluble short acyl chain esters. It is also a biocatalyst for interesterification, alcoholysis, acidolysis, and aminolysis.[1] It showed higher activities against short-chain fatty acid methyl esters compared with activities against various triacylglycerols and long-chain fatty acid methyl esters: Among triacylglycerols with different fatty acids, Lip8 was found to have the highest activity against triacetin (C2). While methyl acetate (C2), methyl propionate (C3), and methyl butyrate (C4) are the substrates with higher hydrolytic activity among different fatty acid methyl esters. Among four Natural oils, Lip8 showed highest activity against tung oil, whose principal fatty acid component is eleostearic acid (C18: 3)[1].
Lip8 gene consists of 1,173 nucleotides, encoding a protein of 391 amino acids[1]. It is a lipolytic enzyme belonging to family VIII, thus have serine residues in the -Ser-Xaa-Xaa-Lys- motif which acts as a catalytic nucleophile[1]. No foldase is needed to assist the non-covalent folding of Lip8 when it is overexpressed. As it is a cytoplasmic gene, it is not accessible to its substrate unless the substrate passes through the outer membrane, or it leaks out of the cell in a significant amount.
Fig.1 Hydrolytic activities of Lip8 against various triacylglycerols, fatty acid methyl esters, and natural oils. The activities of the enzyme against triacylglycerols and fatty acid methyl esters are shown as values relative to that against tri-n-caproin(C6).
The optimum temperature and heat stability of Lip8 are lower than those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain[2]. It showed maximal activity at 30 ° C and pH 7, and is inactivated at more than 40 ° C and at pH 6[1].
Fig.2 Effect of temperature and pH on the activity of Lip8
The part is the gene of interest of the composite part BBa_K3077100.[2] The ordered plasmid is transformed in E.coli and is verified through colony PCR with prefix forward and suffix reverse. We tested its catalytic activity as an esterase through the lipid hydrolysis assay.
[1]: Ogino H1, Mimitsuka T, Muto T, Matsumura M, Yasuda M, Ishimi K, Ishikawa H.Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03. J Mol Microbiol Biotechnol. 2004;7(4):212-23.
[2]: BBa_K3077100
BBa_K3077202
This part, BBa_K3077202, is a new heterologous lipase identified by a metagenome library encoded as LipIAF5-2. It is a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa[1]. The genomic sequence is referenced from ENA (Gene ID EU660533)[2]. It is a newly discovered novel gene sequence that showed no more than 52% identity with other lipases[1].
Being a true lipase, LipIAF5-2 catalyses the hydrolysis of ester bonds, especially that of complex water-insoluble substrates at oil-water interface. It showed high activity with long-length acyl chains, maximal activity with p-NPM (C14), and about 70% activity with p-NPM (C16) and p-NPM (C18)[1].
Fig.3 Effect of chain length on specificity of LipIAF5.2
LipIAF5-2 is also able to efficiently synthesize short chain esters which produces sweet-smelling aromas by transesterification and esterification reactions in organic media. It showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. [3]
Fig.4 Reaction scheme for the transesterification of glyceryl triacetate into isoamyl acetate in presence of isoamyl alcohol.
While transesterification is favored by short acyl donor chains, esterification is compromised by acetic acid (C2:0). This means that very short FFA acyl donors will inhibit the esterification activity of the lipase. One explanation is the possible dead-end inhibition conferred by the carboxylic acid reacting with the active site serine residue of the hydrolase [4]. Furthermore, esterification is much slower than transesterification in the conditions tested, suggesting that LipIAF5-2 has higher affinity for glycerol esters. In transesterification, methanol is the best acyl acceptor for LipIAF5-2. Nevertheless, other alcohol including ethanol, propanol, butanol or pentanol can also efficiently substitute methanol, demonstrating the high potential of the LipIAF5-2 for the synthesis of most short-chain flavor esters.
Fig.5 A) The full bars represent the conversion of triglyceride substrates and the shaded gray bars the conversion of equivalent free fatty acids (FFA). B) Impact of acyl acceptors on transesterification by LipIAF5-2 WCB.
It is solvent-tolerant and thermostable. One paper states that it has maximum activity at 40℃ when acting on triglycerides, with a drastic decrease observed above this temperature[2]. While another paper states that it has a maximum activity of 60℃[1] , and only 30% activity at 40℃ when acting on p-nitrophenyl esters. This discrepancy could be explained by the difference in substrates tested. It is documented that various substrates can shift the observed optimal temperature of enzymes by creating protective effects on their thermal denaturation [5]. As triglycerides take up a high percentage of lipid in food waste, we dominantly take the former as reference, which suggests a high rate of ester synthesis at low- to mid-range temperatures.
Fig.6 Effect of temperature on the hydrolytic activity of the LipIAF5-2 on triglycerides(1) and on p-nitrophenyl esters(2) .
The LipIAF5.2 exhibited a range of activity at alkaline pH from 9 to 11 with a maximum at 10.5.[1]
Fig.7 Effect of pH on the hydrolytic activity of the LipIAF5-2
It’s activity is enhanced by Calcium and manganese divalent cations, and completely inhibited by calcium specific chelating agent EGTA, Triton X-100, SDS detergents and 10mM of Tween 80 under incubation. [1] It is extracellular and is secreted out of the cell[1].
The part is the gene of interest of the composite part BBa_K3077200.[6] The ordered plasmid is transformed in E.coli. However, we don’t have sufficient time to verify the E.coli with Colony PCR as planned.
[1]: Meilleur C., Hupe J.F., Juteau P., Shareck F(2009). "Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library."J. Ind. Microbiol. Biotechnol. 36:853-861(2009)
[2]: https://www.ebi.ac.uk/ena/data/view/EU660533
[3]: Brault G, Shareck F, Hurtubise Y, Lépine F, Doucet N (2014) Short-Chain Flavor Ester Synthesis in Organic Media by an E. coli Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase. PLoS ONE 9(3): e91872. Link
[4]: Abbas H, Comeau L (2003) Aroma synthesis by immobilized lipase from Mucor sp. Enzyme Microb Technol 32: 589–595.
[5]: Paiva AL, Balcao VM, Malcata FX (2000) Kinetics and mechanisms of reactions catalyzed by immobilized lipases. Enzyme Microb Technol 27: 187–204.
[6]: BBa_K3077200