Part I - Keratinases

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Part II - Esterases

In our project we used esterases for degrading the layer of grease and waxes on hair which increases the accessibility for the keratinases. We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared.

EstCS2

EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay.

LipB

EstCS2 from the iGEM Imperial College 2013 was proved to be active. LipB showed an enzyme activity of 2,8 U/mL in the supernatant. First, we repeated the enzyme activity assay from the iGEM TU Darmstadt 2012 to determine the esterase with the highest enzyme activity.

Part III - Lipases

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Part IV - The rose fragrance

The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production. In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose. For our project we discussed different approaches and choose two different scents: rose and Limonene. As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor. Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli.1 This pathway comprised four steps (Fig.1): Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate, 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde, aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol, alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.

Part IV.2 The limonene fragrance

Limonene is a well-known cyclic monoterpene which can occur in two optical forms.2 (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy.2 The (L)-Limonene form has a more harsh turpentine-like odor with a lemon-note.2 For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli.