� Figure 2 – Molecular dynamics simuation of TPL with 50 g / L phenol
� Figure 3 – Binding mode of desired ( a ) & wrong ( b ) ( 2S , 3R ) - diastereomers stability under the desired reaction conditions . Both substrate and product caused significant inhibition of its activity . Recently , we reported that this decarboxylase lost its activity within four hours from the start of the reaction and the conversion obtained was < 30 % under the given conditions of 100 g / L substrate and 20 g / L wet cell loading ( pH 7.0 , 40 ). 4 However , after ten rounds of directed evolution employing our BioEngine * platform , an industrially applicable enzyme with high reaction conversion (> 95 % after 24 hours ) was generated . This facilitates production at a scale of about 2,000 tonnes / year of β-alanine .
Amino acids via TPL
Tyrosine phenol-lyase ( TPL ) is a pyridoxal 5 ’ -phosphate ( PLP ) - dependent enzyme which catalyses the reversible β-elimination reaction of L-tyrosine to phenol , pyruvate and ammonium . Enzymaster has used isolated TPL to synthesise L-tyrosine by linking the aromatic C4-atom of phenol with pyruvate ( Figure 1b ).
However , TPL tends to be unstable under high phenol loading . Based on our experiments , the wild-type TPL only provides 1 % conversion within 24 hours under 200 g / L phenol loading when fed in ten 20 g / L portions over the reaction time , while the enzyme is inactivated with 3 g / L phenol . To find an effective way to evolve the wild-type TPL , we first carried out a molecular dynamics ( MD ) simulation of TPL with 50 g / L phenol ( Figure 2 ). From these results , we found that the active site tunnel of TPL is very prone to being blocked by phenol . Based on the MD analysis , we have designed a targeted combinatorial library for smart directed evolution using our BioNavigator * bioinformatic tools . After six rounds of evolution , we received a variant with a conversion of > 95 % after 24 hours . This is stable at 20 g / L phenol for 24 hours . TPL has also been used to produce L-DOPA ( Levodopa , 3,4-dihydroxy- L-phenyl-L-alanine ), which is a precursor of dopamine and is used to treat Parkinson ’ s disease among other applications . A few
years ago , Ajinomoto developed an industrial process for L-DOPA . Catechol , pyruvate and ammonia are coupled by whole cells from Erwinia herbicola , which is reported as the most favourable strain for L-DOPA production with high TPL activity . 5
Chiral intermediates using DERA
The aldol reaction is an important transformation for enantioselective formation of C-C bonds in organic chemistry . Natural aldolases are powerful tools to catalyse such reactions for the synthesis of chiral molecules . 2-Deoxy- D-ribose 5-phosphate aldolase ( DERA ) has been proven to be an efficient biocatalyst the industrial synthesis of drug precursors , but it usually requires optimisation due to its poor tolerance towards high aldehyde concentrations . 6 As reported by Dick et al ., L167 ( catalytically active residue ) and C47 are two important residues of E . coli DERA that form covalent bonds with an intermediate caused by aldol
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