the first round of active-site combinatorial libraries resulted in the identification of several variants with the desired activity on the target substrate . Building on the best-performing variant from round 1 ( V1 ), activity was enhanced approximately 60-fold over five subsequent rounds of bioinformatics-guided directed evolution , employing targeted mutagenesis and focused combinatorial libraries ( Figure 2 ).
During the evolutionary process , molecular dynamics ( MD ) simulations were utilised to predict amino acid substitutions , generating enzyme variants capable of withstanding additional selection pressures introduced by incrementally increasing DMSO concentrations to up to 40 %.
This adjustment aimed to provide balanced solubility for both substrates - the indole derivative and L-serine . Further optimisation included reducing the amount of L-serine in the reaction from four equivalents to one equivalent and increasing the substrate loading by a factor of three .
Improving the organic substrate tolerance of TPL
Tyrosine phenol lyase ( TPL ), like TrpB , is a PLP-dependent enzyme that catalyses the reversible conversion of L-tyrosine ( Figure 3a , 8 ) into phenol ( 6 ), pyruvate ( 7 ) and ammonia .
In biocatalytic applications , TPL can be utilised for the production of L-tyrosine . To drive the reversible reaction towards C-C bond formation , substrate concentrations must remain high , making it essential to have an enzyme capable of tolerating phenol . Consequently , the primary objective
Figure 5 - Reaction catalysed by PAL
Figure 4 - TPL 3D model with bound PLP
was to engineer an enzyme that could withstand a high phenol load , thereby ensuring stability during the entire process .
In a subsequent step , the substrate scope could be expanded based on alternative substrates ( Figure 3b ) that are known to be accepted by TPL . This enables the generation of noncanonical tyrosine derivatives and beyond , like in the case of substrate 12 .
Through four rounds of directed evolution , a variant containing nine amino acid substitutions compared to its ancestral enzyme was developed . The evolution process was guided by in silico screening and MD simulations under process conditions , which identified critical hot spots in highly flexible regions of the enzyme .
In Figure 4 , flexible regions are highlighted in red , while stabilityenhancing hot spots are marked with green circles . The evolved enzyme exhibited a phenol tolerance of 10 g / L for 24 hours , representing an 18-fold improvement . This enhancement also increased the space-time yield to 102 g / L / day .
PAL derivatives at high substrate loading
Phenylalanine ammonia-lyase ( PAL ) catalyses the reversible , non-oxidative deamination of L-phenylalanine to produce trans-cinnamic acid and ammonia . In biocatalytic applications , PAL has attracted significant attention for its ability to synthesise optically pure phenylalanine derivatives ( Figure 5 , 14 ) from corresponding cinnamic acid derivatives ( 13 ).
However , to optimise the efficiency and cost-effectiveness of PAL for industrial processes , challenges such as substrate specificity , enzyme stability and reaction conditions must be addressed . As with TPL ,
34 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981