CATALYSTS
Isoform PLE-1 PLE-2 PLE-3 PLE-4 PLE-5 PLE-6 PLE-7
Activity [ U / mg ]
7.2 ± 0.3 3.3 ± 0.1 6.9 ± 0.5 11.4 ± 0.7 20.3 ± 0.8 4.2 ± 0.7 3.4 ± 0.2
Table 1 - Specific activities of PLE isoforms recombinantly produced in E . coli Expressed in units (µ mol / min ) per mg of total soluble E . coli protein plus the individual isoform using para-nitrophenyl-acetate as assay substrate
In parallel , InnoSyn optimised the fermentative production of the seven PLE isoforms in E . coli . Fast batch fermentations and high cell density yielding fed-batch fermentation strategies were employed to achieve the optimal balance between E . coli biomass production and the expression levels of the individual PLE isoforms . Finally , we routinely achieve biomass yields of E . coli cells containing the PLE enzymes of more than 300 g cell wet weight / kg of fermentation broth . The specific activities of the seven PLE isoforms on the esterase standard substrate para-nitrophenyl acetate that is used for quality control range from 3 to 20 µ mol / minute and mg of total soluble protein ( Table 1 ),
Figure 2 - Selected ester hydrolyses or formation reactions catalysed by CRL
Isoform CRL-1 CRL-2 CRL-3 CRL-4 CRL-5
Activity [ U / mg ] reflecting the differences in substrate scope of the different PLEs . Having such robust and high-yielding fermentation protocols established , we routinely apply these PLE isoforms mainly for pharma but also for fine chemical syntheses .
CRL isoforms
Encouraged by the successful production and application of the PLEs we screened the literature and sequence databases for similar cases of industrially relevant hydrolytic enzymes which exist in various isoforms . In this way , we rapidly arrived at the lipase of Candida rugosa ( CRL ). This fungal microorganism produces at least five lipases that are 78-88 % identical to each other on an amino
1362 5.4 72 190 0.1
Table Table 2 -Specific 2 activities of CRL isoforms recombinantly produced in P . pastoris
N
B acid level . Remarkably , the isoform CRL-3 is identical to the so-called Candida cylindracea cholesterol esterase . This enzyme , which is frequently applied in the synthesis and hydrolysis of lipids and steroid fatty acid esters , is thus also covered by this collection . CRL isoforms have different catalytic behaviours and substrate specificities . The commercial CRL is a mixture of at least three isoenzymes , the proportions of which depend on the supplier . It is used in a wide variety of applications from biodiesel production , food oil refining , flavour and fragrance ester synthesis to enantioselective transformations of pharmaceutical building blocks ( Figure 2 ). 7 We set out to make these isoforms available in efficient and industrially scalable fermentations of recombinant microbial production strains . We compared the bacterial E . coli system as used for the PLE isoforms with the fungal Pichia pastoris production system and found that the E . coli hosts only produced as high volumetric lipase activity as the P . pastoris system for isoform CRL-4 . For all other isoforms the bacterial E . coli did not produce detectable amounts of CRL activities , while for all fungal P . pastoris production strains , CRL activity was produced . Similar amounts of extracellular protein were produced by all five CRL isoform production strains , but the activity determined on a model activity assay substrate varied significantly , also here reflecting the differences in substrate affinities and activities of the different isoforms . They were highest for CRL-1 and CRL- 3 with > 1,000 U / mg total extracellular protein ( Table 2 ).
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