Speciality Chemicals Magazine JAN / FEB 2022 | Page 30

CATALYSTS
‣ High cell density fermentations of the CRL isoform productions strains generally deliver > 400 g of P . pastoris cell wet weight / kg of fermentation broth in combination with these high activity values . The CRL isoforms have been applied for the selective enrichment of omega-3 fatty acids in fish oil . 8
P . pseudoalcaligenes lipase
Another lipase with broad substrate scope and excellent productivity is Pseudomonas pseudoalcaligenes ( formerly known as P . alcaligenes ) lipase . This bacterial enzyme was developed under the trade name Lipomax for laundry applications by Genencor and Gist-Brocades in the 1990s . After self-cloning of the gene in the same organism , the enzyme was recombinantly produced as an extracellular protein at full scale . 9 Since then , it appeared only seldom in scientific literature , but in our labs it frequently ranked among the most efficient and selective enzymes when screening our hydrolase collection . For instance , for pharmaceutical products and intermediates like naproxen , esters and aliskiren precursors the P . pseudoalcaligenes lipase is highly active and enantioselective ( E > 100 , ee > 99 %) 10 ( Figure 2 ). Interestingly it has ( S ) - selectivity and therefore the opposite enantioselectivity for the aliskiren precursors to PLE . Like PLE , it is highly chemoselective in the mono-hydrolysis of non-chiral diesters . It outperformed PLE and commercial formulations of the lipases from C . rugosa ( CRL ), C . antarctica ( CalA and CalB ), Rhizomucor miehei , Rhizopus javanicus , P . fluorescens and Alcaligenes sp . in the conversion of dimethyl-adipate to the mono-ester .
Figure Figure 3 : 3 - Selected ester hydrolyses catalysed by P . pseudoalcaligenes lipase
At a three-fold lower enzyme loading , P . pseudoalcaligenes lipase reached quantitative conversion of 100 g / L dimethyl adipate to monomethyl adipate in less time than the P . fluorescens and Alcaligenes sp . lipases , while the over-hydrolysis product adipic acid remained below detection level . Even at 500 g / L substrate loading 99 % selectivity was achieved at a substrate-to-catalyst ( S / C ) ratio of 300:1 on weight basis , corresponding to a molar S / C ratio of > 50,000 , and with a volumetric productivity of around 50 g / L and hour . Such high productivities and low biocatalyst loadings make this lipase suitable for speciality , polymer and even bulk chemistries . However , a prerequisite for application in large-scale bulk chemistry processes is the ability to produce the enzyme in largescale and low-cost fermentations . The fermentation process for P . pseudoalcaligenes established in the 1990s still relies on self-cloning in the same organism and ill-defined induction times by media components and co-substrates like soybean oil . We have recently constructed a recombinant Pseudomonas putida strain that can be fermented efficiently on low-cost sugars , with defined induction of gene expression by low concentrations of a cheap externally added chemical inducer . In contrast to other recent work in which the fungal P . pastoris system is used to produce this bacterial enzyme , we can make use of the homologous protein production and excretion machinery of a similar Pseudomonas strain . 11
Conclusions
Hydrolase enzymes like lipases and esterases are reliable workhorses in all segments of the chemical industry , from high-value , low-volume pharma intermediates to high-volume , low-value bulk chemicals . Their intrinsically high stability , activity and selectivity in combination with their commercial availability make them readily applicable for a vast variety of chemical syntheses . Being able to ferment these enzymes in recombinant production hosts , we are now able to produce them more efficiently and to improve them for specific applications by directed evolution and protein engineering . •
J j
Iwona Kaluzna
MARKETING & SALES DIRECTOR
INNOSYN BV iwona . kaluzna @ innosyn . com www . innosyn . com
References
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30 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981