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Biocatalysis is a key technology enabling plastic recycling. However, despite advances done in the development of plastic-degrading enzymes, the molecular mechanisms that govern their catalytic performance are poorly understood, hampering the engineering of more efficient enzyme-based technologies. In this work, we study the hydrolysis of PET-derived diesters and PET trimers catalyzed by the highly promiscuous lipase B from Candida antarctica (CALB) through QM/MM molecular dynamics simulations supported by experimental Michaelis-Menten kinetics. The computational studies reveal the role of the pH on the CALB regioselectivity toward the hydrolysis of bis-(hydroxyethyl) terephthalate (BHET). We exploit this insight to perform a pH-controlled biotransformation that selectively hydrolyzes BHET to either its corresponding diacid or monoesters using both soluble and immobilized CALB. The discoveries presented here can be exploited for the valorization of BHET resulting from the organocatalytic depolymerization of PET., (© 2023. The Author(s).)

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n -propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí ( Euterpe oleracea ), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R -isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S -isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S -isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R -isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S -isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes., Competing Interests: The authors declare no conflict of interest.

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.

Carrier-free immobilization of Candida rugosa lipase (CRL) and polymers containing primary amino groups were cross-linked using carbodiimide. To accomplish this, the free carboxyl groups of the enzyme were activated with carbodiimide-succinimide in organic medium, and then the activated proteins were cross-linked with different polyethylenimines (PEIs). The effect of the cross-linker chain length, the amount of added bovine serum albumin (BSA), and carbodiimide concentration on the catalytic properties of resulting cross-linked enzyme aggregates (CLEAs) was investigated. The CLEAs' size, shape, specific activity, activity recovery, thermostability and enantioselectivity significantly varied according to the preparation procedure. The highest thermostable CRL-CLEA preparation was obtained with 1.3 kDa polyethyleneimine as cross-linker, 10 mg of BSA and 28 mM of carbodiimide. This preparation is 1.3-fold more active and thermostable than CLEAs prepared by the traditional method of amino cross-linking with glutaraldehyde, and retains 60% of residual activity after 22 h at 50 °C. Additionally, the CRL-CLEA preparation showed an enantioselectivity of 91% enantiomeric excess (ee). This immobilization procedure provides an alternative strategy for CLEA production, particularly for enzymes where the traditional method of cross-linking via lysine residues leads to enzyme inactivation.

Lipases are widely used in the industry for different purposes. Although these enzymes are mainly produced by submerged fermentation, lipase production by solid-state fermentation (SSF) has been gaining interest due to the advantages of this type of culture. Major advantages are higher production titers and productivity, less catabolite repression, and use of the dried fermented material as biocatalyst. This chapter describes a traditional methodology to produce fungal (Rhizopus homothallicus) lipases by SSF using perlite as inert support. The use of different devices (glass columns or Erlenmeyer flasks) and type of inoculum (spores or growing mycelium) is considered so that lipase production by SSF could be easily performed in any laboratory.

One-pot multi-step conversions are desirable to achieve more efficient and sustainable chemical processes. In this context, the immobilization of multi-enzyme systems allows the reusability of several stabilized biocatalysts working in cascade and orthogonal reactions to access more complex synthetic schemes. Herein, we have shown the one-pot tandem hydrolysis and oxidation of racemic esters (1-phenylethyl acetate) to yield quantitative conversion of prochiral ketones (acetophenone) catalyzed by a consortium of immobilized enzymes. Eukaryotic lipase and catalase, and microbial thermophilic alcohol dehydrogenase and NADH oxidase are covalently, irreversibly and individually immobilized onto four different carriers, achieving high immobilization yields (>95%) for all the enzymes, and residual activities >50% for both thermophilic alcohol dehydrogenase and NADH oxidase, 18% for the catalase and 10% for the lipase. This heterogeneous system efficiently recycles NAD + with a maximum turnover frequency (TOF) of 294 h −1 and can be reused for up to 10 operational cycles, retaining more than 80% of its initial activity. [ABSTRACT FROM AUTHOR]

The enantioselectivity of carrier-bound and carrier-free immobilized lipase from Candida rugosa (CRL) was studied. CRL was immobilized in six agarose-based carriers functionalized with different reactive groups and in two different CRL cross-linked aggregates. Both, activity and enantioselectivity of all the immobilized lipase preparations were evaluated with different racemic esters under different reaction conditions (temperature, pH and solvent polarity). A strong effect of reaction media and immobilization protocol on enzyme activity and selectivity was found. Enzyme immobilization and reaction engineering allowed us obtaining the best immobilization protocol and reaction conditions to achieve high activity and enantioselectivty of CRL as heterogeneous catalyst. CRL immobilized on an agarose-based carrier activated with primary amino groups preferentially hydrolyzed ( S )-phenylethyl acetate with E > 200 under pH 7, 4 °C and 30% of acetonitrile. On the other hand, CRL aggregated and cross-linked through their carboxylic groups preferentially hydrolyzed the ( S )-isomer of ethyl 2-hydroxy-4-phenylbutyrate with an E = 39 under pH 5, 4 °C and 30% of acetonitrile. This work demonstrates the success of the combinatorial enzyme engineering for the production of highly enantioselective heterogeneous biocatalysts by screening different immobilization protocols and reaction media conditions. [ABSTRACT FROM AUTHOR]