Immobilization of Pseudomonas stutzeri lipase through Cross-linking Aggregates (CLEA) for reactions in Deep Eutectic Solvents

Highlights

• Lipase from Pseudomonas stutzeri was immobilized through cross-linking aggregates.

• The new derivative is active in a synthesis reaction using DES as reaction media.

• The biocatalyst is stable and can be reused several times.

• Cross-linking aggregates are a simple tool to obtain active biocatalysts in DES.

Abstract

The use of deep eutectic solvents (DES) with buffer as cosolvent (up to 10 % v/v) leads to low-viscous media in which lipases can perform synthetic reactions, instead of hydrolysis. This paper explores the immobilization of Pseudomonas stutzeri lipase (TL) in cross-linking aggregates (CLEA) to deliver robust derivatives that are active in media like choline chloride – glycerol DES with buffer as cosolvent. While the free TL enzyme was markedly inactive in these media, TL-CLEA derivatives perform esterifications and can be reused several times. Overall, results are consistent with previous experiments reported for other lipases in these DES-water media and confirm that CLEA immobilization turns out a very useful and straightforward alternative for generating active (bio)catalysts for DES-aqueous media systems. Immobilized systems open the possibility of performing continuous processes in low-viscous DES-buffer media.

Introduction

Lipases are a group of enzymes widely distributed in nature (Barathi and Rajalakshimi, 2019; Javed et al., 2018; Hasan et al., 2009). The interest in these enzymes is based on their applications as catalysts in synthetic reactions (Guajardo et al., 2020; Razak et al., 2020; Rajagopala and Kroutil, 2011), because they can accept a wide range of substrates (Humble and Berglund, 2011) and can perform synthetic reactions in non-aqueous media (the so-called non-conventional media), such as organic solvents (Kajiwara et al., 2019), ionic liquids (Domínguez de María, 2008), deep eutectic solvents (Guajardo et al., 2016) and other biogenic solvents (Guajardo and Domínguez de María, 2020). Lipases follow the mechanism of "interfacial activation", in which the lid that cover the active site can be open in contact with hydrophobic surfaces and enable the contact of substrate with the active site to promote the catalysis (Maraite et al., 2013; Bracco et al., 2020).

In this field, lipase from Pseudomonas stutzeri (TL) (commercialized by Meito Sangyo Co., Ltd., under the trade name of lipase TL) has proven to be a versatile biocatalyst accepting a broad range of substrates, such as wood sterols (Martínez et al., 2004), benzoins (Hoyos et al., 2006), lauric acid (Bernal et al., 2018) and alcohols (Kim et al., 2008). TL lipase has been reported to be active and stable in organic solvents (e.g. toluene, acetone, 1,4 dioxane and THF) (Hoyos et al., 2006; Bernal et al., 2018; Kim et al., 2008; Yamamoto et al., 2008). However, as free enzyme it is hardly active in deep eutectic solvents, just displaying some activity when immobilized on Accurel MP1001 as support, and in some particular DES (e.g. using isosorbide as HBD component, and choline chloride as salt) (Petrenz et al., 2015). The enzyme displayed activity also in other solvents like CPME, 2-MeTHF or toluene (Petrenz et al., 2015).

One well-known strategy to increase the activity and stability and the reuse of the biocatalysts is immobilization (Ortiz et al., 2019; García-Galán et al., 2011; Sheldon, 2007; Cao, 2005; Sheldon et al., 2021). Methodologies may comprise covalent bonding on solid supports (Basso and Serban, 2019; Kumar et al., 2013; Girelli et al., 2019), adsorption on supports (Cao, 2005; Girelli et al., 2019; Fernández- Lafuente et al., 1998), encapsulation (Valldeperas et al., 2019; Ruíz et al., 2019; Guajardo et al., 2020) or entrapment (Tavernini et al., 2020). In this area, immobilization using cross-linked aggregates (CLEA) has many advantages such as the development of a biocatalyst with greater activity and stability without the need for support (Guajardo et al., 2020; Illanes et al., 2012; Sheldon, 2011; Wilson et al., 2004; De Gonzalo and Domínguez de María, 2017; Guajardo et al., 2019a). CLEA are prepared by enzyme precipitation to form aggregates, produced by mixing the protein with precipitating agents (e.g. ammonium sulfate, organic solvents, or polymers) in aqueous solutions. Thus, these physical enzyme-aggregates are crosslinked with a cross-linking agent, such as glutaraldehyde. The cross-linking step generates the covalent bonds between amino acids (specifically lysines) of protein molecules, yielding an insoluble biocatalyst with high stability and activity (Sheldon, 2011; Wilson et al., 2004).

Lipases have been successfully immobilized via cross-linked aggregates (Sheldon, 2007; Guajardo et al., 2020, 2019a; de Rose et al., 2017). However, lipases have a few lysine residues on their surface and thus it is necessary to enrich the preparation with other amino-containing molecules for an efficient covalent bond. There are different types of enriching compounds, such as polyethyleneimine or bovine serum albumin (BSA) (Guajardo et al., 2020, 2019a; Wilson et al., 2006; Guauque Torres et al., 2013). It is important to carefully control the incorporation of BSA, since the high concentration in lysine groups can occlude the active site of the biocatalyst, generating diffusion restrictions, and thus decreasing the activity of the biocatalyst (Guauque Torres et al., 2013).

With a reinforced stability through immobilization, enzymes can efficiently perform reactions in non-conventional media. Herein, the use of hazardous organic solvents may cause environmental issues (Prat et al., 2016), and therefore research is currently being shifted to explore more environmentally friendly solvents as (some) ionic liquids (IL) (Plechkova and Seddon, 2008), and, particularly, deep eutectic solvents (DES) (Abbott et al., 2003, 2004). These solvents are particularly useful when working with substrates with unpaired polarities (Villeneuve, 2007). DES are biodegradable (if starting from biodegradable components), are easy to prepare and are (often) inexpensive, which makes them attractive for the development of new bioprocesses (Abbott et al., 2003, 2004). DES are typically formed by combining an ammonium salt (e.g. choline chloride) with several hydrogen-bond donors like alcohols, acids, etc. The hydrogen-bond donor interacts with the ammonium salt, disrupting the structure and lowering the melting point of the mixture. For instance, the mixture between choline chloride and urea at 1:2 molar ratio (whose melting points are 302 °C and 133 °C respectively), leads to a DES with a melting point of 12 °C (Abbott et al., 2003).

Different examples of DES used in biocatalysis have been reported over the last decade (Huang et al., 2020; Pätzold et al., 2019; Pánic et al., 2021; Shehata et al., 2020; Benworth et al., 2021; Erol and Hollmann, 2021). Particularly, our research group has pioneered the use of continuous processes with DES and esterifications, as well as the immobilization of lipases with CLEA (namely Candida antarctica lipase B) for improved performances in DES (Guajardo et al., 2019a; Guajardo et al., 2020; Guajardo and Domínguez de María, 2019). Herein, a key-aspect is the addition of water (or buffer) as co-solvent (up to 10–20 % v/v), to decrease the viscosity of the DES (Guajardo et al., 2017). At first sight it may be striking to see how esterifications can be performed up to full conversion with such high amounts of water (due to equilibrium pattern of esterifications). However, this can be explained because the DES removes the available water from the media, thus keeping its non-conventional nature. At higher additions of water (>20 % v/v) water starts to be available, and conversion decreases, as hydrolytic reactions also take place (Guajardo et al., 2017; Maugeri et al., 2013). Continuing with our work, in this paper the stabilization of TL lipase by immobilization through CLEA is presented, showing that the obtained crosslinked derivatives (TL-CLEA) remain active in DES. Therefore, a proper immobilization system may lead to active catalysts in these emerging media (Guajardo et al., 2020, 2016; de Rose et al., 2017; Guajardo et al., 2017, 2019a,b).