A Novel Fungal Lipase With Methanol Tolerance and Preference for Macaw Palm Oil

Abstract

Macaw palm is a highly oil-producing plant, which presents high contents of free fatty acids, being a promising feedstock for biofuel production. The current chemical routes are costly and complex, involving highly harsh industrial conditions. Enzymatic processing is a potential alternative; however, it is hampered by the scarce knowledge on biocatalysts adapted to this acidic feedstock. This work describes a novel lipase isolated from the thermophilic fungus Rasamsonia emersonii (ReLip), which tolerates extreme conditions such as the presence of methanol, high temperatures, and acidic medium. Among the tested feedstocks, the enzyme showed the highest preference for macaw palm oil, producing a hydrolyzate with a final free fatty acid content of 92%. Crystallographic studies revealed a closed conformation of the helical amphipathic lid that typically undergoes conformational changes in a mechanism of interfacial activation. Such conformation of the lid is stabilized by a salt bridge, not observed in other structurally characterized homologs, which is likely involved in the tolerance to organic solvents. Moreover, the lack of conservation of the aromatic cluster IxxWxxxxxF in the lid of ReLip with the natural mutation of the phenylalanine by an alanine might be correlated with the preference of short acyl chains, although preserving catalytic activity on insoluble substrates. In addition, the presence of five acidic amino acids in the lid of ReLip, a rare property reported in other lipases, may have contributed to its ability to tolerate and be effective in acidic environments. Therefore, our work describes a new fungal biocatalyst capable of efficiently hydrolyzing macaw oil, an attractive feedstock for the production of "drop-in" biofuels, with high desirable feature for industrial conditions such as thermal and methanol tolerance, and optimum acidic pH. Moreover, the crystallographic structure was elucidated, providing a structural basis for the enzyme substrate preference and tolerance to organic solvents.

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro

“…Expression of LIPR in P. pastoris GS115 Although LIPR from R. emersonii was expressed in E. coli, the expression level was very low. To achieve the efficient expression of LIPR, the gene sequence of LIPR was optimized by codon preference 10 and expressed in P. pastoris GS115. As shown in SDS-PAGE (see Supporting information, Fig.…”

“…The only reported heterologous expression of LIPR was in Escherichia coli, but the expression level was too low. 10 Although the optimal pH value of LIPR was acidic, its function as an acidic lipase has never been studied. Therefore, it is particularly important to achieve high expression of LIPR.…”

Recent trends in metal‐organic frameworks mediated lipase immobilization: A state‐of‐the‐art review

“…Several microbial strains, such as C. rugosa (Thangaraj et al., 2019; Xie & Huang, 2018), Rhizopus oryzae and C. rugosa (Lee et al., 2019), Novozym 435 (Ismail et al., 2017; Marín‐Suárez et al., 2019), Enterobacter (Badoei‐dalfard et al., 2019), C. antarctica (Bencze et al., 2016), P. cepacia (Kumar et al., 2020), and Rhizomucor miehei (Adnan et al., 2018), have been used to immobilize lipase irreversibly through entrapment, cross‐linkage, and covalent bonding for biodiesel production. Various substrates, including oleic acid (Fauzi et al., 2014), macaw palm (Rade et al., 2020), low‐quality fish oil (Marín‐Suárez et al., 2019), Hevea brasiliensis (Arumugam & Ponnusami, 2019), soybean oil (Jambulingam et al., 2019; Lee et al., 2019; Li, Fan, et al., 2017), castor oil (Kumar et al., 2020), sunflower oil (Dumri & Hung Anh, 2014), algae oil (Lee et al., 2019), and waste cooking oil (Ismail et al., 2017; Lee et al., 2019; Mehrasbi et al., 2017), have been utilized with microbial strains for biodiesel synthesis.…”

“…), as well as the high tolerance to several organic solvents After 24 h, the ANL@M-ZIF-8-PDMS showed a slightly higher reaction rate than ANL@M-ZIF-8, reaching 88% of FAME yield compared to 80% for ANL@M-ZIF-8 The initial reaction rate for 3DOM-C@TLL@PDM was 3.3 times greater than that catalyzed by C-C@TLL and 1.8 times higher than that catalyzed by 3DOM-C@TLL P. cepacia (Kumar et al, 2020), and Rhizomucor miehei (Adnan et al, 2018), have been used to immobilize lipase irreversibly through entrapment, cross-linkage, and covalent bonding for biodiesel production. Various substrates, including oleic acid (Fauzi et al, 2014), macaw palm (Rade et al, 2020), low-quality fish oil (Marín-Suárez et al, 2019), Hevea brasiliensis (Arumugam & Ponnusami, 2019), soybean oil (Jambulingam et al, 2019;Lee et al, 2019;Li, Fan, et al, 2017), castor oil (Kumar et al, 2020), sunflower oil (Dumri & Hung Anh, 2014), algae oil (Lee et al, 2019), and waste cooking oil (Ismail et al, 2017;Lee et al, 2019;Mehrasbi et al, 2017), have been utilized with microbial strains for biodiesel synthesis. searchers have reported similar results where the ANL@M-ZIF-8 and ANL@M-ZIF-PDMS were found to be more effective for catalyzing biodiesel synthesis than ANL/ZIF-8, resulting in a higher yield of fatty acid methyl ester (Hu et al, 2020(Hu et al, , 2021 (Figure 14).…”

Exploring Metagenomic Enzymes: A Novel Esterase Useful for Short-Chain Ester Synthesis

“…A classification of the bacterial lipases into eight families has been reported previously by Arpigny and Jaeger (1999), based on their conserved sequence motifs and some fundamental biological properties. Currently, many novel lipolytic enzymes have been discovered from different microorganisms, as yeasts [21,22], fungi [23][24][25] and bacteria [26][27][28][29].…”