Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis

Abstract

Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana

“…However, here we have shown that the lipase 1 (uniprot A0A0A2W0S0) secretion in B. bassiana isolates has a variable baseline of expression with the highest level only observed when the intact cockroach diet regime was used (in Bb813, Figure 2). The lipase 1 found in this study (uniprot A0A0A2W0S0) has not yet been characterized and has a low degree of homology with the recombinant triacylglycerol lipase A (uniprot J4UGV0) used for biodiesel production [15]. The epicuticular layer of insects consists of a complex mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters, and the sets of lipid-degrading enzymes detected here (lipase 1 and other minor lipolytic esterases, Table 2) may have a dominant role in the breakdown of the epicuticle.…”

“…By increasing the information on the secretome of B. bassiana , the use of this fungus as a control agent against insect pests such as P. americana can potentially be supplemented by B. bassiana culture supernatant coming from aerobic fermentation or by using some of the listed exoenzymes discovered here as potential recombinant candidates for a fungus-free control approach, where appropriate. In addition, other uses of B. bassiana exoenzymes can be envisioned, for example, in various biotechnological applications such as recombinant lipases to be used in biodiesel production [15]. …”

Immobilization Effects on the Catalytic Properties of Two Fusarium Verticillioides Lipases: Stability, Hydrolysis, Transesterification and Enantioselectivity Improvement

“…Lipase from Aspergillus fumigatus presented K M of 14 mM and V max of 1.37 mM•mg −1 •min −1 with p-nitrophenyl acetate [27]; even lower values were found to Aspergillus japonicus lipase, for which K M and V max were 0.64 × 10 −3 mmol and 0.25 µmol•min −1 •mL −1 [28]. Vici et al [29] obtained from a recombinant lipase from Beauveria bassiana a K M and V max value of 0.5546 µM and 85.67 µmol•min −1 •mg −1 respectively, against pNPB, pH 6.0 at 50 • C.…”

Cloning of Lipase Gene From Thermomyces langinosus into Pichia pastoris with its Original Signal Peptide

“…Although several different secretion signal sequences, including the native secretion signal present on heterologous proteins, have been used successfully, results have been variable (Cereghino and Cregg 2000). In some case, many studies using α-factor signal peptide in Pichia protein expression system including, Rhizopus chinensis lipase Yu et al (2009); Beauveria bassiana lipase Vici et al(2015); or Rhizopus oryzae lipase (Minning et al 1998).…”