Immobilized Forms of the Ophiostoma piceae Lipase for Green Synthesis of Biodiesel. Comparison with Eversa Transform 2.0 and Cal A

In this work, we analyzed the suitability of a versatile recombinant lipase, secreted by Ophiostoma piceae (OPEr) and produced in Pichia pastoris, as a catalyst of the synthesis of biodiesel. The enzyme was immobilized by five covalent procedures and by hydrophobicity on functionalized nanoparticles of magnetite or of a novel Zn/Mn oxide named G1. Then, they were tested for green production of biodiesel by solventless enzymatic transesterification of discarded cooking oil and methanol (1:4) at 25 °C. The results were compared with those shown by free OPEr and the commercial lipases Eversa® and Cal A®. Several preparations with immobilized OPEr produced high synthesis yields (>90% transesterification), comparable to those obtained with Eversa®, the commercial enzyme designed for this application. Three of the biocatalysts maintained their catalytic efficiency for nine cycles. The process catalyzed by AMNP-CH-OPEr was scaled from 500 µL to 25 mL (50 times), improving its efficiency.

Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

Background: Pseudomonas lipases are widely used in industrial applications due to its unique biochemical properties, but one of the biggest limitations are the low yields obtained in native strains therefore, organisms as E. coli are used for the recombinant lipase overexpression. However, the recombinant lipase is accumulated as inclusion bodies and it affects biological activity, making that researchers evaluate different fermentation conditions to improve the activity of recombinant enzymes. In this study, a statistical experimental design was implemented to evaluate the effects of temperature, agitation rate and osmolyte concentration on the recombinant lipase activity produced in E. coli BL21 (DE3). Once the significant variables were identified, an optimization by a Response Surface Methodology was applied to maximize the lipase production. Results: The Box-Behnken designs revealed different optimal fermentation conditions for each osmolyte experiment. The glycerol showed the highest specific lipase activity compared to the other osmolytes and 0.1 M of osmolyte glycerol,5°C and 110 rpm showed the highest significant increase on the specific lipase activity and the data fitted the model very well. The validation showed that 452.01 U/mg of specific lipase activity was obtained which was significantly higher compared to the group where no glycerol was added (271.38 U/mg). The relative recombinant lipase expression was 2.7-fold lower at 5°C compared to 25 °C, but at 5°C the lipase activity was significantly higher. In addition, when the 3 L shaken Erlenmeyer Bioreactor was used to produce the recombinant lipase based on the power input parameter, the specific lipase activity was not significantly different from that found in Schott (408,4 U/mg and 452 U/mg, respectively), which means that this Bioreactor platform should be used for future scale-up processes.Conclusion: Low temperatures, low agitation rates and 0.1 M of glycerol in the autoinduction media enhanced the activity of the recombinant lipase produced in E. coli BL21(DE3). The optimized conditions and the 3 L shaken Erlenmeyer Bioreactor can be used to produce the recombinant enzyme in a higher volume based on the power input parameter. Further studies using this strategy may lead to the identification of optimal culture conditions for a given recombinant enzyme facilitating the large-scale bioprocess implementation.

Ion-Pair Interaction and Hydrogen Bonds as Main Features of Protein Thermostability in Mutated T1 Recombinant Lipase Originating from Geobacillus zalihae

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 °C and 76.0 °C in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in β-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.

Concentration of Lipase from Aspergillus oryzae Expressing Fusarium heterosporum by Nanofiltration to Enhance Transesterification

Nanofiltration membrane separation is an energy-saving technology that was used in this study to concentrate extracellular lipase and increase its total activity for biodiesel production. Lipase was produced by recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (FHL). A sulfonated polyethersulfone nanofiltration membrane, NTR-7410, with a molecular weight cut-off of 3 kDa was used for the separation, because recombinant lipase has a molecular weight of approximately 20 kDa, which differs from commercial lipase at around 30 kDa for CalleraTM Trans L (CalT). After concentration via nanofiltration, recombinant lipase achieved a 96.8% yield of fatty acid methyl ester (FAME) from unrefined palm oil, compared to 50.2% for CalT in 24 h. Meanwhile, the initial lipase activity (32.6 U/mL) of recombinant lipase was similar to that of CalT. The composition of FAME produced from recombinant concentrated lipase, i.e., C14:1, C16:0, C18:0, C18:1 cis, and C18:2 cis were 0.79%, 34.46%, 5.41%, 45.90%, and 12.46%, respectively, after transesterification. This FAME composition, even after being subjected to nanofiltration, was not significantly different from that produced from CalT. This study reveals the applicability of a simple and scalable nanofiltration membrane technology that can enhance enzymatic biodiesel production.