Application of Rhizomucor miehei lipase-displaying Pichia pastoris whole cell for biodiesel production using agro-industrial residuals as substrate

Sena, Raphael Oliveira; Carneiro, Candida; Moura, Marcelo Victor Holanda; Brêda, Gabriela Coelho; Pinto, Martina Costa Cerqueira; Fé, Luana Xavier Soares Gomes Moura; Fernández‐Lafuente, Roberto; Manoel, Evelin Andrade; Almeida, Rodrigo Volcan; Freire, Denise Maria Guimarães; Cipolatti, Eliane Pereira

doi:10.1016/j.ijbiomac.2021.08.173

Cited by 30 publications

(6 citation statements)

References 60 publications

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“…125 Of particular interest are carrier-free immobilisation strategies. 88,146–151 Without a carrier, the immobilized enzyme occupies a much smaller volume, making it possible to use smaller reactors. Two approaches to preparing aggregates of multiple enzymes to catalyse either cascade or non-cascade reactions deserve a mention here.…”

Section: How Biocatalysts Have Emerged As Enablers In White and Grey ...mentioning

confidence: 99%

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White & grey biotechnologies for shaping a sustainable future

Roy¹,

Gupta²

2023

RSC Sustain.

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Section: How Biocatalysts Have Emerged As Enablers In White and Grey ...mentioning

confidence: 99%

“…199 Earlier, too, it was shown that oils containing high levels of free fatty acids can be efficiently converted into biodiesel by the enzymatic route. 151,200 Burton et al used one of the most expensive immobilized lipases, Novozyme 435, in their work. 199…”

Section: Usefulness Of Coffee Beyond the Last Sipmentioning

confidence: 99%

White & grey biotechnologies for shaping a sustainable future

Roy¹,

Gupta²

2023

RSC Sustain.

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“…Novozyme expressed RM lipase with genetic modified Aspergillus oryzae . RM lipase was also expressed with Pichia pastoris [ 12 , 13 ] or through yeast cell surface display of Pichia pastoris [ 14 ]. However, the yield was relatively low.…”

Section: Introductionmentioning

confidence: 99%

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization

Yang,

Su,

Yang

et al. 2024

Microb Cell Fact

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Background Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. Results The sfGFP(−15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn’t improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. Conclusion The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

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“…7 On the other hand, microbial surface display-based whole-cell biocatalysts have addressed the mass transfer limitation but still face the challenge of low enzyme loading. 8,9 Although cellulosome-derived scaffold protein domains of cohesion and dockerin have been used to assemble enzymes on cell surfaces through noncovalent cohesion-dockerin highaffinity interactions, the high molecular weight of cohesion and dockerin limits the surface display dosage. 10 Additionally, the absence of abundant nanoscale enzyme anchoring structures results in a low specific surface area, which restricts the biocatalysis application of the cohesion and dockerin-based surface display system.…”

Section: Introductionmentioning

confidence: 99%

Genetically Decorating Microbial Surface Curli Fiber with Biocatalytic Properties

Wang,

Shi,

Liu

et al. 2024

ACS Sustainable Chem. Eng.

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Genetically customized microbial surface represents an emerging strategy for creating effective and robust whole cell biocatalysts. In this study, we have genetically engineered new whole cell catalysts that utilize three functional modules: CsgA, SpyTag/SpyCatcher, and Mrcp19k. These modules can programmatically immobilize lipases onto the surface curli fibers of Escherichia coli. The genetically programmed E. coli surface curli fibers exhibited enhanced lipase enzyme dosage, specific activity, and resistance to harsh environments. The application of these genetically tailored whole cell catalysts in the synthesis of fatty acid methyl esters resulted in an 87% yield, with more than 80% relative activity retained after 5 consecutive batches. This genetically programmable immobilization strategy, particularly the integration of Mrcp19k, introduces a concept and technological platform that enables the creation of innovative and highly efficient whole cell biocatalysts and biotransformations.

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Application of Rhizomucor miehei lipase-displaying Pichia pastoris whole cell for biodiesel production using agro-industrial residuals as substrate

Cited by 30 publications

References 60 publications

White & grey biotechnologies for shaping a sustainable future

White & grey biotechnologies for shaping a sustainable future

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization

Genetically Decorating Microbial Surface Curli Fiber with Biocatalytic Properties

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