Maria Dolors Benaiges

Enzymatic synthesis of short-chain esters (flavours) might enable their labelling as natural, increasing their value. Covalently immobilised Rhizopus oryzae lipase (EO-proROL) was used to synthesise isoamyl butyrate and acetate. In cyclohexane, the best performer reaction solvent, 1.8 times higher yield of isoamyl butyrate (ca. 100%) than isoamyl acetate (ca. 55%) was obtained. Optimum initial acid concentration (410 mM) and acid:alcohol mole ratio (0.5) were established by a central composite rotatable design to maximise isoamyl butyrate single-batch and cumulative production with reused enzyme. These conditions were used to scale up the esterification (150 mL) and to assess yield, initial esterification rate, productivity and enzyme operational stability. Commercial isoamyl alcohol and fusel oil results were found to be similar as regards yield (91% vs. 84%), initial reaction rate (5.4 µM min⁻¹ with both substrates), operational stability (40% activity loss after five runs with both) and productivity (31.09 vs. 28.7 mM h⁻¹). EO-proROL specificity for the structural isomers of isoamyl alcohol was also evaluated. Thus, a successful biocatalyst and product conditions ready to be used for isoamyl ester industrial production are here proposed.

Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P_GAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (P_AOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel).

Lipases are biocatalysts with a significant potential to enable a shift from current pollutant manufacturing processes to environmentally sustainable approaches. The main reason of this prospect is their catalytic versatility as they carry out several industrially relevant reactions as hydrolysis of fats in water/lipid interface and synthesis reactions in solvent-free or non-aqueous media such as transesterification, interesterification and esterification. Because of the outstanding traits of Rhizopus oryzae lipase (ROL), 1,3-specificity, high enantioselectivity and stability in organic media, its application in energy, food and pharmaceutical industrial sector has been widely studied. Significant advances have been made in the biochemical characterisation of ROL particularly in how its activity and stability are affected by the presence of its prosequence. In addition, native and heterologous production of ROL, the latter in cell factories like Escherichia coli, Saccharomyces cerevisiae and Komagataella phaffii (Pichia pastoris), have been thoroughly described. Therefore, in this review, we summarise the current knowledge about R. oryzae lipase (i) biochemical characteristics, (ii) production strategies and (iii) potential industrial applications.

Recombinant Rhizopus oryzae lipase (mature sequence, rROL) was modified by adding to its N-terminal 28 additional amino acids from the C-terminal of the prosequence (proROL) to obtain a biocatalyst more suitable for the biodiesel industry. Both enzymes were expressed in Pichia pastoris and compared in terms of production bioprocess parameters, biochemical properties, and stability. Growth kinetics, production, and yields were better for proROL harboring strain than rROL one in batch cultures. When different fed-batch strategies were applied, lipase production and volumetric productivity of proROL-strain were always higher (5.4 and 4.4-fold, respectively) in the best case. rROL and proROL enzymatic activity was dependent on ionic strength and peaked in 200 mM Tris-HCl buffer. The optimum temperature and pH for rROL were influenced by ionic strength, but those for proROL were not. The presence of these amino acids altered lipase substrate specificity and increased proROL stability when different temperature, pH, and methanol/ethanol concentrations were employed. The 28 amino acids were found to be preferably removed by proteases, leading to the transformation of proROL into rROL. Nevertheless, the truncated prosequence enhanced Rhizopus oryzae lipase heterologous production and stability, making it more appropriate as industrial biocatalyst.

A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.

The recombinant 1,3‐positional selective Rhizopus oryzae lipase (rROL) was used to synthesize biodiesel and monoacylglycerols simultaneously. The reaction was carried out in a solvent‐free system with the enzyme immobilized on octadecyl‐Sepabeads. Using response surface methodology, the methyl ester yield was optimized by means of the study of the effect of water, substrate molar ratio (methanol:olive oil) and methanol stepwise addition. It was concluded that in order to prevent enzyme inactivation by methanol, alcohol should be added slowly; otherwise a large amount of water would be present. Taking the best conditions, a 50.3 % yield was achieved in 3 h, which corresponds to 75.4 % of the acyl groups at the 1,3‐position undergoing transesterification. It was also concluded that methyl esters result from the esterification of the free fatty acid hydrolyzed by the enzyme and also from a direct transesterification of oil. In addition, the fatty acid selectivity of rROL was found not to favor one fatty acid in olive oil over another.

New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts