Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

Abstract

Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes.

Keywords: : lipases; biocatalysis; heterologous expression; industrial applications; phospholipases; protein engineering.

Figure 1

Reactions catalyzed by lipases. Lipases catalyze the cleavage of carboxyl ester bonds. This reaction occurs in the presence of water as substrate or product in hydrolysis and esterification, respectively, of an alcohol in alcoholysis, of an organic acid in acidolysis, of ammonia in aminolysis, and between two different esters in interesterification.

Figure 2

Classification of yeast and fungal lipases.

Figure 3

Canonical fold of α/β hydrolases. Strands are indicated by arrows and helices by cylinders. The positions of the histidine (H) and aspartate (D) residues, ad well as the GXSXG and the oxyanion hole are indicated.

Figure 4

Mechanism of the hydrolysis reaction of ester bonds catalyzed by lipases. Aspartate and histidine are shown in blu and green, respectively; serine, substrate and water are shown in black; the oxyanion hole residues are shown in magenta. (a) Nucleophilic attack of the serine hydroxyl on the carbonyl carbon of the susceptible ester bond; (b) tetrahedral intermediate; (c) acyl-enzyme intermediate, released alcohol and nucleophilic attack by water; (d) free enzyme and released acyl product.

Figure 5

Reactions catalyzed by the phospholipases. The black arrows for phospholipases A₁, A₂, B, C, and D (PLA₁, PLA₂, PLB, PLC, and PLD) indicate their site of hydrolysis. PLA₁ and PLA₂ hydrolyze the sn-1 and sn-2 acyl ester bonds, respectively, of the phospholipid. PLB sequentially removes the two fatty acids from the phospholipid. PLC hydrolyzes the glycerophosphate bond, and PLD cleaves the terminal phosphodiesteric bond of the phospholipid. DAG, diacylglycerol; FFA, free fatty acid; PA, phosphatidic acid; P-X, phosphorylated head-group; X, head-group. X can be choline, ethanolamine, glycerol, inositol or serine.

Figure 6

Percentage of use of the microbial hosts for heterologous expression of lipases and phospholipases. Data do not include enzymes autocloned and homologously expressed in the microorganism of origin.