Main Structural Targets for Engineering Lipase Substrate Specificity

Abstract

<jats:p>Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.</jats:p>

A Valuable Product of Microbial Cell Factories: Microbial Lipase

“…A comparison of both conformations is shown in Figure 3 ( Khan et al, 2017 ). Another important feature of lipases is the “oxygen anion hole.” Arpigny and Jaeger divided lipases into three types, GGGX, GX and γ, according to the preference of “oxygen anion hole” in catalyzing different substrates ( Albayati et al, 2020 ). In addition, there are special interfacial recognition sites in the structure of lipase, which will change the conformation in the presence of lipid and amphiphilic, and the stability of lipase on the interface ( Aloulou et al, 2006 ).…”

“…Irrational design is also known as directed evolution. In this design process, protein structural and functional information is not needed (Ollis et al, 2016); random mutations are introduced into the target gene via a means that is meant to simulate natural evolution; targeted screening is then conducted so as to identify the protein that meets the specific structural or functional requirements (Albayati et al, 2020). Generally, this process involves two steps, including first, the construction of the mutant library, which needs to be of adequate size and diversity; the second step involves high-throughput screening, which is fast, sensitive, and directional.…”

Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats

“…Lipase substrate specificity has been a field of research attracting a lot of interest [7][8][9][10], as well as research focused on the chain length selectivity [11]. Compared with esterases, lipases show specifically high activity towards medium to long-chain water-insoluble triglycerides at a two-phase interface [12].…”

Effect of mutation at oxyanion hole residu (H110F) on activity of Lk4 lipase

“…Oxyanion hole consists two residues, the first residue is located between β3-strand and αA-helix. The second residue is located at pentapeptide motive on the C-terminus of catalytic residue of serine [ 8 , 9 ]. Highly conserved pentapeptide motive shows pattern G-X-S-A-G, X refers to second oxyanion residue and S refers to serine as nucleophilic residue [ 10 ].…”

“…Catalytic activity of lipase might be improved by mutation on the catalytic pocket of oxyanion hole [ 9 , 13 ]. There are a few reports showed that mutation on oxyanion hole residues affect features of various lipase [ [14] , [15] , [16] , [17] , [18] ].…”