Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed. KEY POINTS: • The dynamics of lipases contributes to their non-covalent interactions and structural stability. • Thermostability can be enhanced by well-established genetic tools for improved kinetic efficiency. • Molecular dynamics greatly provides structure-function insights on thermodynamics of lipase.

Thermostable lipases and their dynamics of improved enzymatic properties.

Immobilization and bioimprinting strategies to enhance the performance in organic medium of the metagenomic lipase LipC12.

The immobilization protocol greatly alters the effects of metal phosphate modification on the activity/stability of immobilized lipases.

Mining of Phospholipase D (PLD) with high activity and stability has attracted strong interest for investigation. A novel PLD from marine Moritella sp. JT01 (MsPLD) was biochemically and structurally characterized in our previous study; however, the short half-life time (t1/2) under its optimum reaction temperature seriously hampered its further applications. Herein, the disulfide bond engineering strategy was applied to improve its thermostability. Compared with wild-type MsPLD, mutant S148C-T206C/D225C-A328C with the addition of two disulfide bonds exhibited a 3.1-fold t1/2 at 35 °C and a 5.7 °C increase in melting temperature (Tm). Unexpectedly, its specific activity and catalytic efficiency (kcat/Km) also increased by 22.7% and 36.5%, respectively. The enhanced activity might be attributed to an increase in the activation entropy by displacing more water molecules by the transition state. The results of molecular dynamics simulations (MD) revealed that the introduction of double disulfide bonds rigidified the global structure of the mutant, which might cause the enhanced thermostability. Finally, the synthesis capacity of the mutant to synthesize phosphatidic acid (PA) was evaluated. The conversion rate of PA reached about 80% after 6 h reaction with wild-type MsPLD but reached 78% after 2 h with mutant S148C-T206C/D225C-A328C, which significantly reduced the time needed for the reaction to reach equilibrium. The present results pave the way for further application of MsPLD in the food and pharmaceutical industries.

/pdf/improving-both-the-thermostability-and-catalytic-efficiency-1iv98xoq.pdf

Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy

The regioselectivity characteristic of lipases facilitate a wide range of novel molecule unit constructions and fat modifications. Lipases can be categorized as sn-1,3, sn-2, and random regiospecific. Geobacillus zalihae T1 lipase catalyzes the hydrolysis of the sn-1,3 acylglycerol chain. The T1 lipase structural analysis shows that the oxyanion hole F16 and its lid domain undergo structural rearrangement upon activation. Site-directed mutagenesis was performed by substituting the lid domain residues (F180G and F181S) and the oxyanion hole residue (F16W) in order to study their effects on the structural changes and regioselectivity. The novel lipase mutant 3M switches the regioselectivity from sn-1,3 to only sn-3. The mutant 3M shifts the optimum pH to 10, alters selectivity toward p-nitrophenyl ester selectivity to C14-C18, and maintains a similar catalytic efficiency of 518.4 × 10−6 (s−1/mM). The secondary structure of 3M lipase comprises 15.8% and 26.3% of the α-helix and β-sheet, respectively, with a predicted melting temperature (Tm) value of 67.8 °C. The in silico analysis was conducted to reveal the structural changes caused by the F180G/F181S/F16W mutations in blocking the binding of the sn-1 acylglycerol chain and orientating the substrate to bond to the sn-3 acylglycerol, which resulted in switching the T1 lipase regioselectivity.

/pdf/altering-the-regioselectivity-of-t1-lipase-from-geobacillus-3amrc397.pdf

Altering the Regioselectivity of T1 Lipase from Geobacillus zalihae toward sn-3 Acylglycerol using a Rational Design Approach

In order to improve the thermostability of lipases derived from Rhizopus chinensis, we identified lipase (Lipr27RCL) mutagenesis sites that were associated with enhanced flexibility based upon B-factor analysis and multiple sequence alignment. We found that two mutated isoforms (Lipr27RCL-K64N and Lipr27RCL-K68T) exhibited enhanced thermostability and improved residual activity, with respective thermal activity retention values of 37.88% and 48.20% following a 2 h treatment at 50°C relative to wild type Lipr27RCL. In addition, these Lipr27RCL-K64N and Lipr27RCL-K68T isoforms exhibited 2.4- and 3.0-fold increases in enzymatic half-life following a 90 min incubation at 60°C. Together these results indicate that novel mutant lipases with enhanced thermostability useful for industrial applications can be predicted based upon B-factor analysis and constructed via site-directed mutagenesis.

/pdf/improving-the-thermostability-of-rhizopus-chinensis-lipase-4mwmto082f.pdf

Improving the Thermostability of Rhizopus chinensis Lipase Through Site-Directed Mutagenesis Based on B-Factor Analysis

The invention discloses a method for preparing influenza vaccine lipidosome. The method comprises the steps of preparation of a phospholipid membrane, preparation of a blank lipidosome, preparation of a single layer of lipidosome, preparation of lipidosome packaged influenza vaccine protein and preparation of influenza vaccine lipidosome freeze-dried powder. The method comprises the following steps: adding 25ml to 35ml of a phosphate buffer solution of which the pH is 7.0 into the phospholipid membrane; performing ultrasonic treatment for 4-8 times at the temperature of 50 DEG C to prepare the single layer of lipidosome; mixing the single layer of lipidosome with 1500mcg to 2500mcg of an influenza antigen solution; quickly freezing at a low temperature, melting at room temperature and repeatedly freezing and thawing for 2-4 times; pre-freezing at low temperature and freeze-drying to obtain the influenza vaccine lipidosome, and storing at the temperature of 4 DEG C. The method disclosed by the invention has the advantage of overcoming the defects that a protein type vaccine is degenerated due to over-high temperature of the generally-prepared vaccine lipidosome and the immunogenicity is affected. By controlling each parameter, the particle size of the lipidosome is uniform; the lipidosome is relatively low in permeability and stable in solution; the packaging rate of the vaccine is increased, and the effectiveness of the temperature-sensitive protein type vaccine is guaranteed as well.

Method for preparing influenza vaccine lipidosome

The invention discloses a method for detecting purity of a CpG ODN sequence by virtue of a reversed-phase column, a proper mobile phase and an elution process. One basic group (n-1), two basic groups (n-2), or one additional basic group (n+1) and two additional basic groups (n+2), which are deleted in a CpG ODN synthesis process, can be separated from a main product (n). Moreover, the method is automatic in operation, can be qualitative and quantitative, is high in precision and good in reproducibility. The invention further discloses application of the method for detecting purity of the CpG ODN sequence by virtue of the reversed-phase column in detection of the purity of the CpG ODN sequence.

Method for detecting purity of CpG ODN sequence and application of method

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Improving the Thermostability of Rhizopus chinensis Lipase Through Site-Directed Mutagenesis Based on B-Factor Analysis

Method for preparing influenza vaccine lipidosome

Method for detecting purity of CpG ODN sequence and application of method