Engineering surface hydrophobicity improves activity of Bacillus thermocatenulatus lipase 2 enzyme

Bacillus thermocatenulatus lipase 2 (BTL2) is a promising industrial enzyme used in biodiesel production. Although BTL2 has high thermostability and good resistance to organic solvents, the activity of BTL2 is suboptimal for industrial processes. To improve BTL2 activity, we engineered BTL2 lipase by modulating hydrophobicity of its lid domain. Through site‐directed mutagenesis, we constructed three mutants, namely Y225F+S232A, S232A+T236V and Q185L, to cover all uncharged hydrophilic amino acids within the lid domain. Activities of these mutants were characterized. Our findings suggest that one mutant (Y225F+S232A) showed ～35% activity increase in catalyzing heterogeneous hydrolytic reactions relevant for industrial applications. A mathematical framework was established to account for different molecular events that contribute to the observed apparent catalytic activities. Increases in hydrophobicity of lid domains were associated with increased interfacial adsorption of lipases and lower molecular enzymatic activities. The measured apparent activities of lipases include contributions from both events. Lid hydrophobicity can thus result in different changes in lipase activities depending on the mutation site. Our work demonstrates the feasibility of increasing BTL2 activity by modulating the hydrophobicity of lid domains and provides some guidelines for further improving BTL2 activity.     

Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase

The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-A resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases. The zinc-coordinated extra domain makes tight interactions with the loop extended from the C terminus of the lid helix, suggesting that the activation of the family I.5 lipases may be regulated by the strength of the interactions. The unusually long lid helix makes strong hydrophobic interactions with its neighbors. The structural information together with previous biochemical observations indicate that the temperature-mediated lid opening is triggered by the thermal dissociation of the hydrophobic interactions.     

Substitution of Asp189 residue alters the activity and thermostability of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. NTU 03 lipase

Error-prone PCR was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant of the α6 helix (lid domain), with an 189E to V substitution at residue 189, lost its thermostability but exhibited higher activity than that of its wild-type predecessor (r03Lip). Site-saturation mutagenesis was used to explore the sequence-function relationship. Five other mutants also lost thermostability (20–40%) but exhibited enhanced thermoactivity (6.3–79-fold). The mutant E189I showed the highest activity retaining 50% activity after maintaining it at 65°C for 24 h. In comparison to r03Lip, the mutant E189I had a higher affinity for p-nitrophenyl palmitate and p-nitrophenyl stearate (61 and 56% decreased Km) and catalytic efficiency (42-fold and 18-fold increased kcat/Km). The mutant lipase retained its tolerance to n-hexane, but had an improved transesterification activity. The results suggest that residue Glu189 plays a significant role in the thermostability and activity of this thermoalkalophilic lipase.     

Directed evolution of Bacillus licheniformis lipase for improvement of thermostability

Lipases catalyze the hydrolysis of carboxylic acid esters and owing to their vast substrate specificity, they have many industrial applications. Due to the demand of thermostable lipases in industrial applications, we have enhanced the thermostability of lipase from Bacillus licheniformis RSP-09. The thermostable mutant lipases of Bacillus licheniformis RSP-09 were isolated following two rounds of directed evolution using error-prone PCR. The best mutant lipases obtained after first and second round of error-prone PCR were purified and characterized. The mutant lipases showed increased thermostability and retained catalytic function. The best mutant lipase (eP-231-51) showed 13.5-fold increase in percentage thermal stability (% remaining activity after incubation of purified enzyme at 60 °C for 1 h) than wild-type lipase. Also, this mutant lipase (ep-231-51) showed 30% improved catalytic efficiency compared with the wild-type which is due to significant decrease in K_m and marginal increase in k_cat. In addition, the thermostable mutant lipases have shown resistance to hydrophobic organic solvents. The role of mutations in the best mutant lipases of second round i.e. eP-231-51 (Asp72Gly, Asp61Gly, Tyr129His, and Thr101Pro) and eP-231-137 (Leu49Arg, Thr101Pro, Asp72Gly), that led to thermostability have been postulated after the comparison of molecular models of wild-type and mutated enzymes.     

Probing the roles of two tryptophans surrounding the unique zinc coordination site in lipase family I.5

A unique zinc domain found in all of the identified members of the lipase family I.5 is surrounded by two conserved tryptophans (W61 and W212). In this study, we investigated the role of these hydrophobic residues in thermostability and thermoactivity of the lipase from Bacillus thermocatenulatus (BTL2) taken as the representative of the family. Circular dichroism spectroscopy revealed that the secondary structure of BTL2 is conserved by the tryptophan mutations (W61A, W212A, and W61A/W212A), and that W61 is located in a more rigid and less solvent exposed region than is W212. Thermal denaturation and optimal activity analyses pointed out that zinc induces thermostability and thermoactivity of BTL2, in which both tryptophans W61 and W212 play contributing roles. Molecular explanations describing the roles of these tryptophans were pursued by X‐ray crystallography of the open form of the W61A mutant and molecular dynamics simulations which highlighted a critical function for W212 in zinc binding to the coordination site. This study reflects the potential use of hydrophobic amino acids in vicinity of metal coordination sites in lipase biocatalysts design. Proteins 2016; 84:129–142. © 2015 Wiley Periodicals, Inc.     

In silico characterization of thermostable lipases

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.     

Discrimination of thermostable and thermophilic lipases using support vector machines

Discriminating thermophilic lipases from their similar thermostable counterparts is a challenging task and it would help to design stable proteins. In this study, the distributions of N (N=2, 3) neighboring amino acids and the non-adjacent di-residue coupling patterns in the sequences of 65 thermostable and 77 thermophilic lipases had been systematically analyzed. It was found that the hydrophobic residues Leu, Pro, Met, Phe, Trp, as well as the polar residue Tyr had higher occurrence in thermophilic lipases than thermostable ones. The occurrence frequencies of KC EE KE RE, VE, YI, EK, VK, EV, YV, EY, KY, VY and YY in thermophilic proteins were significantly higher, while the occurrence frequencies of QC, QH, QN, HQ, MQ, NQ, QQ, TQ, QS and QT were significantly lower. CXP or CPX showed significantly positive to lipase thermostability, while XXQ or QXX showed significantly negative to lipase thermostability. Non-adjacent di-residue coupling patterns of PR14, RY32, YR47, LE53, LE64, PP64, RP70 and PP101 were significantly different in thermophilic lipases and their thermostable counterparts. The composition of dipeptide, tripeptide and non-adjacent di-residue patterns contained more information than amino acid composition. A statistical method based on support vector machines (SVMs) was developed for discriminating thermophilic and thermostable lipases. The accuracy of this method for the training dataset was 97.17?. Furthermore, the highest accuracy of the method for testing datasets was 98.41?. The influence of some specific patterns on lipase thermostability was also discussed.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design.

The thermoalkalophilic lipase from Bacillus thermocatenulatus BTL2 exhibits a low phospholipase activity (lecithin/tributyrin ratio 0.03). A single round of random mutagenesis of the BTL2 gene followed by screening of 6000 transformants on egg-yolk plates identified three variants with 10-12-fold increased phospholipase activities, corresponding to lecithin/tributyrin ratios of 0.16-0.36. All variants were specific for the sn-1 acyl ester bond of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Mutations occurred predominantly in the N-terminal part of BTL2 with regions surrounding the predicted helix alpha(4) and lid as hotspots. Two mutations, L184P located in the predicted helix alpha(4) and H15P found in the highly conserved oxy-anion hole motif among hydrolases, were identified to account for increased phospholipase activity. Two of the three variants showed reduced activities towards medium- and long-chain fatty acyl methyl esters compared to the wild-type enzyme. Substitution of Leu353 with Ser, which is located adjacent to the active site histidine and is important for phospholipase activity in the Staphylococcus hyicus lipase, increased the absolute phospholipase activities of the variants, but not of BTL2, approximately 2-fold. The engineered best variant displayed a lecithin/tributyrin ratio of 0.52, corresponding to a 17-fold increase compared to the wild-type enzyme. Moreover, this variant exhibited a 1.5-4-fold higher activity towards long-chain fatty acyl methyl ester (C18:1, C18:2, C18 and C20) compared to BTL2. A second round of mutagenesis and screening on lecithin-plates yielded no new variants with further increased phospholipase/lipase activity ratios, but instead one variant with a 5-fold increased expression rate and two variants with a 3-fold reduced activity towards triolein were obtained.     

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca²⁺-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter P_lac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.     

Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase

A thermophilic lipase (lipGRD) from Geobacillus sp. RD-2, isolated from a hot spring in Yunnan, China, was cloned and over-expressed in Escherichia coli. The function of the conserved residue, Tyr224, near the presumed temperature switch site was analyzed by site-directed saturation mutagenesis. The activity of the wild type lipGRD was optimal at 55??C and pH?7.5, but that from mutant Y224C was optimally active at 35??C, whereas Y224P lipase was optimally active at 65??C. Furthermore, the latter lipase retained 60% of its activity after incubation at 65??C for 5?h. The conserved residue Tyr224, which is close to the lid helix, is the key amino acid residue determining the thermostability of the thermostable lipase.     

Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved

Lip2 lipase from Yarrowia lipolytica is a very promising lipase with many potential applications (e.g. resolution of racemic mixtures, production of fine chemicals). Unfortunately this potential is impeded by a very low thermostability for temperatures higher than 40 °C. Error-prone PCR and screening of the library in a high-performance yeast expression system (Y. lipolytica) enabled a thermostable variant to be identified. This variant presents only one mutation, the free cysteine 244 is changed into an alanine. At 60 °C, the half-life time of the purified variant was 127-fold increased compared to the WT enzyme (from 1.5 min to 3 h). Saturation mutagenesis experiment at position 244 demonstrated that the presence of a cysteine at this position was responsible for the thermal denaturation. It was demonstrated that WT Lip2 and the thermostable variant are both inactivated through aggregation mechanisms, but that the kinetics and the nature of the aggregation were different. For the WT enzyme, rapid intermolecular disulphide bridge interchanges triggered by the free cysteine 244 mediates aggregation. For the variant C244A, aggregation still occurred but much slower than for the WT lipase and was mainly driven by hydrophobic forces.     

Engineering of Yarrowia lipolytica lipase Lip8p by circular permutation to alter substrate and temperature characteristics

Applications of lipases are mainly based on their catalytic efficiency and substrate specificity. In this study, circular permutation (CP), an unconventional protein engineering technique, was employed to acquire active mutants of Yarrowia lipolytica lipase Lip8p. A total of 21 mutant lipases exhibited significant shifts in substrate specificity. Cp128, the most active enzyme mutant, showed higher catalytic activity (14.5-fold) and higher affinity (4.6-fold) (decreased K _m) to p-nitrophenyl-myristate (pNP-C₁₄) than wild type (WT). Based on the three-dimensional (3D) structure model of the Lip8p, we found that most of the functional mutation occurred in the surface-exposed loop region in close proximity to the lid domain (S112–F122), which implies the steric effect of the lid on lipase activity and substrate specificity. The temperature properties of Cp128 were also investigated. In contrast to the optimal temperature of 45 °C for the WT enzyme, Cp128 exhibited the maximal activity at 37 °C. But it is noteworthy that there is no change in thermostability.     

Expression, purification, and aggregation studies of His-tagged thermoalkalophilic lipase from Bacillus thermocatenulatus

The His-tagged lipase BTL2 from Bacillus thermocatenulatus was expressed in Escherichia coli and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography. The success of protein separation and purification was pH-dependent and increased with decreasing pH. The purified BTL2 lipase showed a strong tendency to aggregate upon concentration, which prevented a reproducible crystallization. Aggregation studies using dynamic light-scattering (DLS) analysis were performed to improve the purification and concentration of BTL2 lipase. Different chemical classes of additives were tested to manipulate the aggregation behaviour of BTL2 lipase with the aim of obtaining a monodisperse sample to use for crystallization. For the process of concentration of BTL2 lipase in monomeric form, the alcohol 2-propanol and the ionic detergent dodecyl dimethylamine-N-oxide (LDAO) were found to be necessary. For the concentrated lipase, the availability of 5% 2-propanol was sufficient to hold the lipase in monomeric form and no additional detergent was needed.     

Molecular Dynamics of Thermoenzymes at High Temperature and Pressure: A Review

Lipases are known for their versatility in addition to their ability to digest fat. They can be used for the formulation of detergents, as food ingredients and as biocatalysts in many industrial processes. Because conventional enzymes are frangible at high temperatures, the replacement of conventional chemical routes with biochemical processes that utilize thermostable lipases is vital in the industrial setting. Recent theoretical studies on enzymes have provided numerous fundamental insights into the structures, folding mechanisms and stabilities of these proteins. The studies corroborate the experimental results and provide additional information regarding the structures that were determined experimentally. In this paper, we review the computational studies that have described how temperature affects the structure and dynamics of thermoenzymes, including the thermoalkalophilic L1 lipase derived from Bacillus stearothermophilus. We will also discuss the potential of using pressure for the analysis of the stability of thermoenzymes because high pressure is also important for the processing and preservation of foods.     

Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase

We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.     

Enhanced activity of an immobilized lipase promoted by site-directed chemical modification with polymers

The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site-directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or mono-carboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)-propionyl-dextran-NH₂ reduced the activity to 40%.The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase.     

A novel thermostable and organic solvent-tolerant lipase from <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> YB103: screening,purification and characterization

Thermostable lipases offer major biotechnological advantages over mesophilic lipases. In this study, an intracellular thermostable and organic solvent-tolerant lipase-producing strain YB103 was isolated from soil samples and identified taxonomically as Xanthomonas oryzae pv. oryzae. The lipase from X. oryzae pv. oryzae YB103 (LipXO) was purified 101.1-fold to homogeneity with a specific activity of 373.9 U/mg. The purified lipase showed excellent thermostability, exhibiting 51.1 % of its residual activity after incubation for 3 days at 70 °C. The enzyme showed optimal activity at 70 °C, suggesting it is a thermostable lipase. LipXO retained 75.1–154.1 % of its original activity after incubation in 20 % (v/v) hydrophobic organic solvents at 70 °C for 24 h. Furthermore, LipXO displayed excellent stereoselectivity (e.e._p >99 %) toward (S)-1-phenethyl alcohol in n-hexane. These unique properties of LipXO make it promising as a biocatalyst for industrial processes.     

Selection of effective and highly thermostable <Emphasis Type="Italic">Bacillus subtilis</Emphasis> lipase A template as an industrial biocatalyst-A modern computational approach

Biocatalysts are intrinsically reactive and hence their operational stability is of vital significance for any bioprocess. The setback in biocatalyst stability has been tackled from diverse prospects. Inherently, stable biocatalysts are markedly realized and a regular attempt is being made to seek out new organisms that harbor them. Here, we analyzed the industrial biocatalyst lipase A (Native) of Bacillus subtilis and its six thermostable mutants (2M, 3M, 4M, 6M, 9M and 12M) computationally using conformational sampling technique. Consequently, the various structural events deciphering thermostability like root mean square deviation, root mean square fluctuation, radius of gyration and polar surface area showed mutant 12M to be highly stable with statistical validation. Besides, static model analysis involving intra-molecular interactions, secondary structure, solvent accessibility, hydrogen bond pattern, simulated thermal denaturation and desolvation energy also supported 12M comparatively. Of note, the presence of high secondary structural rigidity and hydrogen bonds increased thermostability and functionality of 12M, thus selecting it as a best template for designing thermostable lipases in future. Also, this study has a significant implication toward a better understanding of conformational sampling in enzyme catalysis and enzyme engineering.