Substrate Specificity Of Lipase Research Articles

Effect of seven baking lipases on the lipid class composition of three different cakes

AbstractLipases are effective clean-label improvers for the baking quality of cake. Insights into lipase activities in different cake formulations in combination with the effect on batter/dough and baking quality are needed to further reveal the underlying mechanisms. Therefore, a method using normal phase high-performance liquid chromatography coupled to an evaporative light scattering detector was adapted and validated for the five most abundant lipid classes in three common cake recipes, namely triacylglycerols, diacylglycerols, monoacylglycerols, glycerophosphocholine and lysoglycerophosphocholine. The method revealed total changes of 0.2 mg/g to 186.5 mg/g in lipid class content per dry weight after lipase treatments. Comparative investigations on batter/dough and products of basic cake, pound cake and brioche without or with addition of seven lipases showed that the substrate specificity of lipases is the decisive factor for their effectiveness regarding improved dough and product quality. A high lipase activity only supported already matching substrate specificities. Graphical abstract

Investigations of substrate specificity of lipase and esterase from Triticum aestivum and their Michaelis-Menten kinetics

Enzymatic screening is screening an enzyme library to identify those that possess enzymatic activity towards defined substrates. In this work we screened a lipase and an esterase from Triticum aestivum – most significant cereal crop. The study aimed to investigate whether long-chain or short-chain fatty acids have higher processing rate by esterase and lipase from Triticum aestivum. In the screening, pH values were varied. Moreover, as substrates, several esters with varying length of fatty acids were used. The lipase and esterase showed higher activity towards substrates with a longer carbon chain. Enzyme activity was measured photometrically by using para-nitrophenol. Inhibitory properties of diisopropylfluorophosphate in relation to the esterase were tested. It was found that diisopropylfluorophosphate acts as a non-competitive inhibitor. pH optimum of pancreatin was determined, the value of which is 7.

Lipidomic insights into the reaction of baking lipases in cakes.

Lipases are promising improvers of cake batter and baking properties. Their suitability for use in various cake formulations cannot be predicted yet, because the reactions that lead to macroscopic effects need to be unravelled. Therefore, the lipidome of three different cake recipes with and without lipase treatment was assessed by ultra high performance liquid chromatography-mass spectrometry before and after baking. By comparing the reaction patterns of seven different lipases in the recipes with known effects on texture, we show that lipase substrate specificity impacts baking quality. Key reactions for the recipes were identified with the help of principal component analysis. In the eggless basic cake, glyceroglycolipids are causal for baking improvement. In pound cake, lysoglycerophospholipids were linked to textural effects. Lipase substrate specificity was shown to be dependent on the recipe. Further research is needed to understand how recipes can be adjusted to achieve optimal lipase substrate specificity for desirable batter and baking properties.

Enhancing the Hydrolytic Activity of a Lipase towards Larger Triglycerides through Lid Domain Engineering.

Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or LipMRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (LipMRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation.

Main Structural Targets for Engineering Lipase Substrate Specificity

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.

Lipase Catalysis in Presence of Nonionic Surfactants.

Lipase can catalyze varieties of reactions at the interface of aqueous and organic phase. Among various alternatives to modify catalytic performance of lipase, the addition of surfactants, particularly nonionic surfactants, has been widely studied. Low concentrations of nonionic surfactants augment lipase catalysis; on increasing surfactant concentration, often the catalytic performance decreases. Mole ratio of water to (nonionic) surfactant also has a profound effect on lipase activity. Catalytic abilities of some lipases are either enhanced or reduced in the presence of all nonionic surfactants of the same type, whereas for some other lipases, nonionic surfactants of the same type have mixed effect. Nonionic surfactant even changes substrate specificity of lipase. Water-in-ionic liquid microemulsion involving nonionic surfactant often performs better than other systems in improving catalytic ability of lipase. Tween and Triton surfactants often enhance enantiomeric separation catalyzed by lipase. Nonionic surfactants significantly affect activities of immobilized lipase, being present either as a component during immobilization or as a component in reaction medium. Lipases coated with nonionic surfactants act better than reverse micelles and microemulsions containing lipase. Thus, nonionic surfactants help lipase catalyzed processes in various media to enhance production of useful compounds like flavor ester, structured lipids, optically pure compounds, and noncrystalline polymers.

Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

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.

Monobody-Mediated Alteration of Lipase Substrate Specificity.

Controlling the catalytic properties of enzymes remain an important challenge in chemistry and biotechnology. We have recently established a strategy for altering enzyme specificity in which the addition of proxy monobodies, synthetic binding proteins, modulates the specificity of an otherwise unmodified enzyme. Here, in order to examine its broader applicability, we employed the strategy on Candida rugosa lipase 1 (CRL1), an enzyme with a tunnel-like substrate binding site. We successfully identified proxy monobodies that restricted the substrate specificity of CRL1 toward short-chain fatty acids. The successes with this enzyme system and a β-galactosidase used in the previous work suggest that our strategy can be applied to diverse enzymes with distinct architectures of substrate binding sites.

Purification and biochemical characterization of a halotolerant Staphylococcus sp. extracellular lipase

We have isolated a lipolytic halotolerant bacterium, designated as CJ3, that was identified as a Staphylococcus sp. Culture conditions were optimized and the highest extracellular lipase production amounting to 5U/ml was achieved after 24h of cultivation. The extracellular lipase was purified 24-fold by ammonium sulfate precipitation and a Sephacryl S-200 chromatography, and its molecular mass was found to be around 38kDa, as revealed by SDS-PAGE and gel filtration. The lipase substrate specificity was investigated using short (tributyrin) and long (olive oil) chain triglyceride substrates. The lipase was inhibited by submicellar concentrations of Triton X-100, and maximum specific activities were found to be 802U/mg on tributyrin and 260U/mg on olive oil at pH 8.0 and 45°C. The lipase was fairly stable in the pH range from 6.0 to 9.0, and about 69% of its activity was retained after incubation at 45°C for 60min. The enzyme showed a high tolerance to a wide range of salt concentration and a good stability in organic solvents, especially in long-chain alcohols.

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade)

While invertebrates store neutral lipids as their major energy source, little is known about triacylglycerol (TAG) class composition and their differential catabolism in aquatic arthropods. This study focuses on the composition of the main energy source and its catabolism by lipase from the midgut gland (hepatopancreas) of the crustacean Macrobrachium borellii. Silver-ion thin-layer chromatography of prawn large TAG deposit (80% of total lipids) and its subsequent fatty acid analysis by gas chromatography allowed the identification of 4 major fractions. These are composed of fatty acids of decreasing unsaturation and carbon chain length, the predominant being 18:1n-9. Fraction I, the most unsaturated one, contained mainly 20:5n-3; fraction II 18:2n-6; fraction III 18:1n-9 while the most saturated fraction contained mostly 16:0. Hepatopancreas main lipase (Mr 72 kDa) cross-reacted with polyclonal antibodies against insect lipase, was not dependent on the presence of Ca(2+) and had an optimum activity at 40°C and pH 8.0. Kinetic analysis showed a Michaelis-Menten behavior. A substrate competition assay evidenced lipase specificity following the order: 18:1n-9-TAG>PUFA-enriched-TAG>16:0-TAG different from that in vertebrates. These data indicate there is a reasonable correspondence between the fatty acid composition of TAG and the substrate specificity of lipase, which may be an important factor in determining which fatty acids are mobilized during lipolysis for oxidation in crustaceans.

Substrate specificity of lipase from Burkholderia cepacia in the synthesis of 3′-arylaliphatic acid esters of floxuridine

Among the five vinyl esters bearing phenyl ring in acyl moiety, the lipase from Burkholderia cepacia was most specific toward vinyl 4-phenylbutyrate. Additionally, 3′-regioselectivity of the enzyme was enhanced with the increment of acyl chain length. The lipophilic 3′-arylaliphatic acid esters of floxuridine, its potential prodrugs, were synthesized with high conversions (>98%) and good to excellent regioselectivities (85–99%) via the enzymatic approach.

Substrate specificity of lipases in alkoxycarbonylation reaction: QSAR model development and experimental validation

Although lipases are known to catalyze alkoxycarbonylation reactions in organic solvents, the existing knowledge base on their substrate specificity in alkoxycarbonylation reaction is sparse. Moreover, models to predict substrate specificity have not been reported. Here, we report the experimentally measured rate constants for 180 acyl donor-alcohol pairs and demonstrate the two-step synthesis of over 70 disubstituted carbonate products from simple precursors such as diphenyl carbonate and alcohols. The efficiency of synthesis was found to be dependent on the order of alcohol addition. This motivated the need to develop a model to predict lipase specificity in alkoxycarbonylation reactions. A partial least square model has been developed to correlate the reaction rate with (i) descriptors of alcohol for a fixed acyl donor, (ii) descriptors of acyl donor for a fixed alcohol, (iii) descriptors of both the acyl donor and the alcohol. The number of descriptors being far greater than the number of observations was a potential limitation in the model development. This was addressed by selecting a subset of descriptors using a systematic procedure based on (a) correlation among the descriptors and step-wise regression methodology, and (b) variable influence on projection methodology. The model was able to accurately predict the reaction rate and the optimal order of addition of alcohols in the two-step synthesis of disubstituted carbonates using the enzyme mixture. The descriptor subset and the relevant model would benefit the users of lipases in synthetic applications while the modeling strategy presented here can have applications in predicting specificity of other enzymes.

有機酸の選択的分離が可能なリパーゼ‐オルガノゲル膜システムの開発

We have developed a lipase-facilitated organogel membrane for selective separation of organic acids. We previously demonstrated that lipase-catalyzed reactions drove transport of organic acids through a supported liquid membrane. In this study, a lipase-facilitated liquid membrane was gelled with 12-hydroxystearic acid to stabilize the liquid membrane phase. The lipase-facilitated organogel membrane exhibited the improved stability of the liquid membrane phase compared with a non-gelled supported liquid membrane, although the permeate flux of an organic acid through the organogel membrane was less than that through the non-gelled liquid membrane. The lipase-facilitated organogel membrane demonstrated the selective separation of 3-phenoxypropionic acid from various organic acids due to the substrate specificity of lipases. Enantioselective separation of (S) -ibuprofen from racemic ibuprofen was also achieved owing to the enantioselectivity of lipase.

Transport of organic acids through a supported liquid membrane driven by lipase-catalyzed reactions

We have developed a lipase-facilitated supported liquid membrane. Lipase-catalyzed reactions were coupled with a supported liquid membrane (SLM) to transport organic acids through the SLM. We succeeded in the rational transport of organic acids through the SLM using lipase-catalyzed reactions and observed that there were differences in the transport behavior of various organic acids due to the substrate specificity of lipase. Subsequently, various parameters, such as the alcohol concentration in the feed phase, the pH in each aqueous phase, an organic solvent in the SLM, and the kind of lipase, were investigated. We found that the optimum conditions were 65 vol% alcohol concentration, pH 6.3 in each aqueous phase, isooctane as the liquid membrane phase and Candida rugosa lipase as the esterification biocatalyst.

Transport of Organic Acids through a Supported Liquid Membrane Driven by Lipase-Catalyzed Reactions

We have developed a lipase-facilitated supported liquid membrane. Lipase-catalyzed reactions were coupled with a supported liquid membrane (SLM) to transport organic acids through the SLM. We succeeded in the rational transport of organic acids through the SLM using lipase-catalyzed reactions and observed that there were differences in the transport behavior of various organic acids due to the substrate specificity of lipase. Subsequently, various parameters, such as the alcohol concentration in the feed phase, the pH in each aqueous phase, an organic solvent in the SLM, and the kind of lipase, were investigated. We found that the optimum conditions were 65 vol% alcohol concentration, pH 6.3 in each aqueous phase, isooctane as the liquid membrane phase andCandida rugosa lipase as the esterification biocatalyst.

Characterisation of pancreatic lipase substrate specificity in organic reaction media by a kinetic method

The possibility of assaying porcine pancreatic lipase specificity in organic media by kinetic modelling of esterification and transesterification reactions has been investigated. The calculated rate constant of the two-substrate reversible model was used as to estimate the chain length specificity in esterification reactions of fatty acids. This simple method is suitable to quantify the specificity in given reaction conditions, needing one single measurement for each substrate.

Substrate specificity of lipase B from Candida antarctica in the synthesis of arylaliphatic glycolipids

Arylaliphatic glycolipids are known for their pharmaceutical and medicinal properties. We found that a great variety of arylaliphatic esters can be synthesized from non-activated substrates like glucose or the natural occurring drug salicin using lipase B from Candida antarctica (CAL-B). However, esters based on aromatic carboxylic acids or unsaturated arylaliphatic acids, like cinnamic acid and its derivatives, which are known to display anticancer activity, could not be obtained. In this work, we performed computer-aided molecular modeling based on data of our work published recently and syntheses of new glycolipids to understand why some substances are accepted by CAL-B while some are not. For this purpose, we investigated the accessibility of the lipase binding site for the arylaliphatic acyl donors as well as the steric interactions between the aglycons of glucosides and the residues of the alcohol binding pocket in order to elucidate potentials and limitations of CAL-B for the synthesis of aromatic glycolipids.

Substrate Specificity of Lipases in Protease Preparations

Commercial protease preparations have been screened for their biocatalytic activity in the esterification of various fatty acids with 1-butanol. Of all protease preparations tested, only those from pineapple (bromelain) and Rhizopus sp. were found to contain active lipases. Similar to lipases from microorganisms, animals, and plants, such as papaya (Carica papaya) latex, lipases in the protease preparations of bromelain and Rhizopus sp. strongly discriminated against fatty acids having a cis-4 unsaturation, for example, all-cis-4,7,10,13,16,19-docosahexaenoic acid, cis-6 unsaturation, for example, petroselinic (cis-6-octadecenoic), γ-linolenic (all-cis-6,9,12-octadecatrienoic), and stearidonic (all-cis-6,9,12,15-octadecatetraenoic) acid, as well as cis-8 unsaturation, for example, dihomo-γ-linolenic (all-cis-8,11,14-eicosatrienoic) acid. Fatty acids having a cis-9 unsaturation, for example, oleic (cis-9-octadecenoic) and α-linolenic (all-cis-9,12,15-octadecatrienoic) acids, were very well accepted as substrates by both protease preparations. Fatty acids having hydroxy groups, for example, ricinoleic (12-hydroxy-cis-9-octadecenoic) and 12-hydroxystearic acid, epoxy groups, for example, trans-9,10-epoxystearic acid, and cyclopentenyl groups, for example, hydnocarpic [(11-(2‘-cyclopentenyl)undecanoic] and chaulmoogric [13-(2‘-cyclopentenyl)tridecanoic] acid, were also well accepted as substrates by both enzyme preparations. Keywords: Protease; lipase, biocatalyst; enzymatic esterification; fatty acid specificity

Human Hepatic and Lipoprotein Lipase: The Loop Covering the Catalytic Site Mediates Lipase Substrate Specificity

Hepatic lipase (HL) and lipoprotein lipase (LPL) are key enzymes that mediate the hydrolysis of triglycerides (TG) and phospholipids (PL) present in circulating plasma lipoproteins. Relative to triacylglycerol hydrolysis, HL displays higher phospholipase activity than LPL. The structural basis for this difference in substrate specificity has not been definitively established. We recently demonstrated that the 22-amino acid loops ("lids") covering the catalytic sites of LPL and HL are critical for the interaction with lipid substrate (Dugi, K.A., Dichek, H.L., Talley, G.D., Brewer, H.B., Jr., and Santamarina-Fojo, S. (1992) J. Biol. Chem. 267, 25086-25091). To determine whether the lipase lid plays a role in conferring the different substrate specificities of HL and LPL, we have generated four chimeric lipases. Characterization of these chimeric enzymes using TG (triolein and tributyrin) or PL (dioleoylphosphatidylcholine (DOPC) vesicles, DOPC proteoliposomes, and DOPC-mixed liposomes) substrates demonstrated marked differences between their relative PL/TG hydrolyzing activities. Chimeric LPL containing the lid of HL had reduced triolein hydrolyzing activity (49% of the wild type), but increased phospholipase activity in DOPC vesicle, DOPC proteoliposome, and DOPC-mixed liposome assay systems (443, 628, and 327% of wild-type LPL, respectively). In contrast, chimeric HL containing the LPL lid was more active against triolein (123% of the wild type) and less active against DOPC (23, 0, and 30%, respectively) than normal HL. Similar results were obtained when the lipase lids were exchanged in chimeric enzymes containing the NH2-terminal end of LPL and the COOH-terminal domain of HL. Exchange of the LPL and HL lids resulted in a reversal of the phospholipase/neutral lipase ratio, establishing the important role of this region in mediating substrate specificity. In summary, the lid covering the catalytic domains in LPL and HL plays a crucial role in determining lipase substrate specificity. The lid of LPL confers preferential triglyceride hydrolysis, whereas the lid of HL augments phospholipase activity. This study provides new insight into the structural basis for the observed in vivo differences in LPL and HL function.

Protein Engineering on Lipases

Since the first reports on X-ray crystallographic determination of the 3 D structure of two lipases in 1990, 3 D structures of many other lipases and their complexes with inhibitors have been clarified to elucidate the lipase reaction mechanism. An approach through protein engineering has also been playing an important role in such studies. Recent development of lipase applications in industry have created demand for new lipases having superior and specific functions, leading to the improvement or alteration of lipase functions by a tool of protein engineering. In this article, recent progress in protein engineering on lipases is reviewed, focusing on four wellstudied families of lipase, i.e.. pancreatic lipase, Rhizomucor miehei lipase from filamentous fungi, Geotrichum candidum lipase and lipases from Pseudomonas sp. An understanding of the molecular basis for the broad substrate specificity of lipases as well as the reaction mechanisms may lead to the design and manufacture of protein-engineered lipases having improved properties for industrial use.