Lipolytica Lipase Research Articles

Tolerance properties and growth performance assessment of Yarrowia lipolytic lipase in broilers

ABSTRACTLipases have attracted more interest for several biotechnological applications due to their unique properties, such as security, high stability at low pH values and substrate specificity. Nevertheless, no systematic studies on its effectiveness in corn-soybean when fed to broilers have been published. In this study, in vitro and in vivo assessment of a lipase from the yeast Yarrowia lipolytica for feed purpose was performed. Results showed that Y. lipolytica lipase (YLL) displayed the highest stability at low pH values when compared with other lipases from different sources. YLL stabilized with skimmed milk powder and starch maintained 55% and 48% of its initial activity after 2 h of incubation at pH 6 with bile salt and proteases, respectively. In addition, using broilers fed as substrate, YLL formulations displayed a higher hydrolysis degree after 6 h reaction in the presence of bile salt and digestive enzymes. Moreover, in vivo effect of YLL on growth performance in broilers was studied. From 1 to 42 d, it appears from this study that there is a significant (P <. 05) effect for YLL to improve feed conversion ratio. Consequently, YLL has a great potential for using as a feed supplement in the poultry industry.

Sugar-Improved Enzymatic Synthesis of Biodiesel with Yarrowia lipolytica Lipase 2

In this research, it was found that in Yarrowia lipolytica Lipase 2 (YLLIP2)-catalyzed biodiesel production, the content of fatty acid methyl esters (FAMEs) was increased by about 10% with the addition of d(+)-glucose. The result indicated that the d(+)-glucose could be used as an effective additive in YLLIP2-catalyzed biodiesel production. In accordance with the results above, the single factor experiments of key parameters in the process were first carried out. On the basis of the single factor experiment results, a five-factor, three-level response surface method was adopted to obtain the optimal reaction conditions: lipase dosage 40 IU/g oil, d(+)-glucose to lipase 1:1.05 (w/w), water content 1.95%, and reaction temperature 39.4 °C. A stoichiometric amount of methanol (11.5%, methanol/waste oil) was added in 6 times (every 4 h for each addition). A subsequent pilot scale production in a 5-ton reactor was carried out to check the performance of this method, and a good biodiesel content of 91.4% was obt...

Facile preparation of magnetic carbon nanotubes-immobilized lipase for highly efficient synthesis of 1,3-dioleoyl-2-palmitoylglycerol-rich human milk fat substitutes

In this study, Candida lipolytica lipase (CLL) was immobilized on magnetic multi-walled carbon nanotubes (mMWCNTs) via hydrophobic and cation-exchange interaction. The resultant immobilized CLL showed much better thermal stability, biocatalyst activity and easier recycling than the free form. A method for efficient enzymatic acidolysis of tripalmitin (PPP) with oleic acid (OA), to produce OPO-rich TAGs, was developed, using the immobilized CLL as the biocatalyst. Under optimized conditions (2% water, 20mg/ml enzyme, 1:6 PPP/OA, 50°C, 2h), the content of OPO in the final product reached 46.5%. CLL@mMWCNTs had a better activity and manipulative stability than commercial lipases. More importantly, the feasibility of CLL@mMWCNTs was also validated in the practical production of OPO-rich TAGs, using lard and restructured palm oil as the raw material. These results suggest that CLL@mMWCNTs is a promising biocatalyst for the OPO-rich TAGs production and will be helpful for the infant formula industry.

Immobilization of Y. lipolytica lipase and the continuous synthesis of geranyl propionate

In this study, Y. lipolytica lipase LIP2 (Lip2) was immobilized on the macroporous adsorptive resin DA201-C in n-heptane and then used to catalyze the continuous synthesis of geranyl propionate in a continuous stirred tank reactor (CSTR). With the lipase loading of 0.01g/g resin, 25°C and adsorption for 3h, the immobilization efficiency can be up to 98.6% and the synthetic activity of the lipase was 690.8U/g after immobilization, representing an increase of 28.5% compared with the free lipase (synthetic activity 537.2U/g). The immobilized Lip2 was used to generate geranyl propionate in CSTR, the continuous production of geranyl propionate was performed with 3g of immobilized Lip2 at 35°C. At the flow rate of 6mL/h the process can maintain its steady state at least for 12h, which was about three times of the average residence time, and the esterification ratio of 72.8% could be attained. Finally, FT-IR and NMR were used to identify the ester.

Enhancement of methanol resistance of Yarrowia lipolytica lipase 2 using β-cyclodextrin as an additive: Insights from experiments and molecular dynamics simulation.

The methanol resistance of lipase is a critical parameter in enzymatic biodiesel production. In the present work, the methanol resistance of Yarrowia lipolytica Lipase 2 (YLLIP2) was significantly improved using β-cyclodextrin (β-CD) as an additive. According to the results, YLLIP2 with β-CD exhibited approximately 7000U/mg specific activity in 30wt% methanol for 60min compared with no activity without β-CD under the same conditions. Molecular dynamics (MD) simulation results indicated that the β-CD molecules weakened the conformational change of YLLIP2 and maintained a semi-open state of the lid by overcoming the interference caused by methanol molecules. Furthermore, the β-CD molecule could directly stabilize "pathway" regions (e.g., Asp61-Asp67) and indirectly stabilize "pathway" regions (e.g., Gly44-Phe50) by forming hydrogen bonds with "pathway" regions and nearby "pathway" regions, respectively. The regions stabilized by the β-CD molecule then prevented the closure of active pockets, thus retaining the enzymatic activity of YLLIP2 with β-CD in methanol solvent.

Novel enzyme/exfoliated bentonite nanohybrids as highly efficient and recyclable biocatalysts in hydrolytic reaction

Bentonite exfoliation liberated the interlayer surface with large area for immobilization of enzyme at high enzyme loading. Upon the electrostatic attraction between positively charged bovine pancreatic lipase (BPL), Yarrawia lipolytica lipase (YLL), trypsin and negatively charged surface of unilaminar bentonite, three nanohybrids with a stably card-like structure named as BPL-B, YLL-B and TB were facilely produced. These nanohybrids showed higher activities in hydrolysis as 115.2% (BPL-B), 154.8% (YLL-B) and 138.2% (TB) of that of the correspondingly free enzyme. Furthermore, BPL-B, YLL-B and TB retained 80%, 90% and 82% of each original activity after repeated use for 10, 12 and 8 times, respectively. The recyclable and active nanohybrids are expected to be widely applied as industrial catalysts in the future.

Immobilization of Yarrowia lipolytica lipase Ylip2 for the biocatalytic synthesis of phytosterol ester in a water activity controlled reactor

In this work, phytosterol ester was synthesized using Yarrowia lipolytica lipase Ylip2 that had been immobilized on inorganic support in a solvent-free system and reacted in a computer-aided water activity controlled bioreactor. The immobilization of Ylip2 on celite led to a remarkable increase in the phytosterol conversion compared to that of free lipase. An investigation of the reaction conditions were oleic acid as the fatty acid variety, 10,000U/g substrate, and a temperature of 50°C for phytosterol ester synthesis. Controlling of the water activity at a set point was accomplished by the introduction of dry air through the reaction medium at a digital feedback controlled flow rate. For the esterification of phytosterol ester, a low (15%) water activity resulted in a considerable improvement in phytosterol conversion (91.1%) as well as a decreased reaction time (78h). Furthermore, Ylip2 lipase immobilized on celite retained 90% esterification activity for the synthesis of phytosterol oleate after reused 8 cycles, while free lipase was only viable for 5 batches with 90% esterification activity remained. Finally, the phytosterol oleate space time yield increased from 1.65g/L/h with free lipase to 2.53g/L/h with immobilized lipase. These results illustrate that the immobilized Yarrowia lipolytica lipase Ylip2 in a water activity controlled reactor has great potential for the application in phytosterol esters synthesis.

Behavior of Yarrowia lipolytica Lipase Lip2 under high hydrostatic pressure: Conformational changes and isokineticity diagram

Despite the large and increasing interest in Yarrowia lipolytica Lipase Lip2 (Lip2) for biotransformation in food processing, the effect of conformational changes and isokineticity diagram on Lip2’s behaviour still remains unclear. In this paper, the catalytic ability of Lip2 under high hydrostatic pressure was investigated. After treated at 450MPa and 40°C for 10min, the maximum enzyme activity was obtained, which was 209% of that at atmospheric pressure. Changes of the secondary and tertiary structure of Lip2 were characterized using circular dichroism and fluorescence spectra. The isokineticity diagram for the combined effects of pressure-temperature on denaturation of enzyme was established. The elliptical shape of the diagram was close to that deduced from thermodynamics, indicating the stabilization of Lip2 against thermal denaturation. These results showed that pressure has a remarkable effect on the stabilization of Lip2.

Yarrowia lipolytica Lipase 2 Is Stable and Highly Active in Test Meals and Increases Fat Absorption in an Animal Model of Pancreatic Exocrine Insufficiency

Pancreatic exocrine insufficiency (PEI) reduces pancreatic secretion of digestive enzymes, including lipases. Oral pancreatic enzyme replacement therapy (PERT) with pancreatin produces unsatisfactory results. The lipase 2 produced by the yeast Yarrowia lipolytica (YLLIP2; GenBank: AJ012632) might be used in PERT. We investigated its ability to digest triglycerides in a test meal and its efficacy in reducing fecal fat in an animal model of PEI. YLLIP2 was produced by genetically engineered Y lipolytica and purified from culture media. YLLIP2 or other gastric (LIPF) and pancreatic (PNLIPD) lipases were added to a meal paste containing dietary triglycerides, at a range of pH values (pH 2-7), with and without pepsin or human bile and incubated at 37°C. We collected samples at various time points and measured lipase activities and stabilities. To create an animal model of PEI, steatorrhea was induced by embolization of the exocrine pancreas gland and pancreatic duct ligation in minipigs. The animals were given YLLIP2 (1, 4, 8, 40, or 80 mg/d) or pancreatin (100,000 US Pharmacopeia lipase units/d, controls) for 9 days. We then collected stool samples, measured fat levels, and calculated coefficient of fat absorption (CFA) values. YLLIP2 was highly stable and poorly degraded by pepsin, and had the highest activity of all lipases tested on meal triglyceride at pH 4-7 (pH 6 with bile: 94 ± 34 U/mg; pH 4 without bile: 43 ± 13 U/mg). Only gastric lipase was active and stable at pH 3, whereas YLLIP2 was sensitive to pepsin hydrolysis after pH inactivation. From invitro test meal experiments, the lipase activity of YLLIP2 (10 mg) was estimated to be equivalent to that of pancreatin (1200 mg; 100,000 US Pharmacopeia units) at pH 6. In PEI minipigs, CFA values increased from 60.1% ± 9.3% before surgery to 90.5% ± 3.2% after administration of 1200 mg pancreatin (P < .05); CFA values increased to a range of 84.6% ± 3.0% to 90.0% ± 3.8% after administration of 4-80 mg YLLIP2 (P < .05). The yeast lipase YLLIP2 is stable and has high levels of activity against test meal triglycerides in a large pH range, with and without bile. Oral administration of milligram amounts of YLLIP2 significantly increased CFA values, similar to that of 1.2 g pancreatin, in a minipig model of PEI.

Immobilizing Yarrowia lipolytica Lipase Lip2 via Improvement of Microspheres by Gelatin Modification.

The purpose of this study was to investigate the feasibility of immobilizing Yarrowia lipolytica lipase lip2 on epoxy microspheres with or without gelatin modifications. The activity of lipase immobilized on gelatin-modified supports was twofold higher than those immobilized on native supports. There was no significant difference in the Michaelis-Menten constant (K M ) between the two immobilized lipases. However, lipase immobilized on gelatin modified supports showed an approximately fourfold higher V max than lipase immobilized on native supports. Lipase immobilization on the gelatin-modified support exhibited a significantly improved operational stability in an esterification system. After it was reused for a total of 35 batches, the ester conversion of lipase immobilized on gelatin-modified and native microspheres was 83 and 60 %, respectively. Furthermore, the immobilized lipase could be stored at 4 °C for 12 months without any loss of activity.

Probing role of key residues in the divergent evolution of Yarrowia lipolytica lipase 2 and Aspergillus niger eruloyl esterase A

Yarrowia lipolytica lipase 2 (YLLip2) and Aspergillus niger feruloyl esterase A (AnFaeA) are enzymes of similar structures but with different functions. They are both classified into the same homologous family in Lipase Engineering Database (LED). The major difference between the two enzymes is that YLLip2 exhibits interfacial activity while AnFaeA does not. In order to better understand the interfacial activation mechanisms of YLLip2, structure guided site-directed mutagenesis were performed, mutants were constructed, kinetics parameters and lipase properties were detected. Mutant enzymes showed enhanced catalytic efficiency towards p-nitrophenyl butyrin (pNPB) but their catalytic efficiency decreased towards p-nitrophenyl palmitate (pNPP), their catalysis behavior was more close to feruloyl esterase. Moreover, the mutant enzymes exhibited enhanced thermostability compared with their wild type. These results indicate that I100 and F129 are probably cut-off point of divergent functions between the two enzymes during evolution.

Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity

We have developed an improved and effective method to immobilize Yarrowia lipolytica lipase Lip2 (YLIP2) on an epoxy poly-(glycidylmethacrylate-triallyisocyanurate-ethyleneglycoldimethacrylate) (PGMA-TAIC-EGDMA) support structure with or without amine or/and protein modifications. Our results show that there is an increase in the activity of the immobilized lipase on n-butylamine (BA) modified support (420U/g support) and the biocompatible gelatin modified support (600U/g support) when compared to the support without modification (240U/g support). To further study the influences of BA and gelatin modification on the activity of the immobilized lipase, gelatin and BA were concurrently used to decorate the support structure. Lipase immobilized on 2% BA/gelatin (1:1) modified support obtained the highest activity (1180U/g support), which was five-fold higher than that on a native support structure. These results suggest that the activity of a support-immobilized lipase depends on the support surface properties and a moderate support surface micro-environment was crucial for elevated activity. Collectively, these data show that a combined gelatin and BA modification regulates the support surface more suitable for immobilizing YLIP2.

Cell surface display of Yarrowia lipolytica lipase Lip2p using the cell wall protein YlPir1p, its characterization, and application as a whole-cell biocatalyst.

The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 10(5) molecules per cell, which is close to the theoretical maximum (2 × 10(6) molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively.

Characterization of chain dimensions of poly(ε-caprolactone) diols in THF by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)

In this work, we studied the chain dimensions, shape, and thermodynamic characteristics of poly(e-caprolactone) diols (HO-PCL-OH) in solution. PCL diol samples of different molecular weights and architectures were synthesized using immobilized Yarrowia lipolytica lipase as catalyst. Gel permeation chromatography (GPC) with online right-angle laser-light scattering (RALLS), differential viscometer (DV), and interferometric refractometer IRS detectors offered a proper way to obtain information on thermodynamic characteristics and chain flexibility. The weight-average molecular weights of the PCL diol samples (M w ) ranged from 2,750 to 13,120 uma. The z-average radius of hydrodynamic volume (<R h 2 > z 1/2 ) vs. z-average molecular weight curve (M z ) could be satisfactorily fitted to a power-law equation. Mark–Houwink–Sakurada parameters (K = 2.74 × 10−3 dL/g and a = 0.64) were derived from the plots of [η] w against M w . The overall results clearly suggest that a flexible geometry is present in tetrahydrofuran (THF) solution of PCL at 33 °C. Unlike other PCL systems, PCL diols are more hydrophilic and expected to form associated species. They behave differently from common hydrophobic polymers and do not strictly conform to thermodynamic relationships generally used in Polymer Science. A more compact geometry is present for Mn lower than 4,000 Da. For higher molecular weights, the chain expands and become more elongated. Plots of ln [η] w against ln M w (MHS equation) and of log R hz against log M z suggest that PCL diols behave as flexible chains in a good solvent.

Improving performance of Yarrowia lipolytica lipase lip2-catalyzed kinetic resolution of (R, S)-1-phenylethanol by solvent engineering

Extracellular Yarrowia lipolytica lipase Lip2 (YLIP2) demonstrated an (R)-enantiopreference for efficient resolution of (R,S)-1-phenylethanol by solvent engineering with different kinds of binary solvent. The enantioselectivity was significantly improved by the addition of 1, 4-dioxane. The reaction parameters including co-solvent concentration, reaction temperature, and the reaction time were optimized. When the reaction was carried out with n-hexane in the presence of 0.8% 1,4-dioxane at 50°C for 72 h, the enantiomeric excess of product markedly increased to 99.1% from 66% in pure n-hexane; the enantiomeric ratio was higher than 200, which was 500-fold compared with that in pure n-hexane. The results indicated that it is very important to design the proper co-solvents, especially to create appropriate micro-environment for YLIP2 for catalyzing the resolution of (R,S)-1-phenylethanol.

Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris

Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (PAOX1 and PFLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter PAOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262g/L, 38,500U/mL and 2.82g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field.

Assortment of carbon sources in medium for Yarrowia lipolytica lipase production: A statistical approach.

Glycerol is considered an important renewable feedstock as well as an undesirable side-product of biodiesel production. The aim of this study was to determine whether supplementing a culture medium with a combination of three different carbon sources (olive oil, glucose and glycerol) would optimize lipase production by the yeast Yarrowia lipolytica. The optimization experiments were conducted with a statistical approach using the mixture design. Analysis of the response surface revealed that it would be possible to compose a medium in which both an an extracellular lipase activity of 0.1 U/mL and up to 37.5 g/L of pure glycerol could be obtained. An YPO-Gl30 medium consisting of 30 g/L glycerol and 19.2 mL/L olive oil was selected for further investigation. Although a high biomass yield was found in all cultures, the glycerol content of the YPO-Gl30 medium slightly influenced yeast growth, but it did not prolong the duration of the lag phase. The hydrolytic activity of the extracellular lipases produced in YPO-Gl30 medium was satisfactory.

De Novo Design and High-throughput Screening Strategy Achieved Over Expression of Yarrowia lipolytica Lipase YLL in Pichia patoris

De Novo Design and High-throughput Screening Strategy Achieved Over Expression of Yarrowia lipolytica Lipase YLL in Pichia patoris

Efficient Kinetic Resolution of (R,S)-2-Octanol Catalyzed by Magnetite-Immobilized Yarrowia lipolytica Lipase in Mixed Ionic Liquids

The resolution of (R,S)-2-octanol catalyzed by magnetite-immobilized Yarrowia lipolytica lipase in ionic liquids was investigated. The optimal activity and enantioselectivity of the lipase were obtained in a mixture of [EMIM]BF4 and [OMIM]BF4 (v/v = 5:5). Excellent reusability of the enzyme-ionic liquid system was observed in consecutive batch reactions.

Lid closure mechanism of Yarrowia lipolytica lipase in methanol investigated by molecular dynamics simulation.

In nonaqueous organic solvents, lipases can catalyze esterification reactions, which increase their application value. Yarrowia lipolytica Lipase (Lip2) possesses potential values in medicine and industrial production. In order to investigate its lid closure mechanism in methanol we performed molecular dynamics (MD) simulations of the open conformation of Lip2 in methanol and hexane, respectively. Simulation results indicated that Lip2 undergoes a greater conformational change in methanol. Principle component analysis showed Lip2 has "double-domain" and "torsion" motion modes in hexane and methanol. By analyzing B-factor and dynamical cross-correlation, region Ser274-Asn288, region Thr106-His126, and region Asp61-Asp67 were found to interact with the lid region (Thr88-Leu105). Furthermore, local restricted MD simulations showed that closure mechanism of Lip2 is "double-lid movement" which is also observed in Pseudomonas aeruginosa Lipase (PAL), and we detected two interaction propagation pathways in Lip2 driven by the interaction between Ser274-Asn288 and methanol.