Yarrowia Lipolytica Lipase Lip2 Research Articles

Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

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.

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.

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.

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.

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

Abstract In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.

A novel oriented immobilized lipase on magnetic nanoparticles in reverse micelles system and its application in the enrichment of polyunsaturated fatty acids

A novel oriented immobilized lipase was derived from Yarrowia lipolytica lipase LIP2 covalently immobilized on functionalized Fe3O4 magnetic nanoparticles (MNPs) in reverse micelles system (RMS). The activity recovery reached 382% compared with 29% in aqueous phase, and further ran up to 1425% under optimum conditions. (3-Aminopropyl) triethoxysilane (APTES) coated Fe3O4 nanoparticles were characterized by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD). A significant alteration in the secondary structure of the lipase in RMS with a 15.5% increase of α-helix content and a 12.5% decrease of β-sheet content was detected by circular dichroism (CD). The immobilized lipase was employed to enrich polyunsaturated fatty acids in fish oil, a 90% increase of DHA content was obtained after 12h, and after 20 cycles of successive usage, it still remained over 80% of relative hydrolysis degree, which shows a good recyclability.

Enhanced catalysis of Yarrowia lipolytica lipase LIP2 immobilized on macroporous resin and its application in enrichment of polyunsaturated fatty acids

An immobilization strategy was employed to improve activity and operational stability of Yarrowia lipolytica lipase LIP2 (YlLIP2) by using macroporous resins as carrier. D152H, a cation-exchange resin, was the best support. Under the optimized conditions, the immobilization efficiency was 89.81% and the specific activity was 809,751U/g, being 2.1-fold higher than that of the free lipase. Bioimprinting and interfacial activation were used to further boost the catalytic activity of YlLIP2, respectively enhanced 21.5-fold, 231.2% and 107.2% compared to the free, non-bioimprinted and non-interfacial-activated lipases. The immobilized lipase exhibited much better thermal and pH stability and broader substrate specificity; when used to enrich docosahexaenoic acid (DHA) from Chlorella protothecoides oil, it could increase 1.66-fold of DHA content and show good operational stability. These indicate that the immobilized YlLIP2 offers a promising approach for the enrichment of DHA.

Improving the thermostability of lipase Lip2 from Yarrowia lipolytica

Yarrowia lipolytica lipase Lip2 (YlLip2) is a highly versatile biocatalyst. However, its practical use is often hampered by its low stability. Here three complementary protein engineering strategies were used to improve the thermostability of this enzyme. The first strategy was error-prone PCR based directed evolution, which resulted in a YlLip2 variant with a 2.5-fold longer half-life of thermal inactivation at 50°C compared to the wild-type enzyme. The second strategy was semi-rational design using the so-called B-factor iterative test (B-FIT), which led to the discovery of two thermostable YlLip2 variants that showed a half-life of thermal inactivation 2-fold and 5-fold longer than that of the wild-type enzyme, respectively, at 50°C. The third strategy was to use site-directed mutagenesis to combinatorially combine all three thermostabilizing mutations identified in the first two strategies, which improved the half-life of thermal inactivation of YlLip2 by 7-fold compared to that of the wild-type enzyme. Such engineered lipases provide not only new insights on the protein structure and function relationship but also potentially useful catalysts for practical applications.

Constitutive Expression of Yarrowia lipolytica Lipase LIP2 in Pichia pastoris Using GAP as Promoter

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315U/ml under the flask culture at 28°C, pH7.0, for 48h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300U/ml and 116g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500U/ml and 120g DCW/l. The total protein concentration in the supernatant reached 3.3g/l and the cell viability remained approximately 99% after 80h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.

Combined strategies for the improvement of heterologous expression of a His-tagged Yarrowia lipolytica lipase Lip2 in Pichia pastoris

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important biocatalyst for ester synthesis, biodiesel production and enantiomer resolution. The YlLip2 with an N-terminal histidine-tag (His 6-YlLip2) was successfully expressed in Pichia pastoris . Three different cultivation strategies had been compared for the production of His 6-YlLip2 by P. pastoris using a 10-l bioreactor. The results showed that His 6-YlLip2 activity and cell viability could be greatly improved by employing the combined strategies. Using a low salt medium (LSM) instead of the basal salt medium (BSM) and lowering the temperature from 28 to 25°C, the maximum His 6-YlLip2 activity and volumetric productivity were respectively increased by 55.3 and 79.8%. The production of His 6-YlLip2 and cell viability was further improved by combining sorbitol co-feeding with methanol. In this culture strategy, the maximum activity of His 6-YlLip2 reached 15,600 U/ml after 114 h of induction. The cell mortality decreased by 11.2% (while the control decreased about 27.6%) after 120 h methanol induction. The N-terminal histidine-tag brought convenience to purification. The molecular weight of His 6-YlLip2 was about 38 kDa. The pure His 6-YlLip2 presented a specific activity of 4,830 U/mg when olive oil was used as the substrate.

Production of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using the nitrogen source-regulated FLD1 promoter

BACKGROUND:Yarrowia lipolytica lipase LIP2 (YlLIP2) is an important industrial enzyme that has many potential applications. Although it has been successfully expressed in Pichia pastoris under the control of the AOX1 promoter (pAOX1), there have been many efforts to develop new alternative promoters to pAOX1 in order to avoid using methanol in the fermentation. Investigation of YlLIP2 production in P. pastoris using the formaldehyde dehydrogenase 1 promoter (pFLD1) is especially attractive, since little is known about its application in methanol-free culture strategies. RESULTS: Three fed-batch cultivations were performed to investigate the production of YlLIP2 in a pFLD1-based system. When methanol was used as the fed-batch feeding substrate, the maximum YlLIP2 activity obtained in a 10-L bioreactor was 30 000 U mL−1 after 143 h of culture, whereas the maximum YlLIP2 activity was further increased to 35 000 U mL−1 by adopting a co-induction strategy with methanol and methylamine as a mixed fed-batch substrate. Furthermore, the maximum YlLIP2 activity reached 13 000 U mL−1 after 80 h of cultivation in a methanol-free culture. CONCLUSION: The expression levels of YlLIP2 in the pFLD1-based system were comparable with those in a pAOX1-based system. The results suggest that pFLD1 is an attractive alternative to pAOX1, and may make it feasible to induce high yields of protein expression. Copyright © 2011 Society of Chemical Industry

Intracellular expression of Vitreoscilla hemoglobin improves production of Yarrowia lipolytica lipase LIP2 in a recombinant Pichia pastoris

The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb +) displayed higher biomass and YlLIP2 activity than control strains (VHb −). Compared with VHb − cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb + in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb − cells than in VHb + cells. The highest YlLIP2 activity of VHb + cells was approximately 1.84-fold higher than that of VHb − cells at lower DO levels. Moreover, the recombinant strain VHb + exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb − cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and industrial enzyme production.

Effect of high frequency currents on kinetics of catalyzed synthesis of ammonia

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 (420 U/g support) and the biocompatible gelatin modified support (600 U/g support) when compared to the support without modification (240 U/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 (1180 U/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.