Search

This study harnesses glutamate decarboxylase (GAD) from Yarrowia lipolytica to improve the biosynthesis of γ-aminobutyric acid (GABA), focusing on boosting the enzyme's catalytic efficiency and stability by immobilizing it on nanofibrous membranes. Through recombinant DNA techniques, two GAD genes, YlGAD1 and YlGAD2, were cloned from Yarrowia lipolytica and then expressed in Escherichia coli. Compared to their soluble forms, the immobilized enzymes exhibited significant improvements in thermal and pH stability and increased resistance to chemical denaturants. The immobilization notably enhanced substrate affinity, as evidenced by reduced K m values and increased k cat values, indicating heightened catalytic efficiency. Additionally, the immobilized YlGAD1 and YlGAD2 enzymes showed substantial reusability, maintaining 50% and 40% of their activity, respectively, after six consecutive cycles. These results underscore the feasibility of employing immobilized YlGAD enzymes for cost-effective and environmentally sustainable GABA production. This investigation not only affirms the utility of YlGADs in GABA synthesis but also underscores the advantages of enzyme immobilization in industrial settings, paving the way for scalable biotechnological processes., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Structured lipids (SL) represent a new generation of lipids, considered bioactive compounds. Medium-chain, oleic (18:1n-9), and medium-chain fatty acid (MCFA) structured lipids (MOM-SL) were produced by acidolysis reaction in solvent-free medium with capric (10:0) and lauric (12:0) free fatty acids (FFAs) and triolein or olive oil, using Yarrowia lipolytica lipase as biocatalyst. MCFAs were rapidly incorporated into sn-1,3 SL in acidolysis reactions with triolein and olive oil, up until 30% of incorporation efficiency of capric and lauric acids in SLs. The kinetics of MCFA incorporation in MOM-SL was influenced by the FFA:TAG molar ratio, and for reactions between triolein and lauric acid, increasing FFA:TAG from 2:1 to 4:1 enhanced MCFA incorporation in SL. Y. lipolytica lipase showed a strictly 1,3-regioselective profile in acidolysis reaction, confirmed by nuclear magnetic resonance spectroscopy. Immobilization of this lipase by microencapsulation in chitosan-alginate beads resulted in similar incorporation efficiency for lauric acid with olive oil TAG and this reaction could be performed for 5 cycles without catalytic activity loss. This lipase showed promising properties as a potential biocatalyst that may be effectively used in production of bioactive structured lipids, which might be applied for prevention of metabolic and inflammatory disorders related to obesity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Magnetic nanoparticles (Fe 3 O 4 ) were used for physical adsorption of lipase from Yarrowia lipolytica IMUFRJ 50682. The optimal adsorption conditions were obtained as follows: enzyme/support 19.3 mg/g and temperature of 20 °C for standard protein. High immobilization efficiency of 99% was obtained for 4 mL of crude lipase extract (containing 0.315 mg protein/mL) and 0.02 g of magnetic nanoparticles and this biocatalyst was recycled 30 times with 70% of lipase activity in the end. Purified lipase extracts were also efficiently immobilized and ultrafiltered lipase extract (ULE) and aqueous two-phase system lipase extract (ATPS_LE) when immobilized revealed higher hydrolytic activity in relation to CLE (2.8 and 4.0 times higher, respectively). Broad pH tolerance and high thermostability could be achieved by immobilization on magnetic nanoparticles, with 40% improvement in thermodynamic parameters at 60 °C. Kinetic parameters V max and K m were also better for ULE (V max : 2.3 times higher; K m 43% reduction) and ATPS_LE (V max : 3.0 times higher; K m : 38% reduction) immobilized on magnetic nanoparticles in relation to CLE. These results showed that the immobilization of lipase onto magnetic nanoparticles by physical adsorption is an efficient and simple way to obtain a great catalyst., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

l-Asparaginase (l-asparagine amidohydrolase; E.C.3.5.1.1) as an anticancer agent is used to treat acute lymphocytic leukemia (ALL), Human Burkitt's lymphoma and non-Hodgkin's lymphoma. The commercial asparaginases are obtained from bacteria Erwinia chrysanthemi and Escherichia coli now which had many side effects. In this study, the effects of a novel l-asparaginase from yeast Yarrowia lipolytica was investigated on human ALL and Burkitt's lymphoma cell lines. The l-asparaginase causes metabolic stress, cytotoxicity, and apoptosis due to the arrest of the G0 cell cycle, the activation of caspase-3 and the modulation of mitochondrial membrane integrity. The RT-PCR analysis showed a significant increase in the pro-apoptosis genes such as Bax, Caspase-3, Caspase-8, Caspase-9 and p53 (P < 0.05) while the anti-apoptotic marker Bcl-2 was significantly decreased (P < 0.05). Furthermore, Y. lipolytical-asparaginase causes autophagy and increased ROS. The l-asparaginase has cytotoxic and anticancer effects higher than commercial asparaginase. In conclusion, Y. lipolytical-asparaginase shows interesting anticancer activity and it can be introduced as a new eukaryotic and therapeutic agent and strategy for ALL and Burkitt's lymphoma treatment after the in vivo and clinical experiments., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Extracellular lipase from Yarrowia lipolytica was immobilized by ionotropic gelation with alginate and chitosan as encapsulating agents. Photomicrographs revealed a collapsed and heterogeneous surface of these microcapsules due to freeze-drying process. The optimum reaction temperature for the microencapsulated lipase (40 °C) was higher than for free lipase (35 °C) as well as the optimum pH (8.0 and 7.5, respectively). The study of the reaction kinetics showed that a higher maximum reaction rate (V max ) (221.1 U/mg) for the free lipase in comparison to the immobilized form (175.3 U/mg). A protective effect of the microcapsule was detected in the storage of the enzyme at room temperature, as after 75 days 35% of activity was maintained for the microcapsules, while no activity remained after 15 days with the free enzyme. Lower values for inactivation constant (k d ) and increase in half-life for immobilized lipase showed that lipase microencapsulation favored the thermostability of this enzyme., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50 °C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42 °C, which was about 5.0 °C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5 U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Asparaginase catalyzes the conversion of asparagine into aspartic acid and ammonia. The enzyme has various industrial applications and it is considered as an anticancer drug for treatment of certain leukemias. In the current study, production of asparaginase was investigated by Yarrowia lipolytica as well as optimized its production and determined its molecular characteristics by in silico analysis. Y. lipolytica DSM3286 produced 17.14 U/ml of asparaginase in flask culture. Optimization of asparaginase production was done by response surface methodology and the enzyme production increases up to 102.85 U/ml. The enzyme production reached 210 U/ml in a bioreactor which is 12-fold more than flask culture containing non-optimized medium. Asparaginase gene of Y. lipolytica was identified and isolated on the basis of comparison with asparaginase gene sequences of other microorganisms. The gene has 981 nucleotides and its protein has 326 amino acids. According to in silico analysis, the secondary structure of the enzyme is composed of 9 α-helixes and 11 β-sheets. Y. lipolytica produces type II asparaginase with high affinity for asparagine which is a suitable eukaryotic asparaginase for treatment of hematopoietic cancers. Hence, Y. lipolytica could be recommended as a new eukaryotic microbial source for the production of this important therapeutic enzyme., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fe 3 O 4 @C core-shell nanoparticles were modified by (3-aminopropyl)triethoxysilane (APTES) to generate amine functionality in the surface. Then, the amine functional groups were converted to dithiocarbamate via post-modification with carbon disulfide. This nanostructure with new functional property was used to immobilize lipase (obtained from Yarrowia lipolytica U6). Biocatalytic activity of the Fe 3 O 4 @C-NHCS-LIP was studied in this project. The interaction of lipase and support though dithiocarbamate binder was examined in the hydrolysis of p-nitrophenyl laurate. In this paper, support showed a unique feature in the immobilization of lipase by maintaining the lipase activity, raising the stability of lipase, and reusability., (Copyright © 2018. Published by Elsevier B.V.)

The production of biopolyols (sustainable, renewable and biodegradable precursors), is exclusively focused on the conventional chemical synthesis pathways, and currently, no polyols are produced by enzymatic processes at an industrial scale, because they are not competitive when compared to petrochemically based polyols due to the overall cost for their production. This can be mainly attributed to the high costs of commercial enzymes used in this process and their use is limited by their lack of stability during the bioprocess. Immobilization of enzymes gives the opportunity to converge two important features of enzymes: to increase the protein stability and to reuse enzyme as a catalyst. By this study, we were aiming to explore alternatives to commercially available enzymes. We investigated an effective hydrolytic route from epoxidized oils to polyols by applying lipolytic enzymes from Yarrowia lipolytica and Candida cylindracea immobilized onto fumed silica nanoparticles (fsNP) and those immobilized by the formation of cross-linked enzyme aggregates (CLEAs). Oxirane ring hydrolysis of epoxidized rapeseed oil catalyzed by lipase-fsNP and CLEAs was investigated with respect to various parameters (temperature, pH, concentrations, time of sorption and cross-linking, etc.). The highest conversions for oxirane ring hydrolysis were estimated for CLEA-Lip-derivatives (45-56%)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Biodiesel, an environment friendly alternative for fuels, contains methyl esters of long-chain fatty acids. Our group has reported a methanol-stable YLIP9 from Yarrowia lipolytica MSR80 that shows poor catalysis of long-chain fatty acids. To shift its substrate specificity, residues within lid and binding pocket were identified for sequential mutations using YLIP2 as the template. Of the two point mutations (Glu116Leu and Ser119Val) introduced in the lid, the former mutation (YLIP9L1) increased the catalytic rate by ∼2-fold without any change in substrate specificity. In this mutant, six binding pocket residues (Bp2-Bp7) were further mutated to obtain six double mutants. YLIP9L1Bp3 showed significant shift in substrate specificity towards long-chain pNPesters with 11-fold increase in catalytic efficiency than YLIP9. Double mutations also led to increased thermostability and lowered activation energy of YLIP9L1Bp3 thereby shifting its optimum temperature from 60°C to 50°C. In silico molecular dynamics simulations revealed improved lid flexibility and increased catalytic triad volume in YLIP9L1Bp3. The enzyme YLIP9L1Bp3 was methanol-stable having selectivity for long-chain fatty acids with improved catalytic efficiency. Its application as a biodiesel enzyme was validated by transesterification of palm oil in presence of methanol, where it showed 8-fold increase in conversion of oil to methyl esters., (Copyright © 2017 Elsevier B.V. All rights reserved.)