Purification and Characterization of an Extracellular Lipase Produced by Aspergillus oryzae ST11 as a Potential Catalyst for an Organic Synthesis

The lipase producing Aspergillus sp. ST11 was identified by molecular and morphological methods. The primers ITS1/ITS4 were used for amplifying the ITS region. It showed that the strain was grouped with Aspergillus oryzae and Aspergillus flavus (98 % bootstrap value). The colony morphology of Aspergillus sp. ST11 on malt extract agar and Czapek yeast agar showed a characteristic of A. oryzae. Therefore, it was identified as Aspergillus oryzae ST11. The lipase produced by the strain was purified and characterized. The purification steps involved precipitation with chilled acetone and separation by column chromatography, with HiTrap® Q HP and Toyopearl Butyl-650M, respectively. After purification, the lipase activity was increased 13 fold and with 7.9 % yield. Its molecular mass was 25 kDa. The purified lipase was stable at a pH between 5.0 - 8.0 and had optimum activity at pH 7.5. It was stable at 30 °C and had optimum activity at 37 °C. Its activity was promoted in the presence of Mg2+ but it was greatly decreased in the presence of Co2+, Cu2+, Hg2+ and Zn2+. Surfactants (Triton X-100, Tween-80, Tween-20, arabic gum, and sodium dodecyl sulfate) showed negative effects on lipase activity, while inhibitors (PMSF, EDTA, and β-mercaptoethanol) did not reduce the activity significantly. Polar solvents, such as methanol and ethanol, had much negative effect on lipase activity compared to non-polar solvents, such as hexane and isooctane. The concentrated lipase from A. oryzae ST11 was used to catalyze the transesterification and gave the highest bioconversion (90 %) after 24 h. HIGHLIGHTS Extracellular lipase produced by Aspergillus oryzae ST11 could be applied in many applications which is more flexible compared with the use of whole-cell biocatalysts High stability toward a wide range of pH and temperature obtained from this extracellular lipase The concentrated lipase shows the capability to produce the high biodiesel from natural oil GRAPHICAL ABSTRACT

Endothelial lipase mediates efficient lipolysis of triglyceride-rich lipoproteins

Triglyceride-rich lipoproteins (TRLs) are circulating reservoirs of fatty acids used as vital energy sources for peripheral tissues. Lipoprotein lipase (LPL) is a predominant enzyme mediating triglyceride (TG) lipolysis and TRL clearance to provide fatty acids to tissues in animals. Physiological and human genetic evidence support a primary role for LPL in hydrolyzing TRL TGs. We hypothesized that endothelial lipase (EL), another extracellular lipase that primarily hydrolyzes lipoprotein phospholipids may also contribute to TRL metabolism. To explore this, we studied the impact of genetic EL loss-of-function on TRL metabolism in humans and mice. Humans carrying a loss-of-function missense variant in LIPG, p.Asn396Ser (rs77960347), demonstrated elevated plasma TGs and elevated phospholipids in TRLs, among other lipoprotein classes. Mice with germline EL deficiency challenged with excess dietary TG through refeeding or a high-fat diet exhibited elevated TGs, delayed dietary TRL clearance, and impaired TRL TG lipolysis in vivo that was rescued by EL reconstitution in the liver. Lipidomic analyses of postprandial plasma from high-fat fed Lipg-/- mice demonstrated accumulation of phospholipids and TGs harboring long-chain polyunsaturated fatty acids (PUFAs), known substrates for EL lipolysis. In vitro and in vivo, EL and LPL together promoted greater TG lipolysis than either extracellular lipase alone. Our data positions EL as a key collaborator of LPL to mediate efficient lipolysis of TRLs in humans and mice.

Orchestration an extracellular lipase production from Aspergillus niger MYA 135: biomass morphology and fungal physiology

The impact of biomass morphology and culture conditions on fungal fermentation was widely reviewed in the literature. In this work, we presented three independent experiments in order to evaluate the influence of some of those input factors on a lipase production separately by using the Aspergillus niger MYA 135 and the two-stage fermentation technique. Regarding the culture modality, the biomass was pre-grown in a first reactor. Then, the washed mycelium was transferred to a second reactor to continue the study. Firstly, linear effects of fungal morphology and several physiological parameters on a lipase production were explored using the Plackett–Burman design. The dispersed fungal morphology was confirmed as a proper quality characteristic for producing an extracellular lipase activity. Concerning the impact of the carbon source on the biomass pre-growth, the sucrose (E = 9.923, p < 0.001) and the l-arabinose (E = 4.198, p = 0.009) presented positive and significant effects on the enzyme production. On the contrary, the supplementation of 0.05 g/L CaCl2 displayed a highly negative and significant effect on this process (E = − 7.390, p < 0.001). Secondly, the relationship between the enzyme production and the input variables N:C ratio, FeCl3 and olive oil was explored applying the central composite design. Among the model terms, the N:C ratio of the production medium had the most negative and significant influence on the enzyme synthesis. Thus, it was concluded that a low N:C ratio was preferable to increase its production. In addition, the bifunctional role of FeCl3 on this fungus was presented. Thirdly, a prove of concept assay was also discussed.

Phylogenomic and biochemical analysis reassesses temperate marine yeast Yarrowia lipolytica NCIM 3590 to be Yarrowia bubula

Yarrowia clade contains yeast species morphologically, ecologically, physiologically and genetically diverse in nature. Yarrowia lipolytica NCIM 3590 (NCIM 3590), a biotechnologically important strain, isolated from Scottish sea waters was reinvestigated for its phenotypic, biochemical, molecular and genomic properties as it exhibited characteristics unlike Y. lipolytica, namely, absence of extracellular lipolytic activity, growth at lower temperatures (less than 20 °C) and in high salt concentrations (10% NaCl). Molecular identification using ITS and D1/D2 sequences suggested NCIM 3590 to be 100% identical with reference strain Yarrowia bubula CBS 12934 rather than Y. lipolytica CBS 6124 (87% identity) while phylogenetic analysis revealed that it clustered with Y. bubula under a separate clade. Further, whole genome sequencing of NCIM 3590 was performed using Illumina NextSeq technology and the draft reported here. The overall genome relatedness values obtained by dDDH (94.1%), ANIb/ANIm (99.41/99.42%) and OrthoANI (99.47%) indicated proximity between NCIM 3590 and CBS 12934 as compared to the reference strain Y. lipolytica. No extracellular lipase activity could be detected in NCIM 3590 while LIP2 gene TBLASTN analysis suggests a low 42% identity with e value 2 e−77 and 62% coverage. Hence molecular, phylogenetic, genomics, biochemical and microbial analyses suggests it belongs to Yarrowia bubula.

Production, Purification and Characterization of Extracellular Lipase from a Mutated Strain of Penicillium Citrinum KU613360

In the present study lipase production, purification and characterization were carried out with a novel fungal strain of Penicillium citrinum KU613360 isolated from vegetable oil contaminated soil samples collected from oil mills located in and around Guntur District, Andhra Pradesh, India. The strain improvement was carried out by subjecting the strain to both UV and Ethidium Bromide treatments. The wild strain of P. citrinum KU613360 showed maximum lipase activity of 1.053±0.32IUmL-1 on optimized medium and while the mutated strain treated with combination of UV (300 sec) and Et Br (200 µgcm3), recorded the enzyme activity of 4.260±0.011IUmL-1, using the optimised medium at 6.5 pH and 40°C temperature. Thus, a 404% enhancement in the activity was achieved by using induced mutation on wild strain of P. citrinum KU613360. The molecular weight of the purified lipase from the mutated strain was found to be 35 kDa, when analysed on SDS PAGE. From our results it was concluded that the mutated strain has considerable capability and potentiality to be used in various industrial applications.

Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production

Background Rhizopus species is among the most well-known lipase producers, and its enzyme is suitable for use in many industrial applications. Our research focuses on the production of lipase utilizing waste besides evaluating its applications. Results An extracellular lipase was partially purified from the culture broth of Rhizopus oryzae R1 isolate to apparent homogeneity using ammonium sulfate precipitation followed by desalting via dialysis. The partially purified enzyme was non-specific lipase and the utmost activity was recorded at pH 6, 40 °C with high stability for 30 min. The constants Km and Vmax, calculated from the Lineweaver-Burk plot, are 0.3 mg/mL and 208.3 U/mL, respectively. Monovalent metal ions such as Na+ (1 and 5 mM) and K+ (5 mM) were promoters of the lipase to enhance its activity with 110, 105.5, and 106.5%, respectively. Chitosan was used as a perfect support for immobilization via both adsorption and cross-linking in which the latter method attained immobilization efficiency of 99.1% and reusability of 12 cycles. The partially purified enzyme proved its ability in forming methyl oleate (biodiesel) through the esterification of oleic acid and transesterification of olive oil. Conclusion The partially purified and immobilized lipase from Rhizopus oryzae R1 approved excellent efficiency, reusability, and a remarkable role in detergents and biodiesel production.