Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

Structural and Functional Characterization of Fibronectin in Extracellular Vesicles From Hepatocytes

Extracellular vesicles (EVs) are membrane-limited nanoparticles that are liberated by cells and contain a complex molecular payload comprising proteins, microRNA, RNAs, and lipids. EVs may be taken up by other cells resulting in their phenotypic or functional reprogramming. In the liver, EVs produced by non-injured hepatocytes are involved in the maintenance of hepatic homeostasis or therapeutic outcomes following injury while EVs produced by damaged hepatocytes may drive or exacerbate liver injury. In this study, we examined the contribution of EV fibronectin (FN1) to the biogenesis, release, uptake, and action of hepatocyte-derived EVs. While FN1 is classically viewed as a component of the extracellular matrix that regulates processes such as cell adhesion, differentiation, and wound healing and can exist in cell-associated or soluble plasma forms, we report that FN1 is also a constituent of hepatocyte EVs that functions in EV uptake by target cells such as hepatocytes and hepatic stellate cells (HSC). FN1 co-purified with EVs when EVs were enriched from conditioned medium of human or mouse hepatocytes and a direct association between FN1 and hepatocyte EVs was established by immunoprecipitation and proteinase protection. FN1 ablation in mouse hepatocytes using CRISPR-Cas9 did not alter EV biogenesis but EV uptake by HSC was significantly reduced for FN1 knockout EVs (EVΔFN1) as compared to EVs from wild type hepatocytes (EVWT). The uptake by hepatocytes or HSC of either EVWT or EVΔFN1 required clathrin- and caveolin-mediated endocytosis, cholesterol, lysosomal acidic lipase activity, and low pH, while macropinocytosis was also involved in EVΔFN1 uptake in HSC. Despite their differences in rate and mechanisms of uptake, EVΔFN1 functioned comparably to EVWT in ameliorating CCl4-induced hepatic fibrosis in mice. In conclusion, FN1 is a constituent of hepatocyte EVs that facilitates EV uptake by target cells but is dispensable for EV-mediated anti-fibrotic activity in vivo.

Thermostable acidic lipase of Bacillus glycinifermentans-MK840989 isolated from contaminated environment; its optimization, purification and exploring potential applications

This study is the first report about isolation, purification and optimization of lipase from Bacillus glycinifermentans. In this study, Bacillus glycinifermentansMK-840989 was isolated from a local petrol pump. The bacterium showed lipolytic zones of 0.19cm, 0.044cm, and 0.28cm on peptone yeast agar, olive oil hydrolysis agar and chromogenic plate agar, respectively. B. glycinifermentans also produced an extracellular lipase (55.1µmol/ml). This bacterium preferred acidic environment (pH 5) for growing optimally at 80˚C when the medium was supplemented with 1% olive oil. The olive oil induced its growth up to 9h. The protein content of the purified lipase was estimated about 75mg/ml as compared to its crude form, i.e. 350mg/ml. The purified lipase was found to be thermostable acidic in nature as its optimum activity was observed at 90˚C (0.08U/ml) and pH 5 (0.02U/ml). Other optimization factors included 1% olive oil (0.065U/ml), 0.1mM maltose (0.023U/ml), 0.1mM Ca (0.025U/ml), 1% yeast extract (16.8U/ml), 1% wheat waste (0.019U/ml), 1% commercial detergent (0.016U/ml) and 1% tween-20 (0.015 U/ml). The purified lipase showed a polypeptide of 26.7kDa on SDS-PAGE. These features such as thermostability, acidic nature, ability to show activity in wheat waste and tolerance to detergents render the lipase of B.glycinifermentans MK-840989 as an attractive choice for biotechnologists to employ it at industrial level. The purified lipase of B.glycinifermentans MK-840989 can be a potential candidate for detergent and oil-remediation industry. It can help to replace conventional synthetic detergent as it is cost-effective and eco-friendly.

Saturation mutagenesis in selected amino acids to shift Pseudomonas sp. acidic lipase Lip I.3 substrate specificity and activity

Several Pseudomonas sp. CR611 Lip I.3 mutants with overall increased activity and a shift towards longer chain substrates were constructed.