Glucose induced hepatic lipase expression and ApoB100/ApoAI ratio changes in cultured HepG2 cells in vitro

Backgroundː Hepatic lipase (HL) plays a very important role in lipoprotein catabolism. The aim of this study was to measure both HL activity and ApoB100/ApoAI ratio changes in cell secretions by incubating HepG2 cells with various amounts of glucose. Methodsː HepG2 cells were cultured in low-, normal- or high-glucose Dulbecco's Modified Eagle Medium (DMEM) (1, 4.5 and 10g/L, respectively). HL activities were determined using the Hepatic Lipase Detection Kit (cat. no. A067) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Levels of ApoAI and ApoB100 were measured with commercial sandwich enzyme-linked immunosorbent assay kits (cat#: H0123 and H0124) from ShangHai MEIXUAN Biological Science and Technology Ltd (Shanghai, China). Experiments were repeated six times for each assay. Resultsː Pearson’s correlation coefficient results showed that ApoB100 and ApoB100/ApoAI ratio have positive and significant correlations with HL activity, and ApoAI has a negative and significant correlation with HL activity. Conclusionsː Glucose may increase or decrease ApoB100/ApoAI ratio through upregulation or downregulation of hepatic lipase activity, which suggests a new regulatory pathway in lipoprotein catabolism. This finding may lead to novel therapeutic ways for diagnosis and treatment for coronary artery disease.

Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

Background: Pseudomonas lipases are widely used in industrial applications due to its unique biochemical properties, but one of the biggest limitations are the low yields obtained in native strains therefore, organisms as E. coli are used for the recombinant lipase overexpression. However, the recombinant lipase is accumulated as inclusion bodies and it affects biological activity, making that researchers evaluate different fermentation conditions to improve the activity of recombinant enzymes. In this study, a statistical experimental design was implemented to evaluate the effects of temperature, agitation rate and osmolyte concentration on the recombinant lipase activity produced in E. coli BL21 (DE3). Once the significant variables were identified, an optimization by a Response Surface Methodology was applied to maximize the lipase production. Results: The Box-Behnken designs revealed different optimal fermentation conditions for each osmolyte experiment. The glycerol showed the highest specific lipase activity compared to the other osmolytes and 0.1 M of osmolyte glycerol,5°C and 110 rpm showed the highest significant increase on the specific lipase activity and the data fitted the model very well. The validation showed that 452.01 U/mg of specific lipase activity was obtained which was significantly higher compared to the group where no glycerol was added (271.38 U/mg). The relative recombinant lipase expression was 2.7-fold lower at 5°C compared to 25 °C, but at 5°C the lipase activity was significantly higher. In addition, when the 3 L shaken Erlenmeyer Bioreactor was used to produce the recombinant lipase based on the power input parameter, the specific lipase activity was not significantly different from that found in Schott (408,4 U/mg and 452 U/mg, respectively), which means that this Bioreactor platform should be used for future scale-up processes.Conclusion: Low temperatures, low agitation rates and 0.1 M of glycerol in the autoinduction media enhanced the activity of the recombinant lipase produced in E. coli BL21(DE3). The optimized conditions and the 3 L shaken Erlenmeyer Bioreactor can be used to produce the recombinant enzyme in a higher volume based on the power input parameter. Further studies using this strategy may lead to the identification of optimal culture conditions for a given recombinant enzyme facilitating the large-scale bioprocess implementation.

Red/ET recombineering enhances extracellular lipase production by Burkholderia glumae

Background: Microbial lipases are utilized widely in industrial fields, and the LipA lipase produced by Burkholderia glumae PG1 has been used for the production of enantiopure pharmaceuticals. However, efficient lipase expression and secretion are still problematic. LipA is encoded in an operon that also contains the lipase-specific foldase gene lipB. The purpose of this study was to enhance the production of active lipase by overexpressing lipAB in B. glumae PG1 using a host-vector system.Results: The lipAB operon, isolated from the genomic DNA of B. glumae PG1, was directly cloned into broad-host-range vectors, with pBBR1 or pRk2 backbones, using the Red/ET recombination system and then transformed into the original host strain. Lipase activity of the derivative strains, including PG1/Bl and PG1/Rl, were evaluated. When 1% olive oil was included in the growth medium, the maximum activities of the resultant strains were approximately 39.5- and 15.3-fold higher, respectively, than that of the wild-type PG1, indicating that increased lipase gene dosage resulted in high expression yields of lipase. To further enhance lipase expression, two recombinant strains, PG1/Ba and PG1/Ra, were generated with the plasmid-borne lipAB operon driven by the strong constitutive promoter Papra. PG1/Ba and PG1/Ra displayed 15.0- and 4.3-fold improvements in lipase production in 0.5% glucose-containing medium compared to PG1/Bl and PG1/Rl, respectively. However, glucose repressed lipase gene expression in strain PG1 when the native lipAB promoter was used.Conclusions: Homologous expression of lipase genes in B. glumae PG1 was achieved by the use of high-copy expression vectors, and the resulting presence of multiple copies of lipAB significantly improved extracellular lipase expression. Our results demonstrate that modification of B. glumae PG1 can improve this host system for lipase production.