Long-term environmental exposure to microcystins increases the risk of nonalcoholic fatty liver disease in humans: A combined fisher-based investigation and murine model study.

Microcystins (MCs) produced by cyanobacteria pose serious threats to human health. However, the contribution of long-term exposure to MCs to the development of nonalcoholic fatty liver disease (NAFLD) remains poorly documented. In this study, we estimated the environmental uptake of MCs by a small population of fishers who have lived for many years on Meiliang Bay of Lake Taihu, where cyanobacterial blooms occur frequently. Serum biochemical indices of liver function and their relationships with MC contamination in these people were also investigated. Moreover, to mimic the long-term effects of MC on the livers of fishers, an animal model was established in which mice were exposed to MC-LR at an environmentally relevant level, a reference level (the no-observed adverse effect level, NOAEL), and three times the NOAEL through drinking water for 12 months. We estimated the total daily intake of MCs by fishers through contaminated lake water and food to be 5.95 µg MC-LReq, far exceeding the tolerable daily intake (2.40 µg MC-LReq) proposed by the World Health Organization (WHO). More than 80% of participants had at least one abnormal serum marker. The indices of aspartate aminotransferase (AST)/alanine aminotransferase (ALT), triglyceride (TG), globulin (GLB), and lactate dehydrogenase (LDH) had close positive associations with MC contamination, indicating that both liver damage and lipid metabolism dysfunction were induced by chronic MC exposure. Furthermore, the animal experimental results showed that long-term exposure to MC-LR at the environmentally relevant level led to hepatic steatosis with molecular alterations in circadian rhythm regulation, lipid metabolic processes, and the cell cycle pathway. Exposure to MC-LR at or above the NOAEL worsened the pathological phenotype towards nonalcoholic steatohepatitis disease (NASH) or fibrosis. These results suggest that prolonged exposure to the reference level (NOAEL) of MC-LR could cause severe liver injury to mammals. People with long-term environmental exposure to MCs might be at high risk for developing NAFLD.

Lipase Immobilized on a Novel Rigid-Flexible Dendrimer-Grafted Hierarchically Porous Magnetic Microspheres for Effective Resolution of (R,S)-1-Phenylethanol.

With the rapid development of biotechnological industry, there is an urgent need for exploiting new materials to immobilize enzymes to improve the performance of biocatalysts. In this paper, hierarchically porous magnetic microspheres (PFMMs) were prepared through solvothermal method and rapidly grafted with a novel rigid-flexible dendrimer first synthesized from monomers of trimesoyl chloride (TMC) and 1,6-hexanediamine (HDA) via interfacial polymerization process for covalent immobilization of Pseudomonas fluorescens lipase (PFL). The maximum PFL loading of the synthesized support reaches 87.5 mgprotein/gsupport, and 864% activity recovery of PFMMs-G3.0-PFL can be achieved at pH 9.0. Then, it was used to catalyze the resolution of (R,S)-1-phenylethanol with vinyl acetate. Under the optimized conditions, 50.0% conversion with 99.0% ees can be reached within 1.5 h. In addition, a conversion of 49.2% and ees of 96.9% can be retained after 10 batches of running, displaying an excellent operational stability. Importantly, a further investigation shows that the obviously improved reusability of the immobilized PFL is ascribed to the increased rigidity in comparison to fully flexible dendrimer. Thus, the newly constructed protocol for lipase immobilization exhibits a great prospect in biochemical engineering.

Surface-Displayed Thermostable Candida rugosa Lipase 1 for Docosahexaenoic Acid Enrichment.

Yeast surface display has emerged as a viable approach for self-immobilization enzyme as whole-cell catalysts. Herein, we displayed Candida rugosa lipase 1 (CRL LIP1) on the cell wall of Pichia pastoris for docosahexaenoic acid (DHA) enrichment in algae oil. After a 96-h culture, the displayed CRL LIP1 achieved the highest activity (380 ± 2.8 U/g) for hydrolyzing olive oil under optimal pH (7.5) and temperature (45 °C) conditions. Additionally, we improved the thermal stability of displayed LIP1, enabling retention of 50% of its initial bioactivity following 6 h of incubation at 45 °C. Furthermore, the content of DHA enhanced from 40.61% in original algae oil to 50.44% in glyceride, resulting in a 1.24-fold increase in yield. The displayed CRL LIP1 exhibited an improved thermal stability and a high degree of bioactivity toward its native macromolecule substrates algae oil and olive oil, thereby expanding its potential for industrial applications in fields of food and pharmaceutical. These results suggested that surface display provides an effective strategy for simultaneous convenient expression and target protein immobilization.

Scaled-up biodiesel synthesis from Chinese Tallow Kernel oil catalyzed by Burkholderia cepacia lipase through ultrasonic assisted technology: A non-edible and alternative source of bio energy.

In East Asia, for thousands of years, the fruit of Chinese tallow tree (Sapium sebiferum) has been used for multiple purposes because of its chemical composition; the presence of high amounts of lipids is remarkable, showing potential to be used as substrate for biodiesel synthesis. Previously have been reported the use of alkaline and enzymatic catalysts, microwave technology and the use of ionic liquids as co-solvents with the lipids of this tree species to produce biodiesel. This study shows the results of the use of Burkholderia cepacia lipase as enzymatic catalyst for transesterification of Chinese Tallow Kernel oil (CTK), extracted from the fruit of Chinese tallow tree, into biodiesel, with the use of ultrasonic assisted technology and without the usage of solvents. The optimal operational parameters were determined and the reactions were developed in a batch reactor with the use of ultrasonic irradiation and emulsification to enhance the mass transfer. The scaled-up experiments, in an especially designed 3 L capacity reactor, showed promising results, obtaining 55.20% biodiesel and a kinematic viscosity of 10.31 mm2.s-1 in only 4 h, in comparison with previously published (in vitro) methods. The valorization of this non-edible source of oil represents an opportunity to use as an alternative source for bioenergy and also to tackle the uncontrolled expansion of this oleaginous tree species in some ecologically fragile ecosystems.

Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity.

Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu3(PO4)2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu3(PO4)2/CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu3(PO4)2/Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ees) reached 98% in only 10 min, and the corresponding Cu3(PO4)2/CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application.

Burkholderia cepacia lipase immobilized on heterofunctional magnetic nanoparticles and its application in biodiesel synthesis.

Biodiesel production using immobilized lipase as a biocatalyst is a promising process. The performance of immobilized lipase is mainly determined by supporting materials and immobilization method. To avoid the shortcomings of adsorption and covalent bonding methods, in this study, we developed a novel heterofunctional carrier of being strengthened anion exchange and weakened covalent binding to avoid activity loss and improve operational stability of the immobilized lipase. 2,3-epoxypropyltrimethylammonium chloride with epoxy and quaternary ammonium group and glutaraldehyde were grafted onto aminated magnetic nanoparticles (AMNPs) to generate a new matrix, named GEAMNP. Then Burkholderia cepacia lipase (BCL) was immobilized on GEAMNP via anion exchange and covalent bonding. The transesterification between soybean oil and methanol was used to test the activities. Activity recovery of the immobilized BCL was up to 147.4% and the corresponding transesterification activity was 1.5-fold than that of BCL powder. The immobilized lipase was further used for biodiesel production to confirm its feasibility. The fatty acid methyl esters conversion yield could reach 96.8% in the first 12 h. Furthermore, the immobilized lipase, BCL-GEAMNP showed markedly improved operational stability, better reusability and higher esters than BCL-GAMNP, where MNPs were only modified with (3-aminopropyl) triethoxysilane and glutaraldehyde.

Preparation of Biodiesel with Liquid Synergetic Lipases from Rapeseed Oil Deodorizer Distillate.

To reduce industrial production cost, cheap and easily available rapeseed oil deodorizer distillates were used as feedstock to prepare biodiesel in this study. As a result, liquid forms of Candida rugosa lipase and Rhizopus oryzae lipase (ROL) were functioned as new and effective catalysts with biodiesel yield of 92.63% for 30 h and 94.36% for 9 h, respectively. Furthermore, the synergetic effect between the two lipases was employed to enhance biodiesel yield with a result of 98.16% in 6 h under optimized conditions via response surface methodology. The obtained conversion rate surpassed both yields of the individual two lipases and markedly shortened the reaction time. The resultant optimal conditions were ROL ratio 0.84, water content 46 wt% (w/w), reaction temperature 34 °C, and reaction time 6 h.

Impairment of endoplasmic reticulum is involved in ß-cell dysfunction induced by microcystin-LR.

Microcystins (MCs) widely distributed in freshwaters have posed a significant risk to human health. Previous studies have demonstrated that exposure to MC-LR impairs pancreatic islet function, however, the underlying mechanisms still remain unclear. In the present study, we explored the role of endoplasmic reticulum (ER) impairment in ß-cell dysfunction caused by MC-LR. The result showed that MC-LR modified ER morphology evidenced by increased ER amount and size at low doses (15, 30 or 60 µM) and vacuolar and dilated ER ultrastructure at high doses (100 or 200 µM). Also, insulin content showed increased at 15 or 30 µM but declined at 60, 100, or 200 µM, which was highly accordant with ER morphological alteration. Transcriptomic analysis identified a number of factors and several pathways associated with ER protein processing, ER stress, apoptosis, and diabetes mellitus in the cells treated with MC-LR compared with non-treated cells. Furthermore, MC-LR-induced ER stress significantly promoted the expression of PERK/eIF2α and their downstream targets (ATF4, CHOP, and Gadd34), which indicates that PERK-eIF2α-ATF4 pathway is involved in MC-LR-induced insulin deficiency. These results suggest that ER impairment is an important contributor to MC-LR-caused ß-cell failure and provide a new insight into the association between MCs contamination and the occurrence of human diseases.