Production, purification, properties and current perspectives for modification and application of microbial lipases.

With the industrialization and development of modern science, the application of enzymes as green and environmentally friendly biocatalysts in industry has been increased widely. Among them, lipase (EC. 3.1.1.3) is a very prominent biocatalyst, which has the ability to catalyze the hydrolysis and synthesis of ester compounds. Many lipases have been isolated from various sources, such as animals, plants and microorganisms, among which microbial lipase is the enzyme with the most diverse enzymatic properties and great industrial application potential. It therefore has promising applications in many industries, such as food and beverages, waste treatment, biofuels, leather, textiles, detergent formulations, ester synthesis, pharmaceuticals and medicine. Although many microbial lipases have been isolated and characterized, only some of them have been commercially exploited. In order to cope with the growing industrial demands and overcome these shortcomings to replace traditional chemical catalysts, the preparation of new lipases with thermal/acid-base stability, regioselectivity, organic solvent tolerance, high activity and yield, and reusability through excavation and modification has become a hot research topic.

Geographic Pattern of Variations in Chemical Composition and Nutritional Value of Cinnamomum camphora Seed Kernels from China.

Cinnamomum camphora (camphor tree) is an important non-conventional edible plant species found in East Asia. Here, a detailed characterization for the chemical composition and nutritional value of C. camphora seed kernels (CCSKs) collected from different regions in China is provided. The results showed that there were significant differences among the CCSK samples in weights (1000 fruits, 1000 seeds and 1000 kernels), proximate composition, minerals, phenolics, flavonoids and amino acid contents. The highest contents of oil (62.08%) and protein (22.17%) were found in the CCSK samples collected from Chongqing and Shanghai, respectively. The highest content of mineral in the CCSK samples was K (4345.05-7186.89 mg/kg), followed by P (2735.86-5385.36 mg/kg), Ca (1412.27-3327.37 mg/kg) and Mg (2028.65-3147.32 mg/kg). The CCSK sample collected from Guizhou had the highest levels of total phenolic and flavonoid contents (TPC and TFC), while that from Chongqing had the lowest levels. In addition, the most abundant fatty acid in the CCSK samples was capric acid (57.37-60.18%), followed by lauric acid (35.23-38.29%). Similarities in the fatty acid composition among the CCSK samples were found. The CCSK sample collected from Guizhou had the highest percentage (36.20%) of essential amino acids to total amino acids, and Chongqing had the lowest value (28.84%). These results indicated that CCSK may be developed as an excellent source of plant-based medium-chain oil, protein, dietary fiber, minerals, phytochemicals and essential amino acids.

Acute Oral Toxicity and Genotoxicity Test and Evaluation of Cinnamomum camphora Seed Kernel Oil.

Cinnamomum camphora seed kernel oil (CCSKO) is one of the important natural medium chain triglycerides (MCT) resources, with more than 95.00% of medium chain fatty acids found in the world, and has various physiological effects. However, CCSKO has not been generally recognized as a safe oil or new food resource yet. The acute oral toxicity test and a standard battery of genotoxicity tests (mammalian erythrocyte micronucleus test, Ames test, and in vitro mammalian cell TK gene mutation test) of CCSKO as a new edible plant oil were used in the study. The results of the acute oral toxicity test showed that CCSKO was preliminary non-toxic, with an LD50 value higher than 21.5 g/kg body weight. In the mammalian erythrocyte micronucleus test, there was no concentration-response relationship between the dose of CCSKO and micronucleus value in polychromatic erythrocytes compared to the negative control group. No genotoxicity was observed in the Ames test in the presence or absence of S9 at 5000 µg/mL. In vitro mammalian cell TK gene mutation test showed that CCSKO did not induce in vitro mammalian cell TK gene mutation in the presence or absence of S9 at 5000 µg/mL. These results indicated that CCSKO is a non-toxic natural medium-chain oil.

Immobilization of lipase on ß-cyclodextrin grafted and aminopropyl-functionalized chitosan/Fe3O4 magnetic nanocomposites: An innovative approach to fruity flavor esters esterification.

The lipase from Bacillus licheniformis NCU CS-5 was immobilized onto ß-cyclodextrin (CD) grafted and aminopropyl-functionalized chitosan-coated Fe3O4 magnetic nanocomposites (Fe3O4-CTS-APTES-GA-ß-CD). Fourier transform infrared spectroscopy, thermogravimetry analysis, X-ray diffraction, scanning electron microscopy and transmission electron microscopy showed that not only the functionalized magnetic nanoparticles were synthesized but also the immobilized lipase was successfully produced. The immobilized lipase exhibited higher optimal pH value (10.5) and temperature (60℃) than the free lipase. The pH and thermal stabilities of the immobilized lipase were improved significantly compared to the free lipase. The immobilized lipase remained more than 80% of the relative activity at temperature of 60 ℃ and pH 12.0. The immobilized lipase also remained over 80% of its relative activity after 28 days of storage and 15 cycles of application. The application of the immobilized lipase in esterification of isoamyl acetate and pentyl valerate showed that maximum esterification efficiency was achieved in n-hexane having 68.0% and 89.2% respectively. Therefore, these results indicated that the Fe3O4-CTS-APTES-GA-ß-CD nanoparticles are novel carriers for immobilizing enzyme, and the immobilized lipase can be used as an innovative green approach to the synthesis of fruity flavor esters in food industry.

Dextran Conjugation Improves the Structural and Functional Properties of Heat-Treated Protein Isolate from Cinnamomum camphora Seed Kernel.

The Cinnamomum camphora seed kernel (CCSK), with high contents of medium-chain oil (~59%) and protein (~19%), is an excellent source for a plant-based food ingredient. To broaden the application of the protein isolate (PI) from CCSK in the food industry, the Maillard reaction products (MRPs) were prepared by PI and dextran (DX) under mild wet-heating conditions (60 °C, 5 h), and the structural and functional properties of the PI-DX conjugates were investigated. The covalent bond between PI and DX was confirmed by the degree of grafting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compared with the heated PI, the PI-DX conjugates had more ordered structure, with the decreased random coil content. The changes in tertiary structure of PI-DX conjugates were reflected by the results of intrinsic fluorescence and surface hydrophobicity. Moreover, PI-DX conjugates showed better solubility, emulsifying properties, thermal stability and antioxidant activities. These results provided a theoretical basis for the development of PI-based MRPs with desirable characteristics.

Lauric Triglyceride Ameliorates High-Fat-Diet-Induced Obesity in Rats by Reducing Lipogenesis and Increasing Lipolysis and ß-Oxidation.

Medium-chain triglycerides (MCTs) are found in limited foods. In these medium-chain oil resources, the abundance of lauric acid (LA) is the highest among medium-chain fatty acids (MCFAs), and its effects on lipid metabolism in obese rats have not been well-studied. This study aimed to determine the anti-obesity effects and mechanisms of lauric triglyceride (LT) in Sprague Dawley (SD) rats. LA and glycerin were used to synthesize LT, then LT was used to treat obese rats for 12 weeks. The results showed that LT significantly reduced the body weight, body mass index, and Lee's index in obese rats. The mRNA expression levels of the anorexic neuropeptide POMC in the hypothalamus between the LT group and the other groups were not different, while the gene expression levels of the orexigenic neuropeptides NPY and AGRP decreased significantly in the LT group. Except serum cholesterol, LT improved the serum triglyceride metabolism in the obese rats and reduced adipocyte and hepatic lipid deposition. Moreover, LT inhibited the expression of lipogenesis-related genes and proteins (SREBP-1c, ACC1, and FASN) and increased the expression of lipolysis (ATGL, HSL, and LPL) and ß-oxidation (PPARα, CPT-1a, and PCG-1α) related genes and proteins in the white fat and liver. Furthermore, LT increased the mRNA expression of mitochondrial-biosynthesis-related genes (SIRT1, NRF1, and TFAM) in the liver. The results indicated that LT ameliorates diet-induced obesity in rats.

High Dietary Intervention of Lauric Triglyceride Might be Harmful to Its Improvement of Cholesterol Metabolism in Obese Rats.

Hypercholesterolemia is often considered to be a major risk factor for atherosclerosis, and medium-chain fatty acids have been found to reduce the total cholesterol (TC) level and maintain low-density lipoprotein cholesterol (LDL-c) stability. However, we unexpectedly found that the levels of TC and LDL-c were increased in obese rats treated with high-dose lauric triglycerides (LT). The study aimed to investigate the effect and mechanism of LT on cholesterol metabolism in obese rats. Our results showed that LT intervention could reduce cholesterol biosynthesis by downregulating the expression of HMG-CoA reductase in obese rats. LT increased the expression levels of PPARγ1, LXRα, ABCA1, and ABCG8 in the liver. These results indicated that LT could improve the lipid transfer and bile acid efflux. However, LT significantly increased the expression of PCSK 9, resulting in accelerated degradation of LDLR, thus reducing the transport of very LDL (VLDL) and LDL to the liver. Together with the increased expression of NPC1L1 protein, LT impaired the uptake of VLDL/LDL by the liver and increased the reabsorption of sterols, leading to an increase in the levels of TC and LDL-c in obese rats.

Characterization of a novel lipase from Bacillus licheniformis NCU CS-5 for applications in detergent industry and biodegradation of 2,4-D butyl ester.

Enzymatic degradation has become the most promising approach to degrading organic ester compounds. In this study, Bacillus licheniformis NCU CS-5 was isolated from the spoilage of Cinnamomum camphora seed kernel, and its extracellular lipase was purified, with a specific activity of 192.98 U/mg. The lipase was found to be a trimeric protein as it showed a single band of 27 kDa in SDS-PAGE and 81 kDa in Native-PAGE. It was active in a wide range of temperatures (5-55 °C) and pH values (6.0-9.0), and the optimal temperature and pH value were 40 °C and 8.0, respectively. The enzyme was active in the presence of various organic solvents, metal ions, inhibitors and surfactants. Both crude and purified lipase retained more than 80% activity after 5 h in the presence of commercial detergents, suggesting its great application potential in detergent industry. The highest activity was found to be towards medium- and long-chain fatty acids (C6-C18). Peptide mass spectrometric analysis of the purified lipase showed similarity to the lipase family of B. licheniformis. Furthermore, it degraded more than 90% 2,4-D butyl ester to its hydrolysate 2,4-D within 24 h, indicating that the novel lipase may be applied to degrade organic ester pesticides.