Design of a Genetically Programmed Biomimetic Lipase Nanoreactor.

Alternative to the traditionally independent production of lipase, chemical synthesis of nano-carriers, and then preparing nanoimmobilized enzymes, we exploit a yeast genetically programmed virus biomimetic lipase nanoreactor in a sustainable manner. The nanoreactor biogenesis process integrated lipase production, protein component (coat-protein subunit and scaffold protein) production, self-assembly of protein components, and the encapsulation of lipase into protein nanocages using a simple process. It included overexpression of nanocage components, coat-protein subunits, and fused lipase-scaffold proteins and subsequent spontaneous self-assembly and encapsulation based on the specific interaction between the coat-protein subunit and the scaffold protein fused in the target lipase enzyme. The genetically programmable lipase nanoreactor showed improved stability under various harsh conditions, and was validated in fatty acid methyl ester synthesis with 86% yield at a high concentration of waste cooking oil (200 mM), which demonstrates the robustness and feasibility of the lipase nanoreactor in biodiesel production.

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.

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.

Heterologous expression and characterization of a new lipase from Pseudomonas fluorescens Pf0-1 and used for biodiesel production.

As a kind of important biocatalysts, Pseudomonas lipases are commonly applied in various industrial fields. Pflip1, a new extracellular lipase gene from Pseudomonas. fluorescens Pf0-1, was first cloned and respectively expressed in Escherichia coli BL21(DE3) and Pichia pastoris KM71, the recombinant proteins Pflip1a and Pflip1b were later purified separately. Then Pflip1a was further characterized. The optimum pH of Pflip1a was 8.0 and its optimal temperature was 70 °C. After incubation at 70 °C for 12 h, Pflip1a could retain over 95% of its original activity. It showed the highest activity towards p-nitrophenyl caprylate. Moreover, its activity was profoundly affected by metal ion, ionic surfactants and organic solvents. Furthermore, the two obtained recombinant lipases were immobilized on the magnetic nanoparticles for biodiesel preparation. The GC analysis showed that for the immobilized lipases Pflip1b and Pflip1a, the biodiesel yield within 24 h respectively attained 68.5% and 80.5% at 70 °C. The activities of the two immobilized lipases still remained 70% and 82% after 10 cycles of operations in non-solvent system. These characteristics and transesterification capacity of the recombinant protein indicated its great potential for organic synthesis, especially for biodiesel production.

[Advances in platform compounds and polymers from vegetable oils].

Vegetable oil is one of the most potential alternatives of petroleum and has become a hot issue in recent years. This review focuses on the influence of vegetable oil structure on platform compounds and polymers properties, and further systematically introduces their developments and the latest progress. Meanwhile, we also summarized the main confronting problems and the future development directions in the research of oil-based platform compounds and polymers. The review provides useful information for readers to fully understand biochemical engineering of vegetable oils and their prospects.

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.

Enhanced catalysis of Yarrowia lipolytica lipase LIP2 immobilized on macroporous resin and its application in enrichment of polyunsaturated fatty acids.

An immobilization strategy was employed to improve activity and operational stability of Yarrowia lipolytica lipase LIP2 (YlLIP2) by using macroporous resins as carrier. D152H, a cation-exchange resin, was the best support. Under the optimized conditions, the immobilization efficiency was 89.81% and the specific activity was 809,751 U/g, being 2.1-fold higher than that of the free lipase. Bioimprinting and interfacial activation were used to further boost the catalytic activity of YlLIP2, respectively enhanced 21.5-fold, 231.2% and 107.2% compared to the free, non-bioimprinted and non-interfacial-activated lipases. The immobilized lipase exhibited much better thermal and pH stability and broader substrate specificity; when used to enrich docosahexaenoic acid (DHA) from Chlorella protothecoides oil, it could increase 1.66-fold of DHA content and show good operational stability. These indicate that the immobilized YlLIP2 offers a promising approach for the enrichment of DHA.

Efficient display of active Geotrichum sp. lipase on Pichia pastoris cell wall and its application as a whole-cell biocatalyst to enrich EPA and DHA in fish oil.

Geotrichum sp. lipase (GSL) was first displayed on the cell wall of Pichia pastoris on the basis of the a-agglutinin anchor system developed in Saccharomyces cerevisiae . Surface display levels were monitored using Western blotting, immunofluorescence miscroscopy, and fluorescence-activated cell sorting analysis. Lipase activity of the yeast whole cells reached a maximum at 273 ± 2.4 U/g of dry cells toward olive oil after 96 h of culture at 30 °C, with optimal pH and temperature at 7.5 and 45 °C, respectively. Displayed GSL exhibited relatively high stability between pH 6.0 and 8.0 and retained >70% of the maximum activity. The surface-displayed lipase retained 80% of its original activity after incubation at 45 °C for 4 h. Moreover, the GSL-displaying yeast whole cells were then used as a biocatalyst to enrich eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil on the basis of selective hydrolysis. As a result, EPA and DHA increased from 1.53 and 24.1% in the original fish oil to 1.85 and 30.86%, which were increases of 1.21- and 1.29-fold, respectively. The total yield of EPA and DHA reached 46.62%.

[Study on chemical composition of ethylacetate fraction from Polygonum amplexicaule var. sinense].

OBJECTIVE: To study chemical composition of ethylacetate fraction from Polygonoum amplexicaule D. Don var. sinense Forb. METHODS: TLC, Normal-phase silica gel column, reveres-phase silica gel column, Sephadex-LH, semi-preparative HPLC column were used to isolate chemical compositions of ethylacetate fraction from Polygonoum var. sinense. RESULTS: Eight compounds were identified as: 1. P-Hydroxybenzoic acid, 2. P-Hydroxybenzoic ethanol, 3. Diisobutyl phthalate, 4. Vanillin, 5. Isovanillic acid, 6.3,4,5-trihydroxy-benzoic acid-butyl ester, 7. 4-hydroxy-3-methoxycinnamic acid, 8. 7-hydroxy-6-methoxycoumarin. CONCLUSION: Except of Diisobutyl phthalate, the others are isolated for the first from this plant, moreover, Vanillin, Isovanillic acid and P-hydro -xyphenethyl alcohol are gained from genus for the first time.

Stimulative effects of Polygonum amplexicaule var. sinense on osteoblastic MC3T3-E1 cells.

CONTEXT: Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a well known traditional herb used to treat some diseases, such as fractures, rheumatoid arthritis, muscle injury, and pain. However, its pharmacological mechanism of promoting the healing of fractures is still unknown. OBJECTIVE: The present study was designed to investigate the effects of PAF ethanol extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro, thereby to illuminate the pharmacological mechanism to promote the healing of fractures. MATERIALS AND METHODS: The effects of PAF ethanol extracts on MC3T3-E1 cell proliferation and differentiation were detected by using CCK-8, cell cycle, alkaline phosphatase (ALP), and prostaglandin E(2) (PGE(2)) assays in vitro. RESULTS: The results showed that PAF ethanol extracts significantly stimulated cell proliferation at 0.1-100 µg/mL and the proportion of cells in S-phase increased from 16.33 to 27.29% in osteoblastic MC3T3-E1 cells. Moreover, PAF ethanol extracts increased ALP expression in MC3T3-E1 cells at the concentration from 0.1 to 100 µg/mL and inhibited PGE(2) production induced by TNF-α in osteoblasts at the concentrations ranging from 10 to 100 µg/mL in MC3T3-E1 osteoblasts. DISCUSSION AND CONCLUSION: These results indicated that PAF directly stimulates cell proliferation and differentiation of osteoblasts; therefore, this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.

Enzymatic enrichment of polyunsaturated fatty acids using novel lipase preparations modified by combination of immobilization and fish oil treatment.

Novel modification methods for lipase biocatalysts effective in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids (PUFAs) were described. Based on conventional immobilization in single aqueous medium, immobilization of lipase in two phase medium composed of buffer and octane was employed. Furthermore, immobilization (in single aqueous or in two phase medium) coupled to fish oil treatment was integrated. Among these, lipase immobilized in two phase medium coupled to fish oil treatment (IMLAOF) had advantages over other modified lipases in initial reaction rate and hydrolysis degree. The hydrolysis degree increased from 12% with the free lipase to 40% with IMLAOF. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipases, while low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment. After five cycles of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.

Preparation of cross-linked lipase-coated micro-crystals for biodiesel production from waste cooking oil.

A dual modification procedure composed of cross-linking and protein coating with K(2)SO(4) was employed to modify Geotrichum sp. lipase for catalyzing biodiesel production from waste cooking oil. Compared to single modification of protein coating with K(2)SO(4), the dual modification of cross-linking and lipase coating improved catalytic properties in terms of thermostable stability, organic solvent tolerance, pH stability and operational stability in biodiesel production process, although biodiesel yield and initial reaction rate for CLPCMCs were not improved. After five successive batch reactions, CLPCMCs could still maintain 80% of relative biodiesel yield. CLPCMCs retained 64% of relative biodiesel yield after incubation in a pH range of 4-6 for 4 h, and 85% of relative biodiesel yield after incubation in a range of 45-50 °C for 4 h. CLPCMCs still maintained 83% of relative biodiesel yield after both treated in polar organic solvent and non-polar organic solvent for 4 h.

Lipase-catalyzed biodiesel production with methyl acetate as acyl acceptor.

Biodiesel is an alternative diesel fuel made from renewable biological resources. During the process of biodiesel production, lipase-catalyzed transesterification is a crucial step. However, current techniques using methanol as acyl acceptor have lower enzymatic activity; this limits the application of such techniques in large-scale biodiesel production. Furthermore, the lipid feedstock of currently available techniques is limited. In this paper, the technique of lipase-catalyzed transesterification of five different oils for biodiesel production with methyl acetate as acyl acceptor was investigated, and the transesterification reaction conditions were optimized. The operation stability of lipase under the obtained optimal conditions was further examined. The results showed that under optimal transesterification conditions, both plant oils and animal fats led to high yields of methyl ester: cotton-seed oil, 98%; rapeseed oil, 95%; soybean oil, 91%; tea-seed oil, 92%; and lard, 95%. Crude and refined cottonseed oil or lard made no significant difference in yields of methyl ester. No loss of enzymatic activity was detected for lipase after being repeatedly used for 40 cycles (ca. 800 h), which indicates that the operational stability of lipase was fairly good under these conditions. Our results suggest that cotton-seed oil, rape-seed oil and lard might substitute soybean oil as suitable lipid feedstock for biodiesel production. Our results also show that our technique is fit for various lipid feedstocks both from plants and animals, and presents a very promising way for the large-scale biodiesel production.

[Cell surface display of Yarrowia lipolytica lipase Lip2 in Saccharomyces cerevisiae with a-agglutinin as carrier protein].

OBJECTIVE: In order to display extracellular.lipase Lip2 from Yarrowia lipolytica on the surface of yeast Saccharomyces cerevisiae for whole cell catalysts. METHODS: The mature Lip2 encoding fragment was amplified from Yarrowia lipolytica total DNA, and was inserted into the 3'terminal of AGA2 to give the plasmid pCTLIP2 for surface display of Lip2. Olive oil, tributyrin and p-nitrophenyl palmitate (pNPP) were used as substrates to measure lipase activity. Moreover, the characterization of displayed lipase and its free form was analyzed. RESULTS: The surface displayed lipase was confirmed to be active towards olive oil, tributyrin and p-nitrophenyl palmitate (pNPP), and reached its highest expression level at 182 U/g dry cell after induced by galactose for 72h. The optimum temperature of cell surface displayed Lip2 was 40 degrees C After incubated at 50 degrees C for 4h, the surface displayed lipase retained 23.2% of its full activity, improved a little compared to free Lip2. The surface displayed lipase showed a preference to medium-chain and long-chain fatty acids p-nitrophenyl esters (C8-C16). CONCLUSION: The cell surface display system based on a-agglutinin is an effective system for displaying Lip2, and the whole cell EBY100-pCTLIP2 will be probably suited to a different range of applications.