Purification and In Situ Immobilization of Papain with Aqueous Two-Phase System

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96–100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33–17.65% (w/w) PEG 6000, 14.27–14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77–6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain

Low-Transition-Temperature Mixtures (LTTMs) for Dissolving Proteins and for Drug Formulation

Several diverse proteins are found to readily dissolve in neat low-transition-temperature mixtures (LTTMs). They undergo no irreversible denaturation in such unusual solvents, and the resistance of hen egg-white lysozyme against thermoinactivation in LTTMs is greater than in aqueous solution at extreme pHs. Separately, the water-sensitive drug aspirin is found to form concentrated transparent LTTMs, where it is some 10-fold more stable against cleavage than in water

Inactivation of Bacteria by Electric Current in the Presence of Carbon Nanotubes Embedded Within a Polymeric Membrane

Uniform conductive composite membranes were prepared using a phase inversion method by blending carboxyl-functionalized multi-walled carbon nanotubes (CNTs) with a polysulfone polymer. At 6 % of the embedded CNTs, the membrane pore size measured by transmission electron microscopy (TEM) was approximately 50 nm. Electric current in the presence of the composite membranes markedly inactivated the model pathogenic bacteria Escherichia coli and Staphylococcus aureus, with the extent of bacterial inactivation rising when the current was increased. Over 99.999 % inactivation of both bacteria was observed in deionized water after 40 min at 5 mA direct current (DC); importantly, no appreciable inactivation occurred in the absence of either the electric field or the CNTs within the membranes under otherwise the same conditions. A much lower, although still pronounced, inactivation was seen with alternating current (AC) in a 25 mM NaCl aqueous solution.Skolkovo Foundation (Infectious Diseases Research Center at MIT)National Natural Science Foundation of China (21277173

<it>In vivo</it> functional expression of a screened <it>P. aeruginosa</it> chaperone-dependent lipase in <it>E. coli</it>

<p>Abstract</p> <p>Background</p> <p>Microbial lipases particularly <it>Pseudomonas</it> lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the <it>Pseudomonas</it> lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to <it>Pseudomonas aeruginosa</it> were overexpressed in <it>E. coli</it> with two dual expression plasmid systems to enhance the production of the active lipase LipA without <it>in vitro</it> refolding process.</p> <p>Results</p> <p>In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as <it>P. aeruginosa</it> on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene <it>lipA</it> (936 bp) and lipase specific foldase gene <it>lipB</it> (1023 bp). One single expression plasmid system <it>E. coli</it> BL21/pET28a-<it>lipAB</it> and two dual expression plasmid systems <it>E. coli</it> BL21/pETDuet-<it>lipA</it>-<it>lipB</it> and <it>E. coli</it> BL21/pACYCDuet-<it>lipA</it>-<it>lipB</it> were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by <it>E. coli</it> BL21/pET28a-<it>lipAB</it> and <it>E. coli</it> BL21/pETDuet-<it>lipA</it>-<it>lipB</it> were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by <it>E. coli</it> BL21/pACYCDuet-<it>lipA</it>-<it>lipB</it> was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively.</p> <p>Conclusions</p> <p>The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-<it>lipA</it>-<it>lipB</it> was more suitable for the expression of active LipA than pET28a-<it>lipAB</it> and pETDuet-<it>lipA</it>-<it>lipB</it>. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.</p

Study on Synergistic Anti-Inflammatory Effect of Typical Functional Components of Extracts of Ginkgo Biloba Leaves

There are some differences in the anti-inflammatory activities of four typical components in EGB (extracts of ginkgo biloba leaves), and there is also a synergistic relationship. The order of inhibiting the NO-release ability of single functional components is OA > GF > OPC > G. Ginkgolide (G), proanthocyanidins (OPC), and organic acids (OA) all have synergistic effects on ginkgo flavonoids (GF). GF:OA (1:9) is the lowest interaction index among all complexes, showing the strongest synergy. The anti-inflammatory mechanism of the compound affects the expression of p-JNK, p-P38, and p-ERK1/2 proteins by inhibiting the expression of iNOS and COX2 genes on NFKB and MAPK pathways. This also provides a research basis for the development of anti-inflammatory deep-processing products of EGB

Tailor-Made Deep Eutectic Solvents for Simultaneous Extraction of Five Aromatic Acids from <i>Ginkgo biloba</i> Leaves

Ginkgo biloba leaves have various health benefits due to the presence of bioactive compounds such as polyprenyl acetates, flavonoids, and terpene trilactones. However, there is little literature reported on the aromatic acids in Ginkgo biloba leaves. In this work, five aromatic acids including shikimic acid (SA), 6-hydroxykynurenic acid (6-HKA), protocatechuic acid (PA), gallic acid (GAA), and p-hydroxybenzoic acid (PHBA) were simultaneously extracted from Ginkgo biloba leaves by employing the green deep eutectic solvents (DESs). A DES tailor-made from xylitol, glycolic acid and 1,5-pentanedioic acid at a molar ratio of 1:3:1 with 50% (w/w) water addition, named as NGG50, gave higher extraction yields for the five aromatic acids. Main factors affecting the extraction process were further optimized. The highest extraction yields of SA, GAA, 6-HKA, PA, and PHBA were 94.15 &#177; 0.96 mg/g, 332.69 &#177; 5.19 &#956;g/g, 25.90 &#177; 0.61 &#956;g/g, 429.89 &#177; 11.47 &#956;g/g and 67.94 &#177; 0.37 &#956;g/g, respectively. The NGG50-based extraction process developed here was a successful attempt of simultaneously extracting five aromatic acids from Ginkgo biloba leaves for the first time, which could provide a new exploitation direction of Ginkgo biloba leaves

Study on Synergistic Antioxidant Effect of Typical Functional Components of Hydroethanolic Leaf Extract from Ginkgo Biloba In Vitro

The predicted anti-oxidation is related to apoptosis, proliferation, lipid metabolism, cell differentiation, and immune response. There are some differences in the antioxidant capacity of the four typical components of ginkgo biloba extract (EGb) including ginkgo flavone (GF), ginkgolide (G), procyanidins (OPC), and organic acids (OA), and any two members of them can exhibit apparent synergistic effects. The order of DPPH scavenging ability was: OPC > GF > OA > G. The scavenging ability of procyanidins was close to that of VC; the scavenging capacity of ABTS was GF > OPC > OA > G. The GF:OPC (1:9) showed the best synergism in scavenging DPPH and ABTS radicals. The 193 kinds of small molecules reported in EGb were obtained by analyzing the properties of EGb. In order to construct a corresponding biological activity target set, molecular docking and the network pharmacology method were employed to build the molecular action mechanism network of a compound target, and the main biological functions and signaling pathways involved with their antioxidant activities were predicted. The results displayed that the top ten compounds which belonged to the two broad categories, ginkgo flavonoids and proanthocyanidins, could interact closely with several important target proteins (CASP3, SOD2, MAPK1, HSPA4, and NQO1). This would be expected to lay a theoretical foundation for the deep development of Ginkgo biloba extract

Synthesis of Isorhamnetin-3-O-Rhamnoside by a Three-Enzyme (Rhamnosyltransferase, Glycine Max Sucrose Synthase, UDP-Rhamnose Synthase) Cascade Using a UDP-Rhamnose Regeneration System

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 &deg;C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 &deg;C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 &mu;M, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 &deg;C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids