Identification of isoliquiritigenin as an activator that stimulates the enzymatic production of glycyrrhetinic acid monoglucuronide.

Glycyrrhetinic acid monoglucuronide (GAMG) is a great value-added and has considerable commercial interest due to its strong pharmacological activities and functional low-calorie sweetener. However GAMG is quite rare in natural plants, and it must be prepared from glycyrrhizin (GL) by hydrolysing one terminal glucuronic acid. ß-Glucuronidase is the key enzyme in the biotransformation of GL to GAMG, but its activities need to be enhanced to facilitate the industrial large-scale production of GAMG. In this study, we identified that isoliquiritigenin (ISL), as one of chemical compositions from the total flavonoids glycyrrhiza (TFG), can significantly enhance ß-glucuronidase activity in vitro. Measurements using high-performance liquid chromatography (HPLC) showed that the activity of ß-glucuronidase could be increased by 2.66-fold via the addition of ISL to a ß-glucuronidase solution that contained GL at a 3:10 molar ratio of ISL to GL. ISL was concluded to be an activator because ISL could reduce the Km and Ea of ß-glucuronidase reacting with GL. This study sheds new light on the mechanism of ß-glucuronidase and helps to make industrial production of GAMG through fermentation feasible.

A novel pathway construction in Candida tropicalis for direct xylitol conversion from corncob xylan.

In this study, an integrated xylitol production pathway, directly using xylan as the substrate, was constructed in Candida tropicalis BIT-Xol-1 which could efficiently convert xylose into xylitol. In order to consolidate this bioprocessing, a ß-1,4-xylanase gene (atn) and a ß-xylosidase gene (atl) were cloned from Aspergillus terreus, and were constructed onto episomal plasmid pAUR123. Additionally, combination of the individual atn and atl expression cassette was also cloned onto pAUR123. After transforming, the positive C. tropicalis transformants co-expressing xylanase and xylosidase produced larger hydrolysis zones than those expressing xylanase alone, when incubated on xylan-congo red plates. The engineered C. tropicalis/pAUR-atn-atl-3 (C. tropicalis PNL3) secrete heterologous xylanase and xylosidase simultaneously, with the activities of 48.17 and 11.56 U/mL, respectively. The xylitol yields by C. tropicalis PNL3 utilizing xylan and corncob were 77.1% and 66.9%, respectively. The integrated pathway of xylitol production was feasible and efficient in utilization of xylan-rich renewable biomass via combining saccharification and transformation of xylan in engineered C. tropicalis.

Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18.

Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5mg/kg were obtained after 5h of enzyme treatment at 40°C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields.

Effects of low-shear modeled microgravity on the characterization of recombinant ß-D-glucuronidase expressed in Pichia pastoris.

In this study, we used a high-aspect-ratio vessel (HARV), which could model environment of microgravity on ground to investigate for the first time the effects of low-shear modeled microgravity (LSMMG) on the characterization of recombinant ß-D-glucuronidase expressed in Pichia pastoris. The ß-D-glucuronidase gene (GenBank accession no. EU095019) derived from Penicillium purpurogenum Li-3 encoding ß-D-glucuronidase (PGUS) was expressed in P. pastoris GS115 in two different environments of LSMMG and normal gravity (NG). Results manifested that both LSMMG and NG conditions had insignificant effects on temperature and pH activity (optimal temperature and pH were 55 and 5.0 °C, respectively) and characteristic stability of recombinant PGUS. However, the catalytic activity of recombinant PGUS expressed under LSMMG was less affected by metal ions and EDTA as compared with that of NG. Furthermore, K (m) value of the recombinant PGUS expressed under LSMMG was nearly one fifth of that under NG (1.72 vs. 7.72), whereas catalytic efficiency (k (cat)/K (m)) of PGUS expressed under LSMMG (13.55) was 3.7 times higher than that of NG (3.61). The results initially reveal the significant alterations in catalytic properties of recombinant enzyme in response to LSMMG environment and have potential application in bioprocessing and biocatalysis.

Simulated microgravity affects growth of Escherichia coli and recombinant beta-D-glucuronidase production.

Effects of simulated microgravity (SMG) on bacteria have been studied in various aspects. However, few reports are available about production of recombinant protein expressed by bacteria in SMG. In this study growth of E. coli BL21 (DE3) cells transformed with pET-28a (+)-pgus in double-axis clinostat that could model low shear SMG environment and the recombinant beta-D-glucuronidase (PGUS) expression have been investigated. Results showed that the cell dry weights in SMG were 16.47%, 38.06%, and 28.79% more than normal gravity (NG) control, and the efficiency of the recombinant PGUS expression in SMG were 18.33%, 19.36%, and 33.42% higher than that in NG at 19 degrees C, 28 degrees C, and 37 degrees C, respectively (P < 0.05).

Effect of lipase immobilization on resolution of (R, S)-2-octanol in nonaqueous media using modified ultrastable-Y molecular sieve as support.

The lipase from Penicillium expansum PED-03 (PEL) was immobilized onto modified ultrastable-Y (USY) molecular sieve and the resolution of (R, S)- 2-octanol was carried out in a bioreactor in nonaqueous media by the immobilized lipase. It was found that the conversion rate, enantiomeric excess (ee) value, and enantioselectivity (E) value of the resolution catalyzed by PEL immobilized on modified USY molecular sieve were much higher than those of the reaction catalyzed by free PEL and PEL immobilized on other supports. Immobilized on modified USY molecular sieve, the PEL exhibited obvious activity within a wider pH range and at a much higher temperature and showed a markedly enhanced stability against thermal inactivation, by which the suitable pH of the buffer used for immobilization could be "memorized." The conversion rate of the reaction catalyzed by PEL immobilized on modified USY molecular sieve reached 48.84%, with excellent enantioselectivity (average E value of eight batches >460) in nonaqueous media at "memorial" pH 9.5, 50 degrees C for 24 h, demonstrating a good application potential in the production of optically pure (R, S)-2-octanol.

Enhanced production of Penicillium expansum PED-03 lipase through control of culture conditions and application of the crude enzyme in kinetic resolution of racemic Allethrolone.

Alkaline lipase production was performed in submerged fermentation by Penicillium expansum PED-03. It was found that the suitable carbon source and nitrogen source for lipase production were 0.5% starch and 4.0% soybean meal, respectively. The maximal lipase activity (850 U/mL) of production was achieved at initial pH 5.5-6.0, 26 degrees C, 72 h. Tween-80 was an effective enhancer for lipase production. Agitation speed of the fermentor played an important role, and the suitable agitation speed for lipase production was 500 r/min. The lipase was stable within the range of pH 7.0-10.0 and 20-40 degrees C, and the optimum conditions for the enzymatic reaction were 35 degrees C and pH 9.5. The enzymatic resolution of racemic allethrolone (4-hydroxy-3-methyl-2-(2-propenyl)-2- cyclopenten-1-one) was carried out by the lipase from P. expansum PED-03, and the conversion reached 48% with excellent enantioselectivity (E > 100), which showed a good application potential in the production of optically pure allethrolone.