Continuous synthesis and anti-myocardial injury of tanshinone IIA derivatives.

A series of tanshinone IIA derivatives were synthesized through sulfonation, slat-forming, chlorination, and amidation reactions. Meanwhile, anti-myocardial injury activity was evaluated in vitro. D8 and D9 exhibited a slightly higher anti-myocardial injury (5.78, 7.46 µM) activity compared with esmolol (8.12 µM). In addition, they also displayed a concentration-dependent inhibition on the anti-myocardial injury.

Fungal arachidonic acid-rich oil: research, development and industrialization.

Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.

Bioavailability comparison of a new form of vilazodone XVII to IV in beagles using liquid chromatography/mass spectrometry.

Vilazodone hydrochloride (CAS 163521-12-8) is polymorphic and has 15 crystal forms, referred to as I-XI and XIII-XVI. In the study, we prepared and performed structural identification of a new crystal form named XVII. To investigate this in vivo, a rapid and sensitive method based on liquid-liquid extraction, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the determination of vilazodone hydrochloride in dog plasma. This HPLC-MS/MS method was successfully applied to a bioavailability comparison of two crystal forms of vilazodone hydrochloride (IV and XVII) in six healthy beagles using a single-dose, two-way crossover design. The maximum plasma concentration (C(max)), the time taken to reach C(max), and the area under the concentration-time curve were determined following oral administration of 10 mg vilazodone hydrochloride (IV or XVII) to beagles. These analyses revealed no significant bioavailability differences between vilazodone hydrochloride forms IV and XVII in dogs.

An oxidoreduction potential shift control strategy for high purity propionic acid production by Propionibacterium freudenreichii CCTCC M207015 with glycerol as sole carbon source.

The effects of oxidoreduction potential (ORP) regulation on the process of propionic acid production by Propionibacterium freudenreichii CCTCC M207015 have been investigated. Potassium ferricyanide and sodium borohydride were determined as ORP control agents through serum bottle experiment. In batch fermentation, cell growth, propionic acid and by-products distribution were changed with ORP levels in the range of 0-160 mV. Based on these analysis results, an ORP-shift control strategy was proposed: at first 156 h, ORP was controlled at 120 mV to obtain higher cell growth rate and propionic acid formation rate, and then it was shifted to 80 mV after 156 h to maintain the higher propionic acid formation rate. By applying this strategy, the optimal parameters were obtained as follows: the propionic acid concentration 45.99 g L(-1), productivity 0.192 g L(-1) h(-1), the proportion of propionic acid to total organic acids 92.26 % (w/w) and glycerol conversion efficiency 76.65 %. The mechanism of ORP regulation was discussed by the ratio of NADH/NAD(+), ATP levels, and metabolic flux analysis. The results suggest that it is possible to redistribute energy and metabolic fluxes by the ORP-shift control strategy, and the strategy could provide a simple and efficient tool to realize high purity propionic acid production with glycerol as carbon source.

Ultrasonic pretreatment and acid hydrolysis of sugarcane bagasse for succinic acid production using Actinobacillus succinogenes.

Immense interest has been devoted to the production of bulk chemicals from lignocellulose biomass. Diluted sulfuric acid treatment is currently one of the main pretreatment methods. However, the low total sugar concentration obtained via such pretreatment limits industrial fermentation systems that use lignocellulosic hydrolysate. Sugarcane bagasse hemicellulose hydrolysate is used as the carbon and nitrogen sources to achieve a green and economical production of succinic acid in this study. Sugarcane bagasse was ultrasonically pretreated for 40 min, with 43.9 g/L total sugar obtained after dilute acid hydrolysis. The total sugar concentration increased by 29.5 %. In a 3-L fermentor, using 30 g/L non-detoxified total sugar as the carbon source, succinic acid production increased to 23.7 g/L with a succinic acid yield of 79.0 % and a productivity of 0.99 g/L/h, and 60 % yeast extract in the medium could be reduced. Compared with the detoxified sugar preparation method, succinic acid production and yield were improved by 20.9 and 20.2 %, respectively.

Clostridium beijerinckii mutant with high inhibitor tolerance obtained by low-energy ion implantation.

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l(-1) of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g(-1). When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l(-1) of ABE with a yield of 0.34 g g(-1), including 2.2 g l(-1) acetone, 6.8 g l(-1) butanol, and 0.5 g l(-1) ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.

(4R*,5R*)-2-(4-Meth-oxy-phen-yl)-1,3-dioxolane-4,5-dicarboxamide.

In the title compound, C(12)H(14)N(2)O(5), the five-membered 1,3-dioxolane ring has a twisted conformation. In the crystal, N-H⋯O and C-H⋯O hydrogen bonds link the mol-ecules into a two-dimensional network lying parallel to the ab plane. There are also C-H⋯π inter-actions present in the crystal structure.

(4R*,5R*)-Diethyl 2-(4-nitro-phen-yl)-1,3-dioxolane-4,5-dicarboxyl-ate.

In the title compound, C(15)H(17)NO(8), the nitro group is essentially coplanar with the aromatic ring [dihedral angle = 6.4 (3) Å]. The five-membered ring has a twist conformation. In the crystal, C-H⋯O inter-actions link the mol-ecules into a helical chain propagating along [010].

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose-xylose mixtures.

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.

Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields.

With 30 g glucose/l as carbon source, Clostridium beijerinckii ART124, a mutant created by atmospheric pressure glow discharge, produced 13.7 g total solvent/l (containing 3.1 g acetone/l, 10.4 g butanol/l and 0.2 g ethanol/l) in 72 h. The mutant could also use sucrose or xylose or a mixture of glucose/xylose/arabinose with nearly equal yields.

Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428.

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (µ), specific glucose consumption rate (q (s)), and specific welan formation rate (q (p)), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high µ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L(-1) with a yield of 0.53 ± 0.003 g g(-1) and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.

Strategies for efficient repetitive production of succinate using metabolically engineered Escherichia coli.

Escherichia coli AFP111, a pflB, ldhA, ptsG triple mutant of E. coli W1485, can be recovered for additional succinate production in fresh medium after two-stage fermentation (an aerobic growth stage followed by an anaerobic production stage). However, the specific productivity is lower than that of two-stage fermentation. In this study, three strategies were compared for reusing the cells. It was found when cells were aerobically cultivated at the end of two-stage fermentation without supplementing any carbon source, metabolites (mainly succinate and acetate) could be consumed. As a result, enzyme activities involved in the reductive arm of tricarboxylic acid cycle and the glyoxylate shunt were enhanced, yielding a succinate specific productivity above g⁻¹(DCW)h⁻¹ and a mass yield above 0.90 g g⁻¹ in the subsequent anaerobic fermentation. In addition, the intracellular NADH of cells subjected to aerobic cultivation with metabolites increased by more than 3.6 times and the ratio of NADH to NAD+ increased from 0.4 to 1.3, which were both favorable for driving the TCA branch to succinate.

Biosynthetic pathway of sugar nucleotides essential for welan gum production in Alcaligenes sp. CGMCC2428.

Welan gum is a microbial polysaccharide produced by Alcaligenes sp. CGMCC2428 that has D-glucose, D-glucuronic acid, D-glucose, and L-rhamnose as the main structural unit. The biosynthetic pathway of sugar nucleotides essential for producing welan gum in this strain was established in the following ways: (1) the detection of the presence of several intermediates and key enzymes; (2) the analysis of the response upon addition of precursors to the culture medium; (3) the correlation of the activities between several key enzymes with the yields of welan gum. With addition of 200-microM glucose-6-phosphate and fructose-6-phosphate, the production of welan gum was improved by 18%. The activities of phosphoglucomutase, phosphomannose isomerase, UDP-glucose pyrophosphorylase, and dTDP-glucose pyrophosphorylase, correlated well with the yields of welan gum. According to these findings, the biosynthetic pathway was proposed to involve the metabolism of glucose via two discrete systems. The first involves conversion of glucose to glucose-6-phosphate, with further reactions producing glucose-1-phosphate and fructose-6-phosphate, which are metabolized to the nucleotide sugar precursors of welan gum. The second system involves metabolism of glucose to synthesize the basic structural skeleton of the cell via central metabolic pathways, including the Entner-Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle.

Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp.

The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1,500-L bioreactor using fed-batch fermentation. Six parameters, including specific growth rate, specific glucose consumption rate, specific lipid accumulation rate, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient, were used to understand the relationship between aeration and the fermentation characteristics. Based on the information obtained from the parameters, a stepwise aeration control strategy was proposed. The aeration rate was controlled at 0.4 volume of air per volume of liquid per minute (vvm) for the first 24 h, then shifted to 0.6 vvm until 96 h, and then switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L), and high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained by constant aeration rate.

Production of D-arabitol by a newly isolated Kodamaea ohmeri.

A new yeast, isolated from natural osmophilic sources, produces D-arabitol as the main metabolic product from glucose. According to 18S rRNA analysis, the NH-9 strain belongs to the genus Kodamaea. The optimal culture conditions for inducing production of D-arabitol were 37 degrees C, neutral pH, 220 rpm shaking, and 5% inoculum. The yeast produced 81.2 +/- 0.67 g L(-1) D-arabitol from 200 g L(-1) D-glucose in 72 h with a yield of 0.406 g g(-1) glucose and volumetric productivity Q(P) of 1.128 g L(-1) h(-1). Semi-continuous repeated-batch fermentation was performed in shaker-flasks to enhance the process of D-arabitol production by Kodamaea ohmeri NH-9 from D-glucose. Under repeated-batch culture conditions, the highest volumetric productivity was 1.380 g L(-1) h(-1).

Propionic acid fermentation by Propionibacterium freudenreichii CCTCC M207015 in a multi-point fibrous-bed bioreactor.

Propionic acid was produced in a multi-point fibrous-bed (MFB) bioreactor by Propionibacterium freudenreichii CCTCC M207015. The MFB bioreactor, comprising spiral cotton fiber packed in a modified 7.5-l bioreactor, was effective for cell-immobilized propionic acid production compared with conventional free cell fermentation. Batch fermentations at various glucose concentrations were investigated in the MFB bioreactor. Based on analysis of the time course of production, a fed-batch strategy was applied for propionic acid production. The maximum propionic acid concentration was 67.05 g l(-1) after 496 h of fermentation, and the proportion of propionic acid to total organic acids was approximately 78.28% (w/w). The MFB bioreactor exhibited excellent production stability during batch fermentation and the propionic acid productivity remained high after 78 days of fermentation.

Isolation of NH4+-tolerant mutants of Actinobacillus succinogenes for succinic acid production by continuous selection.

Actinobacillus succinogenes, a typical succinic acid producing microorganism, was inhibited seriously by ammonium ion, which hampered industrialization of A. succinogenes with ammonium ion based material as the pH controller. In this study, we have isolated an ammonium ion-tolerant mutant of A. succinogenes by continuous-culture technique in which all environmental factors beside the stress (ammonium ion) were maintained constant. In this technique, the mutant-generating system was not operated as a nutrient-limited chemostat, but as a nutrient-unlimited system where cells were continuous cultured at the maximum specific growth rate. Mutants were isolated on agar plates containing a acid-base indicator bromothymol blue and high level of ammonium ion which gives 100% killing of the parent strain. When cultured in anaerobic bottles at ammonium ion concentration of 354 mmol/L, the mutant YZ0819 could produce succinic acid 40.21 g/L with yield 80.4%, while the parent strain NJ113 did not grow. With NH4OH being used to buffer the culture pH in 3.0 liter stirred bioreactor, YZ0819 produced 35.15 g/L succinic acid with yield 70.3%, 155% higher than that obtained by NJ113. In addition, the morphology of YZ0819 in fermentation broths was changed. Cells of YZ0819 were aggregated from the beginning to the end of fermentation. These results indicate that YZ0819 would be used to be the potential strain produced efficiently succinic acid with NH4OH as the pH controller and the formation of aggregates may be useful as an aid in the transferring of cells from a cultivation medium for various industrial applications.

Simultaneous Determination of Four Active Components in Tobacco Wastes by LC.

A liquid chromatographic method was developed for the simultaneous quantification of four major active components in tobacco (Nicotiana tobaccum L.) wastes. Samples were extracted with 70% v/v aqueous methanol, four compounds including chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and caffeic acid were identified and determined by using LC coupled to electrospray tandem mass spectrometry and LC-UV method, respectively. Separation in LC-UV was on an Alltima C18 column (250 mm × 4.6 mm i.d.; 5 µm) with a mobile phase consisting acetonitrile: ammonium acetate buffer (pH 4.5) (5:95 v/v), at a flow rate of 1.0 mL min-1, detected at 327 nm. Four regression equations showed good linear relationships (r 2 > 0.999) between the peak area of each marker and concentration. The method has good repeatability and precision, the intra-day and inter-day RSD for both retention time and peak area was less than 1.0%. The recoveries, measured at three concentration levels, varied from 96.33 to 101.10%. The LOD (S/N = 3) and LOQ (S/N = 6) were less than 0.010 and 0.795 µg·mL-1, respectively. This assay was successfully applied to the determination of four active compounds in ten samples. The results indicated that the developed assay method was rapid, accurate, reliable and could be readily utilized as a quantitative analysis method for various of tobacco wastes.

New gene cluster from the thermophile Bacillus fordii MH602 in the conversion of DL-5-substituted hydantoins to L-amino acids.

The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-alpha-amino acids. Since the reaction at higher temperature, the advantageous for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile was firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2 kb DNA fragment by Restriction Site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein hyuP, hyperprotein hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-alpha-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 comparing respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.

Clavuridins A and B, two new trinor-guaiane sesquiterpenes isolated from the Xisha soft coral Clavularia viridis.

In the present study, two new trinor-guaiane sesquiterpenes, named clavuridins B (1), and A (2), along with three known sesquiterpenes (3-5), were isolated from the Xisha soft coral Clavularia viridis. Their structures and absolute configurations were determined on the basis of spectroscopic analysis, X-ray diffraction analysis with Cu Kα radiation and by comparison with related model compounds. Compounds 1 and 3-5 were evaluated for their cytotoxic activity.