Search

Finger citron pomace is a cheap and renewable by-product of the citrus processing industry, representing up to 60% of the fruit biomass. In this study, a pectinase-based and ultrasonic-assisted method was firstly used to extract pectic oligosaccharides (POS) from finger citron pomace. Using the orthogonal experiment design (OED), the maximum conversion rate of up to 64.5% from pomace to POS was obtained under the extraction conditions of 0.25 mg mL-1 pectinase and 50 mg mL-1 pectin at 45 °C and pH 4.5 for 2 h. The extracted POS was then fractionated and purified to homogeneous oligosaccharides (FCPOS-1) with a molecular weight of 2.15 kDa, and the analyses of monosaccharide composition, FTIR, NMR and ESI-MS indicated that FCPOS-1 consisted of GalA and a small amount of mannose, galactose and arabinose. Multiple antioxidant activity assays in vitro revealed that FCPOS-1 possessed remarkable antioxidant properties, especially scavenging activity against DPPH radicals up to 94.07%. FCPOS-1 has the potential to be an effective natural antioxidant for applications in the food and pharmaceutical industries.

In this study, a novel lipase M5 derived from Aspergillus oryzae WZ007 was prone to exhibit high hydrolytic activity and stereoselectivity towards racemic substrate (R,S)-ethyl 2-bromoisovalerate. (R)-ethyl 2-bromoisovalerate was obtained by enzymatic resolution, which is the key chiral intermediate for highly efficient enantiomerically fluvalinate. The results showed that the enzymatic reaction was carried out in 120mM racemic substrate for 3 hours, the enantiomeric excess reached 98.6%, the conversion was 51.7%, and E value above 120. Therefore, the novel lipase M5 has the ability to efficiently produce (R)-ethyl 2-bromoisovalerate, which greatly reduces the industrial production cost of the highly efficient counterpart of fluvalinate.

An intracellular esterase (BCE) from Bacillus cereus WZZ006 was purified to homogeneity with an 89.5-fold purification, specific activity of 1.79 U/mg, and 26.7% recovery. The estimated molecular weight of BCE was 96 kDa which was analyzed by SDS-PAGE and MALDI-TOF-MS. Activity staining denotes that BCE has an unexplored new carboxyl esterase characteristic. BCE enzyme activity was maximum at pH 8.5 and also at 50 °C with pNP-caproate as a substrate. This indicates that the studied BCE as a thermoalkaliphilic esterase. The kinetic properties like Km, Vmax, kcat and kcat/Km value for BCE was found to be 0.98 mM, 0.03 mM/min, 69.47 min−1 and 70.89 mM−1 min−1, respectively. Synthesis of (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indole-2-carboxylic acid methyl ester ((S)-CODHCM) by BCE can be shortened to 3 h compared to 36 h with whole-cell catalysis. The e.e.s achieved was 93.83%, and conversion around 52.78% with E being 39.95. These features render BCE as a promising biocatalyst for the synthesis of a key chiral intermediate for indoxacarb.

Brivaracetam is a structural derivative of the chiral drug levetiracetam and has been approved for the adjuvant treatment of partial epilepsy. As a new antiepileptic drug, it is widely used in a variety of epilepsy models. In this study, a novel lipase M16 derived from Aspergillus oryzae WZ007 was cloned, expressed, and used for chiral resolution. Lipase M16 has a high enantioselectivity to the racemic substrate (R,S)-methyl 2-propylsuccinate 4-tert-butyl ester, and the intermediate (R)-2-propylsuccinic acid 4-tert-butyl ester of brivaracetam was obtained efficiently. Under optimal conditions, the enantiomeric excess of substrate was up to 99.26%, and the e.e.p was 96.23%. The conversion and apparent E value were 50.63% and 342.48, respectively. This study suggests a new biocatalytic resolution via lipase M16 for preparing the brivaracetam chiral intermediate and its potential application in the pharmaceutical industry.

The kinetic resolution of (R,S)-1-(4-chlorophenyl)ethylamine was accomplished using a commercial lipase from Candida antarctica (Novozym 435). The performance of this lipase was investigated for the enantioselective amidation of (R,S)-1-(4-chlorophenyl)ethylamine, leaving the target product (S)-1-(4-chlorophenyl)ethylamine in its unreacted form. The effects of various types of solvents and an acyl donor, the molar ratio of the substrate to the acyl donor, and the reaction temperature were studied. The optimum reaction conditions were found to result in amidation with methyl 2-tetrahydrofuroate at 40°C in methyl tert-butyl ether, with a substrate/acyl donor molar ratio of 1:2.4. The conversion rate of (R,S)-1-(4-chlorophenyl)ethylamine was 52%, with an enantiomeric excess of 99% towards the unreacted substrate in a reaction time of 22 hours. Finally, using optically pure (S)-1-(4-chlorophenyl)ethylamine as the raw material, the chemical synthesis of (S)-N-(1-(4-chlorphenyl)ethyl)-2-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)acetamide, a novel triazolopyrimidine herbicide, was achieved, and the total yield and purity were 83.5% and 95.3%, respectively.

Only a few esterases have been used for the synthesis of optically pure fungicide. For example, (R)-metalaxyl synthesized using esterase-involved bioreaction displays fungicide activity, whereas (S)-enantiomer is redundant. However, the biosynthesis of (R)-metalaxyl is currently hampered by the lower activity, selectivity and thermostability of esterase. Therefore, to obtain a better biocatalyst, several esterase genes were cloned from Pseudochrobactrum asaccharolyticum WZZ003. The esterase PAE07, among eight enzymes, was selected because it exhibited the highest hydrolysis activity toward (R,S)-DMPM. The DNA and amino acid sequence analysis suggested that PAE07 is a new member of lipolytic enzyme family V. The enzymatic properties of PAE07 toward (R,S)-DMPM and model substrate (p-nitrophenyl acetate) were investigated. PAE07 was found to be a highly active esterase with excellent enantioselectivity. The reaction conditions including temperatureand pH were optimized, and the effects of metal ions, organic solvents and detergents were also investigated. Results indicated that PAE07 is a competitive candidate for (R)-metalaxyl manufacturing.

Nutrient reduction was an important but costly strategy to control harmful algal blooms (HABs) in eutrophic lakes. It is still unclear that when and where to reduce nutrients for controlling HABs in a large eutrophic lake. This study proposed a nutrient loading contribution index (NLCI) to evaluate the contribution of nutrient loading on HABs in a large eutrophic lake (Lake Chaohu, China). The index was calculated using a hydrological, hydrodynamic and water quality model. A multi-site and multi-variable calibration revealed that the coupled model captured the spatio-temporal pattern of chlorophyll a in the lake representing HABs. The evaluation results revealed that effective HABs controlling in the lake depended on reducing P loading. Both internal and external P loading contributed significantly to HABs in the lake. Nanfei and Hangbu Rivers were the hot spots for external P reduction. Aug., May and Jul. were the hot moments for external P reduction. The new index (NLCI) can be potentially used in other large eutrophic lakes to make a better strategy for nutrient reduction in water management practice.

A novel enzyme immobilization method employing metal–organic framework (MOF) encapsulation and macroporous resin adsorption was developed in this study. Candida antarctica lipase B (CALB) was firstly encapsulated onto metal–organic frame structures (Zeolitic imidazole framework-8, ZIF-8) and further bonded to macroporous resin by physical adsorption. Under optimized immobilization conditions, the activity of the prepared immobilized lipase (CALB-ZIF-8@D101) determined via the methyl esterification of oleic acid was 38.4 U/mg. Compared with free lipase, the immobilized lipase exhibited improved thermal and operational stability and organic solvent tolerance. These results demonstrate that the immobilization method of ZIF-8 encapsulation and macroporous resin adsorption enhanced enzyme properties at a superior level.

In this study, a newly isolated strain screened from the indoxacarb-rich agricultural soils, Bacillus cereus WZZ006, has a high stereoselectivity to racemic substrate 5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester. (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester was obtained by bio-enzymatic resolution. After the 36-hour hydrolysis in 50-mM racemic substrate under the optimized reaction conditions, the e.e.s was up to 93.0% and the conversion was nearly 53.0% with the E being 35.0. Therefore, B cereus WZZ006 performed high-level ability to produce (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester. This study demonstrates a new biocatalytic process route for preparing the indoxacarb chiral intermediates and provides a theoretical basis for the application of new insecticides in agricultural production.

Jerusalem artichoke was first investigated as a non-food and cheap feedback for the production of docosahexaenoic acid (DHA) through Aurantiochytrium sp. YLH70. Using the Response surface method, up to 143.8 g/L of reducing sugar was obtained in the acidic hydrolysate of Jerusalem artichoke at 80 °C and 6% (v/v) sulphuric acid after 40 min of hydrolysis. The Plackett-Burman experiment showed that natural components of the hydrolysate of Jerusalem artichoke without the addition of any micronutrient, which is essential in traditional media, were suitable for DHA production by Aurantiochytrium sp. YLH70. Moreover, compared to the fructose or glucose medium, the Jerusalem artichoke medium resulted in a higher biomass (32.71 g/L) and DHA content (46.9% of the total fatty acid), which could be associated with the rich nutrition and high osmotic pressure (1306 mOsm/kg H2O) in the Jerusalem artichoke medium. During fed-batch fermentation under the optimal Jerusalem artichoke medium (695 mL/L hydrolysate of Jerusalem artichoke, 15 g/L yeast extract and 25 g/L sea salt), both a productivity of 3.35 g/L/d and a yield of 0.094 g/g reducing sugar for DHA production were achieved. These results demonstrate that Jerusalem artichoke, when sufficiently utilised by Aurantiochytrium sp. YLH70, could indeed be an efficient and cheap option for DHA production.

Harmful algal blooms (HABs) can have dire repercussions on aquatic wildlife and human health, and may negatively affect recreational uses, aesthetics, taste, and odor in drinking water. The factors that influence the occurrence and magnitude of harmful algal blooms and toxin production remain poorly understood and can vary in space and time. It is within this context that we use machine learning (ML) and two 14-year (2005–2018) data sets on water quality and meteorological conditions of China’s lakes and reservoirs to shed light on the magnitude and associated drivers of HAB events. General regression neural network (GRNN) models are developed to predict chlorophyll a concentrations for each lake and reservoir during two study periods (2005–2010 and 2011–2018). The developed models with an acceptable model fit are then analyzed by two indices to determine the areal HAB magnitudes and associated drivers. Our national assessment suggests that HAB magnitudes for China’s lakes and reservoirs displayed a decreasing trend from 2006 (1363.3 km2) to 2013 (665.2 km2), and a slightly increasing trend from 2013 to 2018 (775.4 km2). Among the 142 studied lakes and reservoirs, most severe HABs were found in Lakes Taihu, Dianchi and Chaohu with their contribution to the total HAB magnitude varying from 89.2% (2013) to 62.6% (2018). HABs in Lakes Taihu and Chaohu were strongly associated with both total phosphorus and nitrogen concentrations, while our results were inconclusive with respect to the predominant environmental factors shaping the eutrophication phenomena in Lake Dianchi. The present study provides evidence that effective HAB mitigation may require both nitrogen and phosphorus reductions and longer recovery times; especially in view of the current climate-change projections. ML represents a robust strategy to elucidate water quality patterns in lakes, where the available information is sufficient to train the constructed algorithms. Our mapping of HAB magnitudes and associated environmental/meteorological drivers can help managers to delineate hot-spots at a national scale, and comprehensively design the best management practices for mitigating the eutrophication severity in China’s lakes and reservoirs.

More than 30% of the registered pesticides are chiral with one or more chiral centers and exist as two or more enantiomers. The frequency of chiral chemicals and their environmental safety has been considered in their risk assessment in recent decades. Despite the fact that metabolic disturbance is an important sensitive molecular initiating event of toxicology effects, the potential mechanisms of how chiral compounds affect metabolism phenotypes in organisms remain unclear. As a typical chiral pesticide, metalaxyl is an acylalanine fungicide with systemic function. Although the fungicidal activity almost comes from the R-enantiomer, the toxicity of both enantiomers in animals and human beings is not yet clear. In this study, a nuclear magnetic resonance (NMR)-based metabolomics approach was adopted to evaluate the enantioselectivity in metabolic perturbations in adolescent rats. On the basis of multivariate statistical results, stable and evident metabolic profiles of the enantiomers were obtained. When rats were exposed to R-metalaxyl, the significantly perturbed metabolic pathways were biosynthesis of valine, leucine, and isoleucine, synthesis and degradation of ketone bodies, and metabolism of glycerolipid. In contrast, more significantly perturbed metabolic pathways were obtained when the rats were exposed to S-metalaxyl, including glycolysis, biosynthesis of valine, leucine, and isoleucine, metabolism of glycine, serine, and threonine, synthesis and degradation of ketone bodies, metabolism of glycerophospholipid and glycerolipid. These abnormal metabolic pathways were closely related to liver metabolism. These results offer more detailed information about the enantioselective metabolic effects of metalaxyl in adolescent development and provide data for the health risk assessment of metalaxyl at molecular level.

This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λex 330 nm and λem 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process.

A water transfer project has been ongoing since 2002 to alleviate severe phytoplankton aggregation in Lake Taihu. This study aimed to quantify the effectiveness of the water transfer project on alleviation of phytoplankton aggregation in Lake Taihu on a short-term scale. In this study, a spatially distributed hydrodynamic-phytoplankton model was used to predict the short-term (3–4 days) changes in phytoplankton distribution (represented by chlorophyll a) in Lake Taihu. Four simulations with different water transfer strategies were carried out based on this model. During the water transfer period, phytoplankton aggregation was alleviated in some areas, suggesting that the water transfer project has the potential to alleviate algal blooms on a short-term scale. However, the effectiveness of the water transfer project on alleviating severe algal blooms was strongly affected by other environmental factors (e.g. wind conditions, chlorophyll a distribution, and the amount and quality of the transfer water). This study demonstrates the success of the hydrodynamic-phytoplankton model in evaluating the contribution of the water transfer project to alleviation of phytoplankton aggregation. These evaluation results could assist managers in decision-making before conducting a water transfer plan in Lake Taihu.

Trimethylhydroquinone-1-monoacetate (TMHQ-1-MA) is a valuable synthetic intermediate for vitamin E acetate. Immobilized Lipozyme RM IM from Mucor miehei was shown to be the best biocatalyst for the production of TMHQ-1-MA through regioselective transesterification between trimethylhydroquinone diacetate (TMHQ-DA) and alcohol. The effects of lipase-catalyzed reaction conditions including solvent, acyl receptor, substrate mole ratio, reaction temperature and agitation speed were investigated. The optimum conditions for Lipozyme RM IM catalyzed regioselective transesterification were achieved at a substrate mole ratio of 1:1, an agitation of 200 rpm at 50 °C in MTBE/ n -hexane (3:7). Under the above conditions, Lipozyme RM IM exhibited high substrate tolerance (substrate concentrations of 1.06 M). Recycling experiments demonstrated that Lipozyme RM IM was quite steady under the reaction conditions. The analysis of kinetic experiment showed that the enzymatic reaction obeys the Ping–Pong bi–bi mechanism with n -butanol inhibition.

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD+, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.

A strain of Aspergillus oryzae WZ007 producing intracellular lipase with preference for the formation of the d -biotin intermediate lactone ((3 aS ,6 aR )-tetrahydro-1,3-dibenzylhexahydro-1 H -Furo[3,4- d ]imidazole-2,4-dione) 1 was screened from soil samples. The microbial cells fermented by the strain were used as biocatalysts after lyophilization, to resolve the racemic biotin intermediate lactone 1 by enantioselective lactonization in organic solvents. The enantioselective lactonization afforded optically active lactone 1 with high enantiomeric excess (e.e. ≥ 98%) and conversion ratio ( c ≥ 40%) when the biotransformation was carried out in dichloromethane. The e.e. of product was up to 99% after purification by inducing crystallization in acetone and basically satisfied the industrial application. The biosynthesis has been employed to produce kilograms supplies of optically active lactone 1 with high optical purity.

The title compound, C13H11IN2, crystallizes with two independent molecules (A and B) in the asymmetric unit. The two aromatic rings are inclined to one another by 73.3 (2)° in molecule A, and by 74.4 (1)° in molecule B. In molecule A, the iodophenyl and the phenyl rings are inlclined to the N=C—N plane by 88.0 (4) and 19.0 (4)°, respectively. In molecule B the corresponding angles are 85.0 (4) and 20.7 (4)°, respectively. In the crystal, the two molecules are not parallel but have a dihedral angle between the iodophenyl rings of 8.6 (1)°, and 44.5 (2)° between the phenyl rings. The A and B molecules are linked vvia N—H...N hydrogen bonds to form –A–B–A–B– chains propagating along direction [100].

A simple and reliable procedure was developed to screen biocatalysts with high alcohol dehydrogenase activity, efficient internal coenzyme regeneration, and high stereoselectivity. The strategy of activity screening in a microtitre plate format was based on the detection of fluorescence of NAD(P)H originating from the oxidation of alcohols. The primary and secondary screenings from soil samples yielded a versatile bacterial biocatalyst Rhodococcus erythropolis WZ010 demonstrating potential for the preparation of chiral aryl secondary alcohols. In terms of activity and stereoselectivity, the optimized reaction conditions in the stereoselective oxidation were 30 °C, pH 10.5, and 250 rpm, whereas bioreduction using glucose as co-substrate was the most favorable at 35 °C and pH 7.5 in the static reaction mixture. Under the optimized conditions, fresh cells of the strain stereoselectively oxidized the (S)-enantiomer of racemic 1-phenylethanol (120 mM) to acetophenone and afforded the unoxidized (R)-1-phenylethanol in 49.4 % yield and >99.9 % enantiomeric excess (e.e.). In the reduction of 10 mM acetophenone, the addition of 100 mM glucose significantly increased the conversion rate from 3.1 to 97.4 %. In the presence of 800 mM glucose, acetophenone and other aromatic ketones (80 mM) were enantioselectively reduced to corresponding (S)-alcohols with excellent e.e. values. Both stereoselective oxidation and asymmetric reduction required no external cofactor regeneration system.

The asymmetric unit of the title compound, C14H2Cl4F3NO2, contains two independent molecules. In each molecule, the phthalimide ring system is nearly planar [maximum atomic deviation = 0.031 (2) or 0.038 (2) Å] and oriented with respect to the benzene ring at 65.04 (7) or 71.76 (10)°. Weak intermolecular C—H...O and C—H...F hydrogen bonding is present in the crystal structure.