[Clinical significance of negative fluid balance and infection management in the treatment of acute respiratory distress syndrome caused by severe novel coronavirus infection].

OBJECTIVE: To explore the clinical significance of negative fluid balance and infection management in the treatment of acute respiratory distress syndrome (ARDS) caused by severe novel coronavirus infection. METHODS: A retrospective survey was conducted. Patients with ARDS caused by severe novel coronavirus infection who were hospitalized in the department of critical care medicine of the Third Affiliated Hospital of Gansu University of Chinese Medicine and received non-invasive ventilator assisted ventilation were selected as the research objects. The fluid intake and output of all patients were accurately counted every day, and the fluid intake of the next day was adjusted according to the output of the previous day. According to the fluid negative balance, and whether the hospital infection management measures were complied with during the treatment and inspection of the patients, 45 patients with a negative fluid balance of more than 200 mL/d and strict management of nosocomial infection were taken as the observation group, and 48 patients with a negative fluid balance of less than 200 mL/d and no strict management of nosocomial infection were taken as the control group. The general data, weaning success rate, endotracheal intubation rate, mortality, as well as laboratory indicators such as white blood cell count (WBC), procalcitonin (PCT), C-reactive protein (CRP) after treatment were compared between the two groups. RESULTS: There were no significant differences in gender (male: 51.1% vs. 52.1%), age (years old: 66.31±15.92 vs. 67.50±13.59), acute physiology and chronic health evaluation II (APACHE II: 18.98±4.81 vs. 18.54±4.35) between the observation group and the control group (all P > 0.05), indicating that the baseline data were balanced and comparable. Compared with the control group, the weaning success rate of the observation group significantly increased [53.3% (24/45) vs. 31.2% (15/48), P = 0.031], endotracheal intubation rate significantly decreased [22.2% (10/45) vs. 43.8% (21/48), P = 0.028], mortality significantly reduced [20.0% (9/45) vs. 41.7% (20/48), P = 0.024], laboratory indicators WBC, PCT and CRP levels were significantly reduced [WBC (×109/L): 8.085±4.136 vs. 16.898±7.733, CRP (mg/L): 82.827±52.680 vs. 150.679±74.625, PCT (µg/L): 3.142±2.323 vs. 7.539±5.939, all P < 0.01]. CONCLUSIONS: Fluid negative balance and infection management have significant clinical significance in the treatment of severe novel coronavirus infection with ARDS.

Improved titer and stability of selenium nanoparticles produced by engineered Saccharomyces cerevisiae.

Selenium nanoparticles (SeNPs) have gained significant attention in the fields of medicine and healthcare products due to their various biological activities and low toxicity. In this study, we focused on genetically modifying the Saccharomyces cerevisiae strain YW16 (CICC 1406), which has the ability to efficiently reduce sodium selenite and produce red SeNPs. By overexpressing genes involved in glutathione production, we successfully increased the glutathione titer of the modified strain YJ003 from 41.0 mg/L to 212.0 mg/L. Moreover, we improved the conversion rate of 2.0 g/L sodium selenite from 49.3% to 59.6%. Furthermore, we identified three surface proteins of SeNPs, and found that overexpression of Act1, one of the identified proteins, led to increased stability of SeNPs across different acid-base and temperature conditions. Through a 135-h feed fermentation process using 5.0 g/L sodium selenite, we achieved an impressive conversion rate of 88.7% for sodium selenite, and each gram of SeNPs contained 195.7 mg of selenium. Overall, our findings present an efficient method for yeast to synthesize SeNPs with high stability. These SeNPs hold great potential for applications in nanomedicine or as nutritional supplements to address selenium deficiency.

Enhancing the Catalytic Activity of Glycolate Oxidase from Chlamydomonas reinhardtii through Semi-Rational Design.

Glycolate oxidase is a peroxisomal flavoprotein catalyzing the oxidation of glycolate to glyoxylate and plays crucial metabolic roles in green algae, plants, and animals. It could serve as a biocatalyst for enzymatic production of glyoxylate, a fine chemical with a wide variety of applications in perfumery, flavor, and the pharmaceutical and agrochemical industries. However, the low catalytic activity of native glycolate oxidase and low levels of active enzyme in heterologous expression limit its practical use in industrial biocatalysis. Herein, the glycolate oxidase from Chlamydomonas reinhardtii (CreGO) was selected through phylogenetic tree analysis, and its low level of soluble expression in E. coli BL21(DE3) was improved through the use of the glutathione thioltransferase (GST), the choice of the vector pET22b and the optimization of induction conditions. The semi-rational design of the fusion enzyme GST-Gly-Ser-Gly-CreGO led to the superior variant GST-Gly-Ser-Gly-CreGO-Y27S/V111G/V212R with the kcat/Km value of 29.2 s-1·mM-1, which was six times higher than that of the wild type. In contrast to GST-Gly-Ser-Gly-CreGO, 5 mg/mL of crude enzyme GST-Gly-Ser-Gly-CreGO-Y27S/V111G/V212R together with 25 µg/mL of catalase catalyzed the oxidation of 300 mM of methyl glycolate for 8 h, increasing the yield from 50.4 to 93.5%.

Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone.

d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B5. The commercial synthesis of d-pantolactone is carried out through the selective resolution of dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for the deracemization of dl-pantolactone, using l-pantolactone dehydrogenase from Amycolatopsis methanolica (AmeLPLDH), conjugated polyketone reductase from Zygosaccharomyces parabailii (ZpaCPR), and glucose dehydrogenase from Bacillus subtilis (BsGDH). The AmeLPLDH was used to catalyze the dehydrogenated l-pantolactone into ketopantolactone; the ZpaCPR was used to further catalyze the ketopantolactone into d-pantolactone; and glucose dehydrogenase together with glucose fulfilled the function of coenzyme regeneration. All three enzymes were co-expressed in E. coli strain BL21(DE3), which served as the whole-cell biocatalyst. Under optimized conditions, 36 h deracemization of 1.25 M dl-pantolactone d-pantolactone led to an e.e.p value of 98.6%, corresponding to productivity of 107.7 g/(l·d).

The Protective Effect of Selenium Nanoparticles in Osteoarthritis: In vitro and in vivo Studies.

Introduction: Osteoarthritis (OA) is a common chronic joint disease characterized by articular cartilage degeneration. OA usually manifests as joint pain, limited mobility, and joint effusion. Currently, the primary OA treatment is non-steroidal anti-inflammatory drugs (NSAIDs). Although they can alleviate the disease's clinical symptoms and signs, the drugs have some side effects. Selenium nanoparticles (SeNPs) may be an alternative to relieve OA symptoms. Materials and Results: We confirmed the anti-inflammatory effect of selenium nanoparticles (SeNPs) in vitro and in vivo experiments for OA disease in this study. In vitro experiments, we found that SeNPs could significantly reduce the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the major inflammatory factors, and had significant anti-inflammatory and anti-arthritic effects. SeNPs can inhibit reactive oxygen species (ROS) production and increased glutathione peroxidase (GPx) activity in interleukin-1beta (IL-1ß)-stimulated cells. Additionally, SeNPs down-regulated matrix metalloproteinase-13 (MMP-13) and thrombospondin motifs 5 (ADAMTS-5) expressions, while up-regulated type II collagen (COL-2) and aggrecan (ACAN) expressions stimulated by IL-1ß. The findings also indicated that SeNPs may exert their effects through suppressing the NF-κB p65 and p38/MAPK pathways. In vivo experiments, the prevention of OA development brought on by SeNPs was demonstrated using a DMM model. Discussion: Our results suggest that SeNPs may be a potential anti-inflammatory agent for treating OA.

Engineering a Bacillus subtilis esterase for selective hydrolysis of d, l-menthyl acetate in an organic solvent-free system.

Esterase/lipase-catalyzed selective hydrolysis of d, l-menthyl esters has become one of the promising approaches for producing l-menthol, one of the most important flavoring chemicals with extensive uses. However, the activity and l-enantioselectivity of the biocatalyst are not sufficient for meeting the industrial requirements. Herein, a highly active para-nitrobenzyl esterase from Bacillus subtilis 168 (pnbA-BS) was cloned and then engineered to enhance its l-enantioselectivity. On the basis of the strategy tailoring the steric exclusion effect and structural flexibility of the region adjacent to the substrate, the substitution of Ala400 to Pro caused a remarkable improvement in the E value from 1.0 to 466.6. The variant A400P was purified and further confirmed with strict l-enantioselectivity in the selective hydrolysis of d, l-menthyl acetate, whereas the improved l-enantioselectivity caused decreased activity. To develop an efficient, easy-to-use, and green methodology, organic solvent was omitted and substrate constant feeding was integrated into the whole-cell catalyzed system. During the catalytic process, the selective hydrolysis of 1.0 M d, l-menthyl acetate in 14 h offered a conversion of 48.9%, e.e.p value of >99%, and space-time yield of 160.52 g (l d)-1.

Effects of several flavonoids on human gut microbiota and its metabolism by in vitro simulated fermentation.

Introduction: Flavonoids have antiviral, antitumor, anti-inflammatory, and other biological activities. They have high market value and are widely used in food and medicine fields. They also can regulate gut microbiota and promote human health. However, only a few flavonoids have been reported for their regulatory effects on human gut microbiota. Methods: The effects of hesperidin, hesperetin-7-O-glucoside, hesperetin, naringin, prunin, naringenin, rutin, isoquercitrin, and quercetin on gut microbiota structural and metabolic differences in healthy subjects were studied by means of in vitro simulated fermentation technology. Results: Results showed that the nine kinds of flavonoids mentioned above, especially hesperetin-7-O-glucoside, prunin, and isoquercitrin, were found to have more effect on the structure of human gut microbiota, and they could significantly enhance Bifidobacterium (p < 0.05). After 24 h of in vitro simulated fermentation, the relative abundance of intestinal probiotics (e.g., Lactobacillus) was increased by the three flavonoids and rutin. Furthermore, the relative abundance of potential pathogenic bacteria was decreased by the addition of hesperetin-7-O-glucoside, naringin, prunin, rutin, and isoquercitrin (e.g., Lachnoclostridium and Bilophila). Notably, prunin could also markedly decrease the content of H2S, NH3, and short-chain fatty acids. This performance fully demonstrated its broad-spectrum antibacterial activity. Discussion: This study demonstrates that flavonoids can regulate the imbalance of gut microbiota, and some differences in the regulatory effect are observed due to different structures. This work provides a theoretical basis for the wide application of flavonoids for food and medicine.

Heterologous expression of cyclodextrin glycosyltransferase from Bacillus stearothermophilus in Bacillus subtilis and its application in glycosyl rutin production.

In this paper, the cgt gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus stearothermophilus was cloned into pWB980 plasmid for extracellular expression in Bacillus subtilis SCK6. Through adding a six-histidine affinity tag fused to the C-terminus, the recombinant CGTase could be purified by nickel ion affinity chromatography, and its molecular weight was approximately 76 kDa on SDS-PAGE. Then, the enzymatic properties were determined, and results were as follows: the optimum temperature and pH were identified as 40 ℃ and pH 5.0, respectively. CGTase had good tolerance to metal ions of Mn2+, Ca2+, and Mg2+. The enzyme activity was activated by Na+, Al3+, Fe3+, and Ni+, and it was remarkably inhibited by Cu2+ and Zn2+. To improve the aqueous solubility of rutin, CGTase was used to catalyze the transglycosylation reaction, and the conversion rate could reach as high as 80.13% under optimal conditions. Furthermore, the reaction mixture was treated with glucoamylase and microporous adsorbent resin. The yield of glycosyl-rutin was 56.1%, and its purity was 74.3%, which further improved the value of the product. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03510-5.

Enantioselective resolution of (R,S)-DMPM to prepare (R)-DMPM by an adsorbed-covalent crosslinked esterase PAE07 from Pseudochrobactrum asaccharolyticum WZZ003.

(R)-N-(2,6-dimethylphenyl) aminopropionic acid methyl ester ((R)-DMPM) is an important chiral intermediate of the fungicide N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester ((R)-Metalaxyl). In this study, (1) D3520 (macroporous acrylic anion resin), selected from the ten resins, was used to immobilize the esterase from Pseudochrobactrum asaccharolyticum WZZ003 (PAE07) for resoluting the (R,S)-DMPM to obtain (R)-DMPM. (2) Up to 20 g/L PAE07 could be immobilized onto D3520 with a high enzymatic activity of 32.4 U/g. Moreover, the Km and Vmax values of 19.1 mM and 2.8 mM/min for D3520-immobilized PAE07 indicated its high activity and stereoselectivity. (3) The optimal temperature and pH for the immobilized PAE07 were 40 ℃ and 8.0, and substrate concentration was up to 0.35 M. After 15 h reaction, the conversion rate from (R,S)-DMPM to (R)-DMPM was 48.0% and the e.e.p and E values were 99.5% and 1393.0, respectively. In scale-up resolution, 200 g/L substrate and 12.5 g immobilized esterase PAE07 condition, a conversion rate from substrate to product of 48.1% and a product e.e.p of 98% were obtained within 12 h, with the activity of immobilized PAE07 retained 80.2% after 5 cycles of reactions. These results indicated that the D3520-immobilized esterase PAE07 had great potential for enzymatic resolution of (R,S)-DMPM to prepare (R)-Metalaxyl.

The Production of Pyruvate in Biological Technology: A Critical Review.

Pyruvic acid has numerous applications in the food, chemical, and pharmaceutical industries. The high costs of chemical synthesis have prevented the extensive use of pyruvate for many applications. Metabolic engineering and traditional strategies for mutation and selection have been applied to microorganisms to enhance their ability to produce pyruvate. In the past decades, different microbial strains were generated to enhance their pyruvate production capability. In addition to the development of genetic engineering and metabolic engineering in recent years, the metabolic transformation of wild-type yeast, E. coli, and so on to produce high-yielding pyruvate strains has become a hot spot. The strategy and the understanding of the central metabolism directly related to pyruvate production could provide valuable information for improvements in fermentation products. One of the goals of this review was to collect information regarding metabolically engineered strains and the microbial fermentation processes used to produce pyruvate in high yield and productivity.

Mechano-Enzymatic Degradation of the Chitin from Crustacea Shells for Efficient Production of N-acetylglucosamine (GlcNAc).

Chitin, the second richest polymer in nature, is composed of the monomer N-acetylglucosamine (GlcNAc), which has numerous functions and is widely applied in the medical, food, and chemical industries. However, due to the highly crystalline configuration and low accessibility in water of the chitin resources, such as shrimp and crab shells, the chitin is difficult utilize, and the traditional chemical method causes serious environment pollution and a waste of resources. In the present study, three genes encoding chitinolytic enzymes, including the N-acetylglucosaminidase from Ostrinia furnacalis (OfHex1), endo-chitinase from Trichoderma viride (TvChi1), and multifunctional chitinase from Chitinolyticbacter meiyuanensis (CmChi1), were expressed in the Pichia pastoris system, and the positive transformants with multiple copies were isolated by the PTVA (post-transformational vector amplification) method, respectively. The three recombinants OfHex1, TvChi1, and CmChi1 were induced by methanol and purified by the chitin affinity adsorption method. The purified recombinants OfHex1 and TvChi1 were characterized, and they were further used together for degrading chitin from shrimp and crab shells to produce GlcNAc through liquid-assisted grinding (LAG) under a water-less condition. The substrate chitin concentration reached up to 300 g/L, and the highest yield of the product GlcNAc reached up to 61.3 g/L using the mechano-enzymatic method. A yield rate of up to 102.2 g GlcNAc per 1 g enzyme was obtained.

Engineering of Yarrowia lipolytica for producing pyruvate from glycerol.

The present study aims to increase pyruvate production by engineering Yarrowia lipolytica through modifying the glycerol metabolic pathway. Results: Wild-type Yarrowia lipolytica (Po1d) was engineered to produce six different strains, namely ZS099 (by over-expressing PYK1), ZS100 (by deleting DGA2), ZS101 (by over-expressing DAK1, DAK2, and GCY1), ZS102 (by over-expressing GUT1 and GUT2), ZS103 (by over-expressing GUT1) and ZSGP (by over-expressing POS5 and deleting GPD2). Production of pyruvate from engineered and control strains was determined using high-performance liquid chromatography (HPLC). Subsequently, the fermentation conditions for producing pyruvate were optimized, including the amount of initial inoculation, the addition of calcium carbonate (CaCO3), thiamine and glycerol. Finally, for scaled-up purposes, a 20-L fermentor was used. It was observed that pyruvate production increased by 136% (8.55 g/L) in ZSGP strain compared to control (3.62 g/L). Furthermore, pyruvate production by ZSGP reached up to 110.4 g/L in 96 h in the scaled-up process. We conclude that ZSGP strain of Y. lipolytica can be effectively used for pyruvate production at the industrial level. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03158-7.

Homologous Expression and Characterization of α-L-rhamnosidase from Aspergillus niger for the Transformation of Flavonoids.

Aspergillus niger has been used for homologous and heterologous expressions of many protein products. In this study, the α-L-rhamnosidase from A. niger (Rha-N1, GenBank XP_001389086.1) was homologously expressed in A. niger 3.350 by Agrobacterium tumefaciens-mediated transformation. The enzyme activity of Rha-N1 was 0.658 U/mL, which was obtained by cultivation of engineered A. niger in a 5-L bioreactor. Rha-N1 was purified by affinity chromatography and characterized. The optimum temperature and optimum pH for Rha-N1 were 60 °C and 4.5, respectively. Enzyme activity was promoted by Al3+, Li+, Mg2+, and Ba2+ and was inhibited by Mn2+, Fe3+, Ca2+, Cu2+, and organic solvents. The result indicated that rutin was the most suitable substrate for Rha-N1 by comparison with the other two flavonoid substrates hesperidin and naringin. The transformed products of isoquercitrin, hesperetin-7-O-glucoside, and prunin were identified by LC-MS and 1H-NMR.

Semi-rational protein engineering of a novel esterase from Bacillus aryabhattai (BaCE) for resolution of (R,S)-ethyl indoline-2-carboxylate to prepare (S)-indoline-2-carboxylic acid.

A gene encoding an esterase from Bacillus aryabhattai (BaCE) was identified, synthesized and efficiently expressed in the Escherichia coli system. A semi-rational protein engineering was applied to further improve the enzyme's enantioselectivity. Under the guidance of the molecular docking result, a single mutant BaCE-L86Q and a double mutant BaCE-L86Q/G284E were obtained, with its Emax value 6.4 times and 13.9 times of the wild-type BaCE, respectively. The recombinant BaCEs were purified and characterized. The overwhelming E value demonstrated that BaCE-L86Q/G284E was a promising biocatalyst for the biological resolution to prepare (S)-indoline-2-carboxylic acid.

Enzymatic extraction of pectic oligosaccharides from finger citron (Citrus medica L. var. sarcodactylis Swingle) pomace with antioxidant potential.

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.

Characterizing the river water quality in China: Recent progress and on-going challenges.

Food production systems, urbanization, and other anthropogenic activities dramatically alter natural hydrological and nutrient cycles, and are primarily responsible for water quality impairments in China's rivers. This study compiled a 16-year (2003-2018) dataset of river water quality (161,337 records from 2424 sites), watershed/landscape features, and meteorological conditions to investigate the spatial water quality patterns and underlying drivers of river impairment (defined as water quality worse than Class V according to China's Environmental Quality Standards for Surface Waters, GB3838-2002) at a national scale. Our analysis provided evidence of a distinct water quality improvement with a gradual decrease in the frequency of prevalence of anoxic conditions, an alleviation of the severity of heavy metal pollution, whereas the cultural eutrophication has only been moderately mitigated between 2003 and 2018. We also identified significant spatial variation with relatively poorer water quality in eastern China, where 17.2% of the sampling sites registered poor water quality conditions, compared with only 4.6% in western China. Total phosphorus (TP) and ammonia-nitrogen (NH3-N) are collectively responsible for >85% of the identified incidences of impaired conditions. Bayesian modelling was used to delineate the most significant covariates of TP/NH3-N riverine levels in six large river basins (Liao, Hai, Yellow, Yangtze, Huai, and Pearl). Water quality impairments are predominantly shaped by anthropogenic drivers (82.5% for TP, 79.5% for NH3-N), whereas natural factors appear to play a secondary role (20.5% for TP, 17.5% for NH3-N). Two indicator variables of urbanization (urban areal extent and nighttime light intensity) and farmland areal extent were the strongest predictors of riverine TP/NH3-N levels and collectively accounted for most of the ambient nutrient variability. We concluded that there is still a long way to go in order to eradicate eutrophication and realize acceptable ecological conditions. The design of the remedial measures must be tailored to the site-specific landscape characteristics, meteorological conditions, and should also consider the increasing importance of non-point source pollution and internal nutrient loading.

[Application of self-made protective clothing in tracheal intubation for patients with severe respiratory infectious diseases].

OBJECTIVE: To explore the effect of self-made protective clothing in tracheal intubation for the patients with respiratory infectious diseases. METHODS: Self-made protective clothing were made by adult model plastic raincoat with sleeve lets and goggles. A prospective randomized controlled study was conducted. Patients with severe respiratory infectious diseases who needed tracheal intubation admitted to the department of intensive medicine of the Third Affiliated Hospital of Gansu University of Chinese Medicine from January 1st 2018 to March 31st 2020 were enrolled. According to the random number table method, they were divided into two groups. The control group was wearing standard protective clothing, while the test group was wearing self-made protective clothing for endotracheal intubation. The wearing time, infection rate of operators and costs of protective clothing were compared between the two groups. The patients were sprayed with trypanosome blue diluent before tracheal intubation, and the whole body of the operator was photographed with fluorescence before wearing self-made protective clothing and after doing tracheal intubation to take off the self-made protective clothing, in order to evaluate the permeability resistance of self-made protective clothing. RESULTS: A total of 86 patients were enrolled. There were 46 cases in the test group, included 28 cases of influenza A (H1N1) virus infection, 11 cases of influenza B virus infection and 7 cases of adenovirus infection. There were 40 cases in the control group, included 15 cases of H1N1 virus infection, 10 cases of influenza B virus infection, 10 cases of adenovirus infection and 5 cases of unknown pathogen. There was no significant difference in respiratory etiology between the two groups (χ2 = 3.789, P = 0.435). The wearing protective clothing time of the control group was 11.6 times than that of the test group (minutes: 22.23±1.45 vs. 1.86±0.24, χ2 = 19.023, P < 0.001). The cost of standard protective clothing was 12.5 times than that of self-made protective clothing (Yuan/set: 500 vs. 40). Fluorescent photography showed that the whole body of the operator was not stained after tracheal intubation, indicating that the protective clothing had good anti permeability and achieved the protective effect. There was no operator infection in the test group and the control group. CONCLUSIONS: Self-made protective clothing has short wearing time, low cost and equivalent isolation effect compared with standard protective clothing, which is worthy of clinical promotion.

Effects of different extraction methods on structural and physicochemical properties of pectins from finger citron pomace.

The functionality and property of pectin are correlated with its structure which is affected by the extraction method used. In this study, three different methods of extracting pectin, the microwave-assisted extraction (MAE), the ultrasonic-assisted extraction (UAE) and the conventional heating extraction (CHE), were used at three different temperatures with both an acid and alkali extraction solution. It was found that temperature mainly influenced pectin yield, while pectin structures and physicochemical properties were affected by the pH condition and extraction technology. The alkali-extraction with MAE and UAE at short time promoted the yield of low-ester pectin. Monosaccharide composition analysis showed a high galacturonic acid (GalA) content in the pectin derived from MAE and UAE. The high viscosity and desirable viscoelastic properties of the acid-MAE pectin were due to its large molecular weight and particle size. The results contribute to our understanding of the association among pectin extraction, structure and properties.

A novel lipase from Aspergillus oryzae WZ007 catalyzed synthesis of brivaracetam intermediate and its enzymatic characterization.

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.

Directed evolution of Aspergillus oryzae lipase for the efficient resolution of (R,S)-ethyl-2-(4-hydroxyphenoxy) propanoate.

Aspergillus oryzae lipase (AOL) is a potential biocatalyst for industrial application. In this study, a mutant lipase AOL-3F38N/V230R was screened through two rounds of directed evolution, resulting in a fourfold increase in lipase activity, and threefold in catalytic efficiency (kcat/Km), while maintaining its excellent stereoselectivity. AOL-3F38N/V230R enzyme activity was maximum at pH 7.5 and also at 40 °C. And compared with wild-type AOL-3, AOL-3F38N/V230R preferentially hydrolyzed the fatty acid ethyl ester carbon chain length from C4 to C6-C10. In the same catalytic reaction conditions, the conversion of (R,S)-ethyl-2-(4-hydroxyphenoxy) propanoate ((R,S)-EHPP) by AOL-3F38N/V230R can be increased 169.7% compared to the original enzyme. The e.e.s of (R,S)-EHPP achieved 99.4% and conversion about 50.2% with E value being 829.0. Therefore, AOL-3F38N/V230R was a potential biocatalyst for obtaining key chiral compounds for aryloxyphenoxy propionate (APP) herbicides.