Enhancing surfactin production in B. velezensis Bs916 combined cumulative mutagenesis and expression key enzymes.

Surfactin is a lipopeptide which has attracted massive attention due to its versatile bioactive properties, although it has less commercial application due to its low yield in wild strains. The B. velezensis Bs916 has enable commercial production of surfactin due to its outstanding capacity to synthesize lipopeptides and amenable to genetically engineering. In this study, 20 derivatives with high surfactin production were obtained firstly by transposon mutagenesis and knockout techniques, and the surfactin yield of the derivative H5 (△GltB) was increased approximately 7-folds, reaching to 1.48 g/L. The molecular mechanism of high yielding surfactin in △GltB was investigated by the transcriptomic and KEGG pathway analysis. The results indicated that △GltB enhanced its ability to synthesize surfactin mainly by promoting transcription of the srfA gene cluster and inhibiting degradation of some key precursors such as fatty acid. Secondly, we obtained a triple mutant derivative BsC3 by cumulative mutagenesis of the negative genes GltB, RapF, and SerA, and it could increase the surfactin titer by twofold, reaching to 2.98 g/L. Thirdly, we achieved overexpression of two key rate-limiting enzyme genes, YbdT, and srfAD, and the derivative BsC5 which further increased the surfactin titer by 1.3-fold, reaching to 3.79 g/L. Finally, the yield of surfactin by derivatives was significantly increased under the optimal medium, particularly the BsC5 increased the surfactin titer to 8.37 g/L. To the best of our knowledge, this is one of the highest yields that have been reported. Our work may pave way for large scale production of surfactin by B. velezensis Bs916. KEY POINTS: • Elucidation of the molecular mechanism of surfactin high-yielding transposon mutant. • Genetically engineering of B. velezensis Bs916 surfactin titer to 8.37 g/L for large scale preparation.

Genome Sequencing and Genetic Engineering Reveal the Contribution of Bacitracin Produced by Bacillus paralicheniformis CPL618 to Anti-Staphylococcus aureus Activity.

Staphylococcus aureus is one of the important pathogens causing human diseases, especially its treatment has great challenges due to its resistance to methicillin and vancomycin. The Bacillus strains are known to be major sources of second metabolites that can function as drugs. Therefore, it is of great value to excavate metabolites with good inhibitory activity against S. aureus from Bacillus strains. In this study, a strain Bacillus paralicheniformis CPL618 with good antagonistic activity against S. aureus was isolated and genome analysis showed that the size was 4,447,938 bp and contained four gene clusters fen, bac, dhb, and lch which are potentially responsible for four cyclic peptides fengycin, bacitracin, bacillibactin, and lichenysin biosynthesis, respectively. These gene clusters were knockout by homologous recombination. The bacteriostatic experiment results showed that the antibacterial activity of ∆bac decreased 72.3% while Δfen, Δdhb, and ΔlchA did not significantly changed as that of wild type. Interestingly, the maximum bacitracin yield was up to 92 U/mL in the LB medium, which was extremely unusual in wild type strains. To further improve the production of bacitracin, transcription regulators abrB and lrp were knocked out, the bacitracin produced by ΔabrB, Δlrp, and ΔabrB + lrp was 124 U/mL, 112 U/mL, and 160 U/ml, respectively. Although no new anti-S. aureus compounds was found by using genome mining in this study, the molecular mechanisms of high yield of bacitracin and anti-S. aureus in B. paralicheniformis CPL618 were clarified. Moreover, B. paralicheniformis CPL618 was further genetically engineered for industrial production of bacitracin.

Extracellular lipopeptide bacillomycin L regulates serial expression of genes for modulating multicellular behavior in Bacillus velezensis Bs916.

In wild strains of Bacillus, a handful of extracellular natural products act as signals that can regulate multicellular behavior, but relatively little is known about molecular mechanisms' detail. We proposed a previously unreported molecular mechanism for triggering multicellularity in B. velezensis Bs916 by an endogenous cyclic lipopeptide, bacillomycin L. The genome-wide effect on gene expression was caused by the disruption of bacillomycin L gene cluster, and 100 µg/mL bacillomycin L was revealed by quantitative transcriptomics. A total of 878 differentially expressed genes among Bs916, Δbl, and Δbl + 100BL were identified and grouped into 9 functional categories. The transcription levels of 40 candidate genes were further evaluated by RT-qPCR analysis. The expression of eight candidate genes regulated by bacillomycin L in a dose-dependent manner was revealed by LacZ fusion experiment. Although the addition of bacillomycin L could not completely restore the expression levels of the differentially regulated genes in △bl, our results strongly suggest that bacillomycin L acts as a tuning signal of swarming motility and complex biofilm formation by indirectly regulating the expression levels of some two-component systems (TCSs) connector genes, particularly including several Raps that potentially regulate the phosphorylation levels of three major regulators ComA, DegU, and Spo0A.Key points• Proposed model for bacillomycin L regulation in B. velezensis Bs916.• Bacillomycin L can act as an extracellular signal to regulate the phosphorylation levels of three major regulators, ComA, DegU, and Spo0A and control the multicellular processes of vegetative growth, competent, motility, matrix production, sporulation, and autolysis.

Platinum(IV) complexes conjugated with chalcone analogs as dual targeting anticancer agents: In vitro and in vivo studies.

For the sake to develop novel platinum(IV) complexes to reverse cisplatin (CDDP) resistence, four multifunctional platinum(IV) prodrugs via conjugating chalcones with the related platinum(IV) complexes derived from cisplatin were designed and evaluated for anti-tumor actyivities in vitro and in vivo. Among them, complex 9 exhibited excellent anticancer activities in vitro with IC50 values at the submicromolar level against the tested human cancer cells, whereas showed low cytotoxicity towards human normal liver cells HL-7702. Further mechanistic studies indicated that complex 9 induced G2/M phase arrest and apoptosis in A549 cells, which was associated with a collapse of the mitochondrial membrane potential (MMP), alterations in the expression of some apoptosis-related proteins, and enhanced level of the intracellular reactive oxygen species (ROS). More importantly, complex 9 significantly suppressed the tumor growth in the A549 xenograft model without obvious hints of toxicity.

Immunomodulatory activities of different solvent extracts from Tricholoma matsutake (S. Ito et S. Imai) singer (higher basidiomycetes) on normal mice.

The immunomodulatory activities of different solvent extracts from the culinary-medicinal mushroom Tricholoma matsutake were studied in vivo in normal mice. The extracts were prepared using different solvents in an order of increasing polarity. The immunomodulatory activities were investigated by measuring the thymus and spleen index, phagocytic rate of macrophage phagocytosis, delayed-type hypersensitivity, plaque-forming cell, and proliferation of splenocytes. Results demonstrated that water extract (WE) and n-butyl alcohol extract (BAE) of T. matsutake could enhance the immunity of mice significantly compared with the control group. Main components of WE and BAE were polysaccharides, proteins, and flavonoids; we presume that these may be the main immunomodulating and immuno-enhancing agents in T. matsutake.

Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation.

Methanol tolerance of lipase is one of the important factors affecting its esterification ability in biodiesel preparation. By B factor indicated prediction of Candida antarctica lipase B (CalB) surface amino acids, eight sites (Val139, Ala146, Leu147, Pro218, Val286, Ala287, Val306, and Gly307) with high B value indicating more flexibility were chosen to perform saturation mutagenesis. High-methanol-tolerant variants, CalB-P218W and -V306N, created larger haloes on emulsified tributyrin solid plate including 15% (v/v) methanol and showed 19% and 31% higher activity over wild-type CalB (CalB-WT), respectively. By modeling, a newly formed hydrogen bond in CalB-V306N and hydrophobic force in CalB-P218W contributing more stability in protein may have resulted in increased methanol tolerance. CalB-P218W and -V306N transesterified the soybean oil into biodiesel at 30 °C by 85% and 89% yield, respectively, over 82% by CalB-WT for 24 h reactions. These results may provide a basis for molecular engineering of CalB and expand its applications in fuel industries. The as-developed semi-rational method could be utilized to enhance the stabilities of many other industrial enzymes.

Smartphone-assisted bioenzyme-nanozyme-chromogen all-in-one test strip with enhanced cascade signal amplification for convenient paraoxon sensing.

Monitoring of pesticide residues in food and environmental matrices is undoubtedly crucial to guarantee food safety and ecological health, yet how to realize their sensitive and convenient detection is still challenging. Herein, we propose an all-in-one test strip that elaborately integrates bioenzyme, nanozyme and chromogen together, and achieve the highly sensitive and convenient sensing of pesticide residues assisted by a smartphone. A sequential self-assembly strategy was first explored to acquire an integrative bioenzyme-nanozyme-chromogen assembly, and then the assembly was confined in a biocompatible hydrogel to construct the test strip. Thanks to both the proximity and confinement effects, a ∼1.2-fold improvement of the cascade catalytic efficiency was gained to benefit high-sensitivity detection. More importantly, since all the sensing elements, including target recognition units and signal amplification modules, were rationally integrated in the test strip, detection operation was significantly simplified, making it possible for in-field rapid analysis. Besides, the microenvironment provided by the alginate hydrogel carrier endowed the test strip with an excellent sensing stability. By taking paraoxon as a typical pesticide, high-performance detection of the target was accomplished via the smartphone-assisted all-in-one test strip. Moreover, the test strip was successfully applied for paraoxon detection in various real samples and exhibited good correlations with commercial kits, demonstrating its great prospect for practical applications. Our work not only offers a new tool for the high-sensitivity and convenient monitoring of pesticide residues, but will also inspire the development of efficient multi-enzyme sensing platforms.

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite.

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30-60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The K (m) and V (max) values of the immobilized enzyme were found to be 0.0619 mmol l(-1) and 0.147 mmol h(-1) mg(-1), respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.

Intensification of biodiesel production using dual-frequency counter-current pulsed ultrasound.

Biodiesel production from soybean oil deodorizer distillate intensified by dual-frequency counter-current pulsed ultrasound and the kinetics were studied. Results indicated that the biodiesel conversions enhanced by single-frequency were lower than those enhanced by dual-frequency. For dual-frequency, the biodiesel conversion of SMM was higher than those of SQM. The biodiesel conversion of the combination of 20/28kHz is the highest. The effects of 20/28kHz SMM on biodiesel production were studied and optimal conditions were: methanol to triglyceride molar ratio 8:1, catalyst content 1.8%, the water content in feedstock should be less than 0.4%, the acid value of feedstock should be less than 2mgKOH·g-1, the biodiesel conversion could reach 96.3%. The kinetics of SMM and SSPU were studied and results showed that the transesterification reaction was pseudo-second order and the energy activation obtained by SMM and SSPU were 18.122kJ·mol-1 and 26.034kJ·mol-1, respectively. These results showed that transesterification reaction intensified by SMM is easier to take place than SSPU.

Optimization for ultrasonic-microwave synergistic extraction of polysaccharides from Cornus officinalis and characterization of polysaccharides.

Ultrasonic-microwave synergistic extraction (UMSE) of polysaccharides from Cornus officinalis was optimized by response surface methodology (RSM). The effect of four different factors on the yield of C. officinalis polysaccharides (COP) was studied. RSM results showed that the optimal conditions were extraction time of 31.49823 min, microwave power of 99.39769 W, and water-to-raw material ratio of 28.16273. The COP yield was 11.38±0.31% using the modified optimal conditions, which was consistent with the value predicted by the model. The crude COP was purified by DEAE-Cellulose 52 chromatography and Sephadex G-100 chromatography. Five fractions, namely, crude COP, COP-1, COP-2, COP-3, and COP-4, were obtained. Monosaccharide composition analysis revealed that the COP was composed of glucose, arabinose, fucose, xylose, mannose, and rhamnose. Preliminary structural characterizations of COP were conducted by scanning electron microscopy and Fourier transform infrared spectroscopy.

Complex Enzyme-Assisted Extraction, Purification, and Antioxidant Activity of Polysaccharides from the Button Mushroom, Agaricus bisporus (Higher Basidiomycetes).

Agaricus bisporus polysaccharides (ABP) were extracted by complex enzyme-assisted extraction methodology. The following were optimal conditions for the extraction of crude ABP: complex enzyme amount, 2.2%; temperature, 62°C; time, 3 h; and pH, 4. Under these conditions, the experimental yield of crude ABP was 6.87%. The crude ABP was purified by diethylaminoethyl-cellulose 52 chromatography and Sephadex G-100 chromatography, and one fraction-namely, ABP-1-was produced. The ABP-1 contained 93.67% carbohydrate, 1.46% protein, and 0.62% uronic acid. The constituent monosaccharides were predominantly glucose, galactose, mannose, and xylose. The antioxidant activities of ABP-1 were investigated by measuring its scavenging ability on 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals, its ferric-reducing activity power, and the reducing power assay. At a concentration of 1.2 mg/mL, ABP-1 seemed to possess good free radical scavenging activity, with a scavenging value of about 56%. The results indicate that ABP-1 has good antioxidant activity.

Biodiesel production from soybean oil deodorizer distillate enhanced by counter-current pulsed ultrasound.

Biodiesel production from soybean oil deodorizer distillate enhanced by counter-current pulsed ultrasound was studied. Effect of static probe ultrasonic enhanced transesterification (SPUE) and counter-current probe ultrasonic enhanced transesterification (CCPUE) on the biodiesel conversion were compared. The results indicated that CCPUE was a better method for enhancing transesterification. The working conditions of CCPUE were studied by single-factor experiment design and the results showed that the optimal conditions were: initial temperature 25 °C, methanol to triglyceride molar ratio 10:1, flow rate 200 mL/min, catalyst content 1.8%, ultrasound working on-time 4 s, off-time 2 s, total working time 50 min. Under these conditions, the average biodiesel conversion of three experiments was 96.1%.

[Studies on supercritical CO2 fluid extraction for ginkgolic acids in the epicarp of Ginkgo biloba].

The technology of supercritical CO2 fluid extraction for ginkgolic acids in the epicarp of Ginkgo biloba L. was studied. The effect of pressure, temperature and extraction time on the yield of the ginkgolic acids was explored. The optimum conditions for supercritical CO2 fluid extraction was 30 MPa, 45 degrees C, extraction time 6 h and the flow rate of CO2 2L/min. The ginkgolic acids were determined by HPLC. Supercritical CO2 fluid extraction exceled the traditional solvent extraction in high yield, high purification and easy operation.

Pulsed counter-current ultrasound-assisted extraction and characterization of polysaccharides from Boletus edulis.

Four methods for extracting polysaccharides from Boletus edulis, namely, hot-water extraction, ultrasonic clearer extraction, static probe ultrasonic extraction, and pulsed counter-current probe ultrasonic extraction (CCPUE), were studied. Results showed that CCPUE has the highest extraction efficiency among the methods studied. Under optimal CCPUE conditions, a B. edulis polysaccharide (BEP) yield of 8.21% was obtained. Three purified fractions, BEP-I, BEP-II, and BEP-III, were obtained through sequential purification by DEAE-52 and Sephadex G-75 chromatography. The average molecular weights of BEP-I, BEP-II, and BEP-III were 10,278, 23,761, and 42,736 Da, respectively. The polysaccharides were mainly composed of xylose, mannose, galactose, and glucose; of these, mannose contents were the highest. The antioxidant activities of the BEPs were further investigated by measurement of their ability to scavenge DPPH and hydroxyl radicals as well as their reducing power. The results indicated that the BEPs have good antioxidant activity.

A comparison study on extraction of polysaccharides from Tricholoma matsutake by response surface methodology.

The Tricholoma matsutake polysaccharides (TMP) extraction was optimized using counter-current probe ultrasonic extraction (CCPUE) by response surface methodology (RSM). The effect of three factors (liquid-solid ratio, extraction time and pulse duration) on the TMP yield was studied. The results showed that the optimal conditions were liquid-solid ratio (15), extraction time (61 min) and pulse duration (6.0 s). Under best conditions, the maximum TMP yield was 8.11 ± 0.35%, which was close with the predicted yield value (8.03%). The five methods, hot-water extraction (HWE), enzyme assisted extraction (EAE), ultrasonic clearer extraction (UCE), static probe ultrasonic extraction (SPUE), and CCPUE for extracting TMP by RSM were further compared. The results indicated that CCPUE had the largest yield of TMP with modest energy consumption.

Extraction, purification, and antioxidant activities of polysaccharides from Tricholoma mongolicum Imai.

Ultrasonic-microwave synergistic extraction (UMSE), purification, and characterization of Tricholoma mongolicum Imai polysaccharides (TMIPs) from fruit bodies were investigated in this study. Response surface methodology (RSM) was employed to optimize the UMSE conditions. The results indicated that the optimal extraction conditions were as follows: extraction time of 24.65 min, microwave power of 109.98 W, and water to raw material ratio of 21.62 ml/g. Under optimized conditions, the yield and purity of TMIPs were 35.41 ± 0.62% and 73.92 ± 0.83%, respectively. Crude TMIPs were purified by DEAE-Cellulose 52 chromatography and Sephadex G-100 chromatography to afford four fractions, namely, TMIP-1, TMIP-2, TMIP-3, and TMIP-4. Preliminary TMIP characterization was conducted by Fourier transform infrared spectroscopy. TMIP antioxidant activities were investigated by measuring its scavenging ability on 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radicals, ferric reducing activity power, and reducing power assay. The results indicated that TMIPs have good antioxidant activity.

Extraction and purification of polysaccharides from pine medicinal mushroom, Tricholoma matsutake (higher Basidiomycetes) fruit bodies.

Ultrasound-assisted extraction, purification, and characterization of antioxidant polysaccharides from Tricholoma matsutake were carried out. On the basis of the results of a single-factor test, three independent and main variables, including extraction time (X1: 130-170 s), ultrasonic power (X2: 340-380 W), and ratio of water to raw material (X3: 45-55 mL/g) were studied by Box-Behnken design. The optimum extraction conditions are as follows: extraction time 160 s, ultrasonic power 365 W, and ratio of water to raw material 53.5 mL/g. Under the optimized conditions, the yield of T. matsutake polysaccharides (TMP) was 7.97±0.31%. The crude TMP was purified by DEAE-cellulose 52 chromatography and Sephadex G-100 chromatography to afford two fractions, TMP-1 and TMP-2. The crude TMP contained 85.76% carbohydrates, 3.57% proteins, and 0.13% uronic acids. The constituent monosaccharides were predominantly glucose, galactose, mannose, xylose, fucose, and glucuronic acid residues.

Enzyme assisted extraction of polysaccharides from the fruit of Cornus officinalis.

Process of enzyme assisted extraction (EAE) of polysaccharides from Cornus officinalis was optimized by response surface methodology (RSM). The influence of four different factors on the yield of C. officinalis polysaccharides (COP) was studied. Results showed that the optimal conditions were compound enzyme amount of 2.15%, extraction pH of 4.2, extraction temperature of 55 °C and extraction time of 97 min. Under these conditions, the COP yield was 9.29±0.31%, which was well in agreement with the value predicted by the model. The three methods, EAE, hot water extraction (HWE), ultrasound-assisted extraction (UAE) for extracting COP by RSM were further compared. Results showed that EAE had the largest yield of polysaccharides with lower equipment cost.

Biodiesel production from Jatropha oil catalyzed by immobilized Burkholderia cepacia lipase on modified attapulgite.

Lipase from Burkholderia cepacia was immobilized on modified attapulgite by cross-linking reaction for biodiesel production with jatropha oil as feedstock. Effects of various factors on biodiesel production were studied by single-factor experiment. Results indicated that the best conditions for biodiesel preparation were: 10 g jatropha oil, 2.4 g methanol (molar ratio of oil to methanol is 1:6.6) being added at 3h intervals, 7 wt% water, 10 wt% immobilized lipase, temperature 35°C, and time 24h. Under these conditions, the maximum biodiesel yield reached 94%. The immobilized lipase retained 95% of its relative activity during the ten repeated batch reactions. The half-life time of the immobilized lipase is 731 h. Kinetics was studied and the Vmax of the immobilized lipases were 6.823 mmol L(-1). This immobilized lipase catalyzed process has potential industrial use for biodiesel production to replace chemical-catalyzed method.