Kinetics of Asymmetric Reduction of Phenylglyoxylic Acid to R-(-)-Mandelic Acid by Saccharomyces Cerevisiae FD11b

The kinetics of asymmetric production of R-(-)-mandelic acid(R-MA)from phenylglyoxylic acid (PGA) catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of glucose and PGA were controlled respectively with a dual feeding system. When the electron donor glucose was supplied at the rate of 0.0833mmol·gdw-1·h-1, the specific production rate (q p ) and the enantiomeric excess of R-MA reached the maxi-mum 0.353mmol·gdw-1·h-1 and 97.1%, respectively. The apparent reduction activity of yeast FD11b was obviously af-fected by both substrate PGA and product MA. The qp value reached the maximum 0.36—0.38mmol·gdw-1·h-1 when the PGA concentration was controlled between 25 and 35mmol·L-1. The obvious substrate inhibition of bioconversion was observed at the PGA concentrations higher than 40mmol·L-1. The accumulation of product MA also caused a severe feed-back inhibition for its production when the product concentration was above 60mmol·L-1. The kinetic model with the inhibition effect of both substrate and product was simulated by a computer-based least-square arithmatic. The estab-lished kinetic model was in good agreement with the experimental data.

Characterization and biological evaluation of a novel silver nanoparticle-loaded collagen-chitosan dressing

Effective coverage and protection is a priority in wound treatment.Collagen and chitosan have been widely used for wound dressings due to their excellent biological activity and biocompatibility.Silver nanoparticles(AgNPs)have a powerful antibacterial effect.In this study,a macromolecular and small-molecular collagen mixed solution,a macromolecular and small-molecular chitosan mixed solution were prepared,and a silver nanoparticle-loaded collagen-chitosan dressing(AgNPCCD)has been proposed.First,the effects of a collagen-chitosan mixed solution on the proliferation of human umbilical vein endothelial cells and the secretion of cytokines were evaluated.Then,the characteristics and antibacterial effects of the AgNP-CCD were tested,and the effects on wound healing and the influence of wound cytokine expression were investigated via a deep second-degree burn wound model.The results showed that at the proper proportion and concentration,the collagen-chitosan mixed solution effectively promoted cell proliferation and regulated the levels of growth factors(vascular endothelial growth factor[VEGF],epidermal growth factor[EGF],plateletderived growth factor[PDGF],transforming growth factor[TGF-b1],basic fibroblastic growth factor[bFGF])and inflammatory factors(TNF-α,IL-1β,IL-6,IL-8).Moreover,AgNP solutions at lower concentrations exerted limited inhibitory effects on cell proliferation and had no effect on cytokine secretion.The AgNP-CCD demonstrated satisfactory morphological and physical properties as well as efficient antibacterial activities.An in vivo evaluation indicated that AgNP-CCD could accelerate the healing process of deep second-degree burn wounds and played an important role in the regulation of growth and inflammatory factors,including VEGF,EGFL-7,TGF-β1,bFGF,TNF-α and IL-1β.This AgNP-CCD exerted excellent biological effects on wound healing promotion and cytokine expression regulation.

Rational and semi-rational engineering of cytochrome P450s for biotechnological applications

The cytochrome P450 enzymes are ubiquitous heme-thiolate proteins performing regioselective and stereoselective oxygenation reactions in cellular metabolism.Due to their broad substrate scope and catalytic versatility,P450 enzymes are also attractive candidates for many industrial and biopharmaceutical applications.For particular uses,enzyme properties of P450s can be further optimized through directed evolution,rational,and semi-rational engineering approaches,all of which introduce mutations within the P450 structures.In this review,we describe the recent applications of these P450 engineering approaches and highlight the key regions and residues that have been identified using such approaches.These“hotspots”lie within critical functional areas of the P450 structure,including the active site,the substrate access channel,and the redox partner interaction interface.

Expression of Drosophila melanogaster acetylcholinesterase(DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides

Acetylcholinesterase(AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method.An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris.The pPIC9 K-Drosophila melanogaster acetylcholinesterase(DmAChE)-like expression vector was constructed by codon optimization of this mutant strain,which was transformed into P.pastoris GS115,and positive clones were selected on yeast peptone dextrose(YPD) plate with G418 at 4.0 mg/mL.The GS115-pPIC9 K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h,with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) pattern of the fermentation supernatant.After preliminary purification by ammonium sulfate precipitation,the enzyme activity was detected to be 76.9 U/(mL·min).In addition,the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.

m4C DNA methylation regulates biosynthesis of daptomycin in Streptomyces roseosporus L30

Despite numerous studies on transcriptional level regulation by single genes in drug producing Actinomyces,the global regulation based on epigenetic modification is not well explored.N4-methylcytosine(m4C),an abundant epigenetic marker in Actinomycetes’genome,but its regulatory mechanism remains unclear.In this study,we identify a m4C methyltransferase(SroLm3)in Streptomyces roseosporus L30 and multi-omics studies were performed and revealed SroLm3 as a global regulator of secondary metabolism.Notably,three BGCs inΔsroLm3 strain exhibited decreased expression compared to wild type.In-frame deletion of sroLm3 in S.roseosporus L30 further revealed its role in enhancing daptomycin production.In summary,we characterized a m4C methyltransferase,revealed the function of m4C in secondary metabolism regulation and biosynthesis of red pigment,and mapped a series of novel regulators for daptomycin biosynthesis dominated by m4C methylation.Our research further indicated that m4C DNA methylation may contribute to a metabolic switch from primary to secondary metabolism in Actinomyces.

A sandwich structure composite wound dressing with firmly anchored silver nanoparticles for severe burn wound healing in a porcine model

Wounds may remain open for a few weeks in severe burns,which provide an entry point for pathogens and microorganisms invading.Thus,wound dressings with long-term antimicrobial activity are crucial for severe burn wound healing.Here,a sandwich structure composite wound dressing anchored with silver nanoparticles(AgNPs)was developed for severe burn wound healing.AgNPs were in situ synthesized on the fibers of chitosan nonwoven fabric(CSNWF)as the interlayer of wound dressing for sustained release of silver ion.The firmly anchored AgNPs could prevent its entry into the body,thereby eliminating the toxicity of nanomaterials.The outer layer was a polyurethane membrane,which has a nanoporous structure that could maintain free transmission of water vapor.Chitosan/collagen sponge was selected as the inner layer because of its excellent biocompatibility and biodegradability.The presence of AgNPs in the CSNWF was fully characterized,and the high antibacterial activity of CSNWF/AgNPs was confirmed by against Escherichia coli,Pseudomonas aeruginosa and Staphylococcus aureus.The superior wound healing effect on deep dermal burns of presented composite wound dressing was demonstrated in a porcine model.Our finding suggested that the prepared AgNPs doped sandwich structure composite wound dressing has great potential application in severe wound care.

iGMDR:Integrated Pharmacogenetic Resource Guide to Cancer Therapy and Research

Current pharmacogenetic studies have obtained many genetic models that can predict the therapeutic efficacy of anticancer drugs.Although some of these models are of crucial importance and have been used in clinical practice,these very valuable models have not been well adopted into cancer research to promote the development of cancer therapies due to the lack of integration and standards for the existing data of the pharmacogenetic studies.For this purpose,we built a resource investigating genetic model of drug response(iGMDR),which integrates the models from in vitro and in vivo pharmacogenetic studies with different omics data from a variety of technical systems.In this study,we introduced a standardized process for all integrations,and described how users can utilize these models to gain insights into cancer.iGMDR is freely accessible at https://igmdr.modellab.cn.