The Au-Pt alloy nanoparticles（Au-PtNPs） were electrochemically deposited on the surface of polyaniline nanotube（nanoPAN） and chitosan（CS） modified glassy carbon electrode（GCE）. The electrochemical behavior of ...The Au-Pt alloy nanoparticles（Au-PtNPs） were electrochemically deposited on the surface of polyaniline nanotube（nanoPAN） and chitosan（CS） modified glassy carbon electrode（GCE）. The electrochemical behavior of lincomycin at Au-PtNPs/nanoPAN/CS modified GCE was investigated by cyclic voltammetry, linear sweep voltammetry and chronocoulometry. Cyclic voltammetric experiments show that lincomycin at the nanocomposite membrane modified electrode exhibited a pair of quasi-reversible redox peaks in pH=6.0 PBS. The membrane could accelerate the electron transfer of lincomycin on the electrode and significantly enhance the peak current. In a range of 3.0-100.0 mg/L, the reductive peak current of lincomycin at 0.42 V was linearly related to its concentration and the linear regression equation was ip,c=0.2703ρ-0.0042（ip, c： μA; ρ： mg/L; r=0.998, n=7） with a detection limit of 1.0 mg/L（S/N =3）. Compared with other methods, this method exhibited many advantages such as high sensitivity, selectivity, wide linear range and low detection limit. The method was used to determine the content of lincomycin in injections commercially available with satisfactory results. Some electrochemical parameters involved in the redox reaction of lincomycin, such as parameter of kinetic ha, standard rate constant ks and the number of H^＋, were also calculated.展开更多
1 INTRODUCTIONLincomycin is an important antibiotic widely used in medicine.Recovery of lincomycin fromfermentation filtrate can be carried out either by solvent extraction with n-butyl alcohol(ormethylene dichloride)...1 INTRODUCTIONLincomycin is an important antibiotic widely used in medicine.Recovery of lincomycin fromfermentation filtrate can be carried out either by solvent extraction with n-butyl alcohol(ormethylene dichloride) or by adsorption with activated carbon.Both approaches are time-consuming,with poor selectivity and high solvent losses.Improved solvent extraction展开更多
The lincosamide family antibiotic lincomycin is a widely used antibacterial pharmaceutical generated by Streptomyces lincolnensis,and the high-yield strain B48 produces 2.5 g/L lincomycin,approximately 30-fold as the ...The lincosamide family antibiotic lincomycin is a widely used antibacterial pharmaceutical generated by Streptomyces lincolnensis,and the high-yield strain B48 produces 2.5 g/L lincomycin,approximately 30-fold as the wild-type strain NRRL 2936.Here,the genome of S.lincolnensis B48 was completely sequenced,revealing a^10.0 Mb single chromosome with 71.03%G+C content.Based on the genomic information,lincomycinrelated primary metabolism network was constructed and the secondary metabolic potential was analyzed.In order to dissect the overproduction mechanism,a comparative genomic analysis with NRRL 2936 was performed.Three large deletions(LDI-III),one large inverted duplication(LID),one long inversion and 80 small variations(including 50 single nucleotide variations,13 insertions and 17 deletions)were found in B48 genome.Then several crucial mutants contributing to higher production phenotype were validated.Deleting of a MarRtype regulator-encoding gene slinc377 from LDI,and the whole 24.7 kb LDII in NRRL 2936 enhanced lincomycin titer by 244%and 284%,respectively.Besides,lincomycin production of NRRL 2936 was increased to 7.7-fold when a 71 kb supercluster BGC33 from LDIII was eliminated.As for the duplication region,overexpression of the cluster situated genes lmbB2 and lmbU,as well as two novel transcriptional regulator-encoding genes(slinc191 and slinc348)elevated lincomycin titer by 77%,75%,114%and 702%,respectively.Furthermore,three negative correlation genes(slinc6156,slinc4481 and slinc6011)on lincomycin biosynthesis,participating in regulation were found out.And surprisingly,inactivation of RNase J-encoding gene slinc6156 and TPR(tetratricopeptide repeat)domain-containing protein-encoding gene slinc4481 achieved lincomycin titer equivalent to 83%and 68%of B48,respectively,to 22.4 and 18.4-fold compared to NRRL 2936.Therefore,the comparative genomics approach combined with confirmatory experiments identified that large fragment deletion,long sequence duplication,along with several mutations of genes,especially regulator genes,are crucial for lincomycin overproduction.展开更多
The biosynthesis of bioactive secondary metabolites,specifically antibiotics,is of great scientific and economic importance.The control of antibiotic production typically involves different processes and molecular mec...The biosynthesis of bioactive secondary metabolites,specifically antibiotics,is of great scientific and economic importance.The control of antibiotic production typically involves different processes and molecular mechanism.Despite numerous efforts to improve antibiotic yields,joint engineering strategies for combining genetic manipulation with fermentation optimization remain finite.Lincomycin A(Lin-A),a lincosamide antibiotic,is industrially fermented by Streptomyces lincolnensis.Herein,the leucine-responsive regulatory protein(Lrp)-type regulator SLCG_4846 was confirmed to directly inhibit the lincomycin biosynthesis,whereas indirectly controlled the transcription of SLCG_2919,the first reported repressor in S.lincolnensis.Inactivation of SLCG_4846 in the high-yield S.lincolnensis LA219X(LA219XΔ4846)increases the Lin-A production and deletion of SLCG_2919 in LA219XΔ4846 exhibits superimposed yield increment.Given the effect of the double deletion on cellular primary metabolism of S.lincolnensis,Plackett-Burman design,steepest ascent and response surface methodologies were utilized and employed to optimize the seed medium of this double mutant in shake flask,and Lin-A yield using optimal seed medium was significantly increased over the control.Above strategies were performed in a 15-L fermenter.The maximal yield of Lin-A in LA219XΔ4846-2919 reached 6.56 g/L at 216 h,55.1%higher than that in LA219X at the parental cultivation(4.23 g/L).This study not only showcases the potential of this strategy to boost lincomycin production,but also could empower the development of high-performance actinomycetes for other antibiotics.展开更多
Aim A novel method has been developed for evaluation of the levels of total residual protein in antibiotics produced by fermentation using gel filtration chromatography (GFC) combined with Bradford assay based on dete...Aim A novel method has been developed for evaluation of the levels of total residual protein in antibiotics produced by fermentation using gel filtration chromatography (GFC) combined with Bradford assay based on determination of residual protein in lincomycin hydrochloride. Methods The chromatographic conditions were SuperdexTM peptide column, 0.01 mol*L-1 phosphate buffer solution as mobile phase, and flow rate of 1 mL·min-1. Five hundred microliters of lincomycin hydrochloride solution (3 g of lincomycin hydrochloride dissolved in 10 mL of mobile phase) was injected into the chromatograph and the eluted solution was collected between 6 min and 14.5 min (protein eluted from column within this period), and the residual content of total protein in the eluted solution was assayed using Bradford assay method. Results The average recovery was more than 90% for bovine serum albumin, the calibration equation for the range of 0-12 μg·mL-1 of protein was y=-0.002 4x2+0.064 2x+0.002 9, r2=0.999 9, RSD=0.1%-0.9%, and the LOD and LOQ were 3 and 10 ng·mL-1 of protein, respectively. Conclusion The novel method for determining the residual protein in ferment antibio-tics is simple, rapid, and precise.展开更多
建立了一种PRIME HLB固相萃取结合高效液相色谱-串联质谱法测定禽蛋中林可霉素残留的分析方法。样品经1%氨化乙腈提取,涡旋、振荡、超声处理后,再进行高速低温离心,Oasis PRIME HLB固相萃取柱净化。收集全部流出液经氮气吹干,复溶,供液...建立了一种PRIME HLB固相萃取结合高效液相色谱-串联质谱法测定禽蛋中林可霉素残留的分析方法。样品经1%氨化乙腈提取,涡旋、振荡、超声处理后,再进行高速低温离心,Oasis PRIME HLB固相萃取柱净化。收集全部流出液经氮气吹干,复溶,供液相色谱质谱分析。结果表明,林可霉素在1~50 ng·mL^(-1)线性关系良好,相关系数r为0.9993。方法定量限为1.0μg·kg^(-1),加标回收率为76.5%~93.3%,相对标准偏差为4.91%~8.57%。该方法准确性较好,可满足禽蛋中林可霉素的日常检测。展开更多
基金Supported by the National Natural Science Foundation of China(Nos.20635020 and 20805025)Doctorial Foundation of the Ministry of Education of China(No.20060426001) Doctorial Fund of Qingdao University of Science and Technology, China(No.0022278)
文摘The Au-Pt alloy nanoparticles（Au-PtNPs） were electrochemically deposited on the surface of polyaniline nanotube（nanoPAN） and chitosan（CS） modified glassy carbon electrode（GCE）. The electrochemical behavior of lincomycin at Au-PtNPs/nanoPAN/CS modified GCE was investigated by cyclic voltammetry, linear sweep voltammetry and chronocoulometry. Cyclic voltammetric experiments show that lincomycin at the nanocomposite membrane modified electrode exhibited a pair of quasi-reversible redox peaks in pH=6.0 PBS. The membrane could accelerate the electron transfer of lincomycin on the electrode and significantly enhance the peak current. In a range of 3.0-100.0 mg/L, the reductive peak current of lincomycin at 0.42 V was linearly related to its concentration and the linear regression equation was ip,c=0.2703ρ-0.0042（ip, c： μA; ρ： mg/L; r=0.998, n=7） with a detection limit of 1.0 mg/L（S/N =3）. Compared with other methods, this method exhibited many advantages such as high sensitivity, selectivity, wide linear range and low detection limit. The method was used to determine the content of lincomycin in injections commercially available with satisfactory results. Some electrochemical parameters involved in the redox reaction of lincomycin, such as parameter of kinetic ha, standard rate constant ks and the number of H^＋, were also calculated.
基金Supported by the National Natural Science Foundation of China.
文摘1 INTRODUCTIONLincomycin is an important antibiotic widely used in medicine.Recovery of lincomycin fromfermentation filtrate can be carried out either by solvent extraction with n-butyl alcohol(ormethylene dichloride) or by adsorption with activated carbon.Both approaches are time-consuming,with poor selectivity and high solvent losses.Improved solvent extraction
基金This study was supported by the National Natural Science Foundation of China(NSFC)(31900059)the China Postdoctoral Science Foundation(2019M650079)the Research Program of State Key Laboratory of Bioreactor Engineering.We thank Dr.Weihong Jiang(Institute of Plant Physiology and Ecology,Chinese Academy of Sciences)for kindly providing the plasmid pKCcas9dO.
文摘The lincosamide family antibiotic lincomycin is a widely used antibacterial pharmaceutical generated by Streptomyces lincolnensis,and the high-yield strain B48 produces 2.5 g/L lincomycin,approximately 30-fold as the wild-type strain NRRL 2936.Here,the genome of S.lincolnensis B48 was completely sequenced,revealing a^10.0 Mb single chromosome with 71.03%G+C content.Based on the genomic information,lincomycinrelated primary metabolism network was constructed and the secondary metabolic potential was analyzed.In order to dissect the overproduction mechanism,a comparative genomic analysis with NRRL 2936 was performed.Three large deletions(LDI-III),one large inverted duplication(LID),one long inversion and 80 small variations(including 50 single nucleotide variations,13 insertions and 17 deletions)were found in B48 genome.Then several crucial mutants contributing to higher production phenotype were validated.Deleting of a MarRtype regulator-encoding gene slinc377 from LDI,and the whole 24.7 kb LDII in NRRL 2936 enhanced lincomycin titer by 244%and 284%,respectively.Besides,lincomycin production of NRRL 2936 was increased to 7.7-fold when a 71 kb supercluster BGC33 from LDIII was eliminated.As for the duplication region,overexpression of the cluster situated genes lmbB2 and lmbU,as well as two novel transcriptional regulator-encoding genes(slinc191 and slinc348)elevated lincomycin titer by 77%,75%,114%and 702%,respectively.Furthermore,three negative correlation genes(slinc6156,slinc4481 and slinc6011)on lincomycin biosynthesis,participating in regulation were found out.And surprisingly,inactivation of RNase J-encoding gene slinc6156 and TPR(tetratricopeptide repeat)domain-containing protein-encoding gene slinc4481 achieved lincomycin titer equivalent to 83%and 68%of B48,respectively,to 22.4 and 18.4-fold compared to NRRL 2936.Therefore,the comparative genomics approach combined with confirmatory experiments identified that large fragment deletion,long sequence duplication,along with several mutations of genes,especially regulator genes,are crucial for lincomycin overproduction.
基金supported in part by the Anhui Provincial Natural Science Foundation for Excellent Young Scholars(grant no.2208085Y09)the National Natural Science Foundation of China(grant no.32170073,31972930).
文摘The biosynthesis of bioactive secondary metabolites,specifically antibiotics,is of great scientific and economic importance.The control of antibiotic production typically involves different processes and molecular mechanism.Despite numerous efforts to improve antibiotic yields,joint engineering strategies for combining genetic manipulation with fermentation optimization remain finite.Lincomycin A(Lin-A),a lincosamide antibiotic,is industrially fermented by Streptomyces lincolnensis.Herein,the leucine-responsive regulatory protein(Lrp)-type regulator SLCG_4846 was confirmed to directly inhibit the lincomycin biosynthesis,whereas indirectly controlled the transcription of SLCG_2919,the first reported repressor in S.lincolnensis.Inactivation of SLCG_4846 in the high-yield S.lincolnensis LA219X(LA219XΔ4846)increases the Lin-A production and deletion of SLCG_2919 in LA219XΔ4846 exhibits superimposed yield increment.Given the effect of the double deletion on cellular primary metabolism of S.lincolnensis,Plackett-Burman design,steepest ascent and response surface methodologies were utilized and employed to optimize the seed medium of this double mutant in shake flask,and Lin-A yield using optimal seed medium was significantly increased over the control.Above strategies were performed in a 15-L fermenter.The maximal yield of Lin-A in LA219XΔ4846-2919 reached 6.56 g/L at 216 h,55.1%higher than that in LA219X at the parental cultivation(4.23 g/L).This study not only showcases the potential of this strategy to boost lincomycin production,but also could empower the development of high-performance actinomycetes for other antibiotics.
文摘Aim A novel method has been developed for evaluation of the levels of total residual protein in antibiotics produced by fermentation using gel filtration chromatography (GFC) combined with Bradford assay based on determination of residual protein in lincomycin hydrochloride. Methods The chromatographic conditions were SuperdexTM peptide column, 0.01 mol*L-1 phosphate buffer solution as mobile phase, and flow rate of 1 mL·min-1. Five hundred microliters of lincomycin hydrochloride solution (3 g of lincomycin hydrochloride dissolved in 10 mL of mobile phase) was injected into the chromatograph and the eluted solution was collected between 6 min and 14.5 min (protein eluted from column within this period), and the residual content of total protein in the eluted solution was assayed using Bradford assay method. Results The average recovery was more than 90% for bovine serum albumin, the calibration equation for the range of 0-12 μg·mL-1 of protein was y=-0.002 4x2+0.064 2x+0.002 9, r2=0.999 9, RSD=0.1%-0.9%, and the LOD and LOQ were 3 and 10 ng·mL-1 of protein, respectively. Conclusion The novel method for determining the residual protein in ferment antibio-tics is simple, rapid, and precise.
文摘建立了一种PRIME HLB固相萃取结合高效液相色谱-串联质谱法测定禽蛋中林可霉素残留的分析方法。样品经1%氨化乙腈提取,涡旋、振荡、超声处理后,再进行高速低温离心,Oasis PRIME HLB固相萃取柱净化。收集全部流出液经氮气吹干,复溶,供液相色谱质谱分析。结果表明,林可霉素在1~50 ng·mL^(-1)线性关系良好,相关系数r为0.9993。方法定量限为1.0μg·kg^(-1),加标回收率为76.5%~93.3%,相对标准偏差为4.91%~8.57%。该方法准确性较好,可满足禽蛋中林可霉素的日常检测。
