Gel Filtration Chromatography Combined with Bradford Method for Determination of Total Residual Protein in Ferment Antibiotics

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.

Electrochemical Study of Lincomycin on Au-PtNPs/nanoPAN/ Chitosan Nanocomposite Membrane and Its Determination in Injections

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.

Extraction Equilibrium of Lincomycin with Long-Chain Alcohols

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

Coupled strategy based on regulator manipulation and medium optimization empowers the biosynthetic overproduction of lincomycin

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.

Effects of periodical application of bioactive peptides derived from cottonseed on performance,immunity,total antioxidant activity of serum and intestinal development of broilers

This experiment aimed to examine the effect of periodical application of bioactive peptides derived from cottonseed(BPC)in comparison with using sub-therapeutic doses of lincomycin and the excessive in-clusion of vitamin E on performance,immunity,total antioxidant capacity of serum and intestinal morphology of broiler chickens.A total of 240 one-d-old male broiler chicks with similar initial weight(Ross strain)were randomly assigned to 6 groups(8 chicks/pen):non-treated group(basal diet),basal diet supplemented with 2 mg/kg lincomycin,basal diet supplemented with 50 IU vitamin E,basal diet supplemented with 6 g BPC/kg in starter period,basal diet supplemented with 6 g BPC/kg in starter and grower periods and basal diet supplemented with 6 g BPC/kg throughout the whole experiment.The highest final body weight was obtained in the group supplemented with BPC in starter and grower periods.In the finisher phase,broilers fed the diet containing BPC in the starter period and in the whole trial had significantly(P<0.05)better feed conversion ratios(FCR).Jejunal villus height was significantly elevated in broilers supplemented with antibiotic(P<0.001),furthermore it tended to be greater in broilers fed BPC in the starter period.The jejunal villus height-to-crypt depth ratio was significantly(P<0.01)higher in broilers fed the diet containing antibiotic in comparison to other groups.Humoral immune response against Newcastle disease vaccine tended to be elevated in broilers fed the diet containing BPC in the whole trial(P>0.05).Broilers supplemented with BPC in starter and grower,and in the whole trial had significantly(P<0.05)higher antibody titers against sheep red blood cells(SRBC).The highest total antioxidant capacity was obtained in broilers supplemented with the excessive level of vitamin E,furthermore it tended to improve in broilers fed the diet containing BPC in the whole trial.In summary,the results of the study indicated that addition of BPC in broiler diets in the whole trial could improve FCR,immune responses and total antioxidant activity of serum,and BPC could be used in broiler diets as an alternative to in-feed antibiotics.

Complete genome sequence of high-yield strain S. lincolnensis B48 and identification of crucial mutations contributing to lincomycin overproduction

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.