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.

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.

盐酸林可霉素结晶工艺研究

为提高盐酸林可霉素收率,本研究主要考察了溶析结晶过程中结晶温度、搅拌转速、溶析剂滴加速率、溶剂与溶析剂体积分数比、晶种加入量、养晶时间等因素对盐酸林可霉素结晶收率的影响。结果显示,当结晶温度为25℃,搅拌转速为200 r/min,溶析剂滴加速率为8 mL/min,溶剂与溶析剂体积分数比为1:10,晶种加入量为2%,养晶时间为2 h时,可以有效提升盐酸林可霉素收率。

抗生素过敏致人死亡1例

临床抗生素中毒和致死的情况,主要是抗生素过量使用或者患者本身体质特别敏感而引发的。本文以一起非法行医案为例,采用液相色谱-串联质谱法对抗生素引起的疑似过敏性死亡病例中林可霉素、头孢曲松和地塞米松成分进行检测分析。结合案件勘查情况、尸体解剖、病理学检验及毒物检验的结果,证实死者死于抗生素过敏反应。本结果填补了液相色谱-串联质谱法同时测定人体血浆中林可霉素、头孢曲松和地塞米松的空白,填充了因抗生素过敏致死的数据量,归纳了抗生素过敏致死的法医学指征及检验,可为抗生素过敏和死亡病例的临床和法医学鉴定提供方法和数据参考。

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.

盐酸林可霉素注射液包装系统中16种元素杂质含量测定及相容性考察

为建立测定盐酸林可霉素注射液包装系统中16种元素杂质的含量方法,考察在稳定性研究过程中盐酸林可霉素注射液与包装材料的相容性风险,提取液及药液以2%硝酸溶液稀释,分别用钪(Sc)、铟(In)、铋(Bi)和锗(Ge)元素为内标,采用电感耦合等离子体质谱法(ICP-MS)测定16种元素杂质含量。结果表明,2批新生产的盐酸林可霉素注射液(0个月)中16种元素杂质检测结果均符合分析评价阈值要求。在加速条件下存放6个月和长期条件下存放12个月和24个月的同批次样品,硅元素检出量在限度的48%~58%,硼和铝两种元素检出量均小于其限度的30%,钒、镍、铅、镉、铅、砷、汞、钴、锂、锑、铜、铁、锌和钡13种元素检出量均小于其限度的10%。加速实验和长期实验的研究结果表明,包装材料与本品间有相互作用,但该作用未随放置时间的增加有明显的增长趋势,说明本品在全生命周期内与包装系统相容性良好,风险较低。

竞争性吸附法降低林可霉素B组分的工艺研究

生产林可霉素的传统工艺为萃取法,该方法不能有效去除B组分杂质,目前较新颖的树脂法虽能将B组分去除,但效率不够高。本文以竞争性吸附原理为指导,通过连续上柱的方式,放大树脂对产品与杂质的分离能力,从而实现了B组分的高效去除。本研究也为竞争性吸附法在其他生化产品分离领域的应用提供了借鉴。

前体物质对林可霉素发酵的影响

为提升林可霉素发酵效价,本文对林可霉素发酵工艺进行优化,考察在发酵过程中补加含硫前体物质、HMP、氨基酸等对林可霉素发酵的影响。结果表明,当添加0.1g/L半胱氨酸、1.5g/L硫酸钠、0.01g/L甲硫氨酸时,林可霉素发酵效价高达9855μ/mL;此外,添加4.0g/L HMP时,林可霉素效价高达9810μ/mL;另外,对5种氨基酸进行浓度组合优化后,林可霉素的发酵效价也得到了显著提升,其效价在氨基酸优化浓度为谷氨酸(0.1g/L)、缬氨酸(0.15g/L)、蛋氨酸(0.2g/L)、亮氨酸(0.5g/L)、酪氨酸(0.05g/L)时达到最高,高达9560μ/mL。因此,通过优化林可霉素前体物质添加工艺,可加快林可霉素的生物合成速率,有效提高林可霉素的发酵效价。

渗透压对林可霉素发酵效价影响的研究

为有效提高林可霉素A产量降低林可霉素B组分含量,提升林可霉素产品纯度,本研究以NaCl作为渗透压调节剂研究渗透压对林可霉素发酵的影响,通过比较不同NaCl质量分数(0、0.5%、1.0%、1.5%、2.5%)下林可霉素A和林可霉素B效价的大小,确定以1.0%NaCl作为渗透压调节剂的最适添加质量分数。同时,在该条件下,比较分析了甘油、脯氨酸和甜菜碱三种渗透压保护剂对林可霉素效价的影响,结果显示,甘油质量分数为0.5%时,林可霉素A效价最高林可霉素B效价最低,脯氨酸和甜菜碱质量分数为0.25%时,林可霉素A效价最高林可霉素B效价最低,比较发现0.25%脯氨酸效果显著高于0.5%甘油和0.25%甜菜碱。综上所述,在1.0%NaCl+0.25%脯氨酸时,可以有效提高林可霉素A效价降低林可霉素B效价,为进一步扩大林可霉素A产量奠定了基础。

黄豆饼粉熟化条件对林可霉素发酵的影响

黄豆饼粉是抗生素发酵最常用的有机氮源之一,黄豆饼粉制作时对大豆不同的处理条件造成不同的熟化度,不同的熟化度对不同抗生素品种的发酵影响不同。本文主要是使用不同熟化条件的黄豆饼粉对林可霉素进行摇瓶发酵考察,并在15 L发酵罐进行验证,最终熟化温度95℃、熟化时间20 min最佳,林可霉素发酵单位7708μg/mL,较对照提高17.1%,为林可霉素发酵生产对黄豆饼粉的选择提供数据支持。

林可霉素生产废水的厌氧生物处理工艺

采用单相中温升流式厌氧污泥床 (UASB)反应器厌氧生物工艺处理含有有毒难降解有机物的林可霉素生产废水 .当进水COD 80 0 0～ 1 4 0 0 0mg/L ,HRT约 1 0h时 ,COD容积负荷可达 2 0～ 35kg/(m3·d) ,COD去除率为50 %～ 55% .适时调整并维持较高的表面水力负荷 [0 .2～ 0 4m3/(m2 ·h) ]、较高的进水有机基质浓度 (COD为2 0 0 0～ 30 0 0mg/L)和污泥COD负荷 [0 2～ 0 5kg/(kg·d) ],并适当延长启动驯化时间可培养出沉降性好、污泥活性较高的颗粒污泥 .废水厌氧生物降解动力学符合Monod方程 ,动力学常数Vmax=1 3d- 1,Ks=81 33mg/L .废水中不可生物降解物质占总COD的比例约为 30 % ,这是废水COD去除率偏低的重要因素 .

含固率和接种比对林可霉素菌渣厌氧消化的影响

通过林可霉素菌渣的中温厌氧消化摇瓶实验,比较不同的含固率(3%、5%、8%、10%)和接种比(0.5、1、2、3)对菌渣产甲烷能力的影响,以确定菌渣厌氧消化的最优工艺条件.结果表明,在研究参数范围内,含固率越低,接种比越高,越有利于甲烷的产生;经过10d的培养,在含固率为3%、接种比为3时的工况中,菌渣的挥发性固体(VS)累计净产甲烷量最高,为106mL/g;而含固率>5%、接种比<2的工况均受到了不同程度的抑制.因此,林可霉素菌渣的厌氧消化应采用含固率<5%、接种比>2的液态发酵工艺,此条件既能保证厌氧消化不受消化产物(胺和挥发性有机酸)积累的抑制,也可以缓冲菌渣中残留林可霉素对消化微生物可能产生的抑制效应.

盐酸林可霉素两种晶型晶体结构的分析与比较

目的:分析并比较盐酸林可霉素两种晶型单晶晶习及其晶体结构,探讨不同晶体结构对盐酸林可霉素胶囊溶出性的影响。方法:扫描电子显微镜、单晶X射线衍射与粉末X射线衍射法、溶出度测定法。结果:盐酸林可霉素晶型Ⅰ属正交晶系P22_12_1空间群;晶型Ⅱ属单斜晶系P2_1空间群,不对称单位中两个分子互为构象异构体。晶型Ⅰ胶囊溶出快于晶型Ⅱ。结论:盐酸林可霉素两种晶型分属不同晶系,不对称单位化学计量式不同,造成两者胶囊溶出度不同。

氨氮和林可霉素对有机物厌氧消化的抑制效应

以含高蛋白质和抗生素的林可霉素生产残渣产沼利用为背景,通过中温厌氧消化批式实验,研究不同的氨氮浓度（300~4500 mg·L-1）和林可霉素浓度（0~100 mg·L-1）对含氮含抗生素类生物质废物的厌氧消化产甲烷能力的抑制作用。结果表明,氨氮和林可霉素浓度均与累积产甲烷量显著负相关。经过240 h的培养,氨氮和林可霉素对厌氧消化产甲烷量的半抑制浓度（EC50）分别为1445 mg·L-1和35 mg·L-1;游离氨的EC50在27~28 mg·L-1之间。在25 mg·L-1林可霉素存在下,氨氮对厌氧消化的抑制趋势和抑制程度并未明显改变,说明氨氮的抑制作用独立于林可霉素。

高效液相色谱-电喷雾串联质谱法测定蜂蜜中的林可胺类抗生素残留

建立了蜂蜜中林可胺类抗生素林可霉素和氯林可霉素的高效液相色谱-电喷雾串联质谱（HPLC／ESI-MS／MS）检测方法。样品经固相萃取提取净化、反相液相色谱分离后进行质谱分析，在选择反应监测模式（SRM）下进行特征母-子离子对信号采集。根据保留时间、母离子和两个特征子离子信息进行定性分析，以共同的基峰离子m／z126进行定量。两种抗生素的检测限（S／N=3）为0．1p．g／kg，定量限为0．5p．g／kg，在1．0～200p．g／L时峰强度与质量浓度的线性关系良好（r^2〉0．996）。在1．0，5．0，20．0p．g／kg3个添加水平，两种抗生素的平均回收率范围为80％～110％，日内测定结果的相对标准偏差小于8％，日间测定结果的相对标准偏差小于15％。结果表明，该法简单、灵敏，特异性强，适用于蜂蜜中林可胺类抗生素残留的分析确证。

纳滤膜分离洁霉素生产废水的试验研究

针对洁霉素生产废水 ,采用国内外多种纳滤膜在不同工况条件下进行了分离试验研究 ,筛选和确定了适用纳滤膜的种类和其最佳工作条件 ,为纳滤膜的进一步应用奠定了基础 .

生物素与氨基酸对林可霉素生物合成的影响

在合成培养基中利用林可链霉菌发酵生产林可霉素。当向培养基中加入生物素和氨基酸时,林可霉素的产量受到很大影响。本研究中首先采用两水平因子设计法筛选出6个显著影响因子,即生物素、谷氨酸、缬氨酸、蛋氨酸、亮氨酸和酪氨酸;然后采用中心组合设计法,得到上述6因子的优化含量分别为30μg/L、100.5、83、29、117.5和58.5mg/L;最后在摇瓶中进行验证实验,优化条件下发酵液的生物效价为2116μg/ml;对照样品I(培养基中无生物素和氨基酸)和II(培养基中按原配方加入6个显著影响因子)的生物效价分别为893和1481μg/ml,与样品I和II比较,生物效价分别提高136.62%和42.88%。

动物可食性组织中林可霉素和大观霉素残留检测方法——气相色谱法

大观霉素、林可霉素两者联合使用是兽医上常用的复方制剂。但是不规范用药可导致在动物组织或其产品中残留,会产生较明显副作用,不仅影响动物性产品的加工生产,而且还会严重影响消费者的身体健康。实验建立了检测动物可食性组织中林可霉素和大观霉素残留量的气相色谱法,线性实验和回收率实验结果表明,该方法测定可食性组织中的林可霉素和大观霉素的残留量准确可靠,组织中添加林可霉素的定量限为30μg/kg,大观霉素定量限为40μg/kg。在组织中添加30~3 000μg/kg林可霉素或40~10 000μg/kg大观霉素,回收率为73.2%~85.7%。该法能同时检测林可霉素和大观霉素两种药物,缩短了分析时间,有机试剂用量少,前处理简单,回收率高。