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De novo biosynthesis of 8‑prenylnaringenin in Saccharomyces cerevisiae improved by screening and engineering of prenyltransferases and precursor pathway
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作者 Chaojie Guo Yongkun Lv +2 位作者 Hongbiao Li Jingwen Zhou Sha Xu 《Systems Microbiology and Biomanufacturing》 EI 2023年第4期647-658,共12页
8-Prenylnaringenin(8-PN)is a valuable medical phytoestrogen,which is a precursor to many prenylated flavonoids.How-ever,the availability of 8-PN is limited by inefficient prenyltransferases(PTs)and inadequate substrat... 8-Prenylnaringenin(8-PN)is a valuable medical phytoestrogen,which is a precursor to many prenylated flavonoids.How-ever,the availability of 8-PN is limited by inefficient prenyltransferases(PTs)and inadequate substrate precursor levels in microbial chassis.First,six PTs from different sources and their truncated cognates were expressed in a(2S)-naringenin producing strain.Only SfN8DT-1 derived from Sophora flavescens and its truncated cognate,tSfN8DT-1,could synthe-size 8-PN.Second,tSfN8DT-1 was engineered by multiple sequence alignment and a mutant tSfN8DT-1^(Q12E)with greater catalytic activity was obtained.Third,key genes,tHMGR and IDI1,of the mevalonate(MVA)pathway were overexpressed using a copy number combinatorial strategy,which greatly improved 8-PN titer by 368.75%.Fourth,a predicted structure of tSfN8DT-1^(Q12E)was used for molecular docking and virtual saturation mutagenesis.Two key residues,P229 and N305,were identified and saturation mutagenesis on these two sites resulted in an improved mutant N305M.The best-performing mutant,tSfN8DT-1^(Q12EN305M),produced 49.35±0.05 mg/L(5.57±0.01 mg/g DCW)8-PN in a shaking flask.Finally,101.40±2.55 mg/L of 8-PN was obtained in a 5-L bioreactor,which is the greatest titer reported to date for 8-PN.This study combined metabolic engineering and protein engineering methods to enhance precursor supplements and improve the catalytic ability of SfN8DT-1.The production of 8-PN in Saccharomyces cerevisiae was greatly increased through these methods,which may provide a feasible strategy for the biosynthesis of prenylated flavonoids. 展开更多
关键词 Saccharomyces cerevisiae PRENYLTRANSFERASES Prenylated flavonoid Protein engineering 8-prenylnaringenin
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啤酒花中8-异戊烯基柚皮素提取及含量测定方法的优化及验证
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作者 崔熠可 赖玉清 +4 位作者 黄惠明 陈静华 周跃平 闫冀 吴磊 《中国生物制品学杂志》 CAS CSCD 2017年第8期850-854,共5页
目的优化啤酒花中8-异戊烯基柚皮素(8-prenylnaringenin,8-PN)的提取及含量测定方法,并进行验证。方法采用L_8(4~1×2~2)正交试验,料液比为1∶15,利用回流法提取啤酒花中的8-PN,反相高效液相色谱法测定啤酒花中8-PN的含量[色谱分离... 目的优化啤酒花中8-异戊烯基柚皮素(8-prenylnaringenin,8-PN)的提取及含量测定方法,并进行验证。方法采用L_8(4~1×2~2)正交试验,料液比为1∶15,利用回流法提取啤酒花中的8-PN,反相高效液相色谱法测定啤酒花中8-PN的含量[色谱分离条件:ODS C18反相柱(4.6 mm×250 mm,0.45μm),用乙腈、1%甲酸进行梯度洗脱,流速:1.2 ml/min,检测波长:288 nm,柱温:30℃]。探讨提取溶剂(甲醇、乙醇、正丁醇、乙酸乙酯)、提取时间(40、60 min)、提取温度(40、60℃)对啤酒花中8-PN提取含量的影响。结果以正丁醇为提取溶剂,料液比为1∶15,利用回流提取,提取温度40℃,时间40 min,测得啤酒花中8-PN的最高提取含量为79.1 mg/kg。该方法的标准曲线相关系数R^2=0.999 8,线性范围为0.001~0.1 mg/ml,检出限为0.122 mg/L;检测浓度为0.03 mg/ml标准液的保留时间和峰面积的相对标准偏差(relative standard deviation,RSD)均小于1%,加标回收率在98%~102%之间。结论优化的方法可简便、准确地测定啤酒花中8-PN含量,为啤酒花中8-PN的进一步分离、纯化提供了实验依据。 展开更多
关键词 啤酒花 8-异戊烯基柚皮素 提取 含量测定
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离子交换树脂分离-分光光度法测定赤泥中钪 被引量:2
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作者 林筑 孔凡军 +1 位作者 郑美娟 何锦林 《冶金分析》 CAS CSCD 北大核心 2016年第8期73-77,共5页
赤泥用硝酸、氢氟酸和硫酸溶解,过强酸型树脂(Dowex50W-X8(H+型))交换树脂柱,用2mol/L盐酸多次淋洗,再用5mol/L盐酸洗脱富集在交换分离树脂柱上的钪,加热蒸干,再加高氯酸冒烟至尽干,依次加入10 mol/L过氧化氢和6 mol/L盐酸,加热至尽干,... 赤泥用硝酸、氢氟酸和硫酸溶解,过强酸型树脂(Dowex50W-X8(H+型))交换树脂柱,用2mol/L盐酸多次淋洗,再用5mol/L盐酸洗脱富集在交换分离树脂柱上的钪,加热蒸干,再加高氯酸冒烟至尽干,依次加入10 mol/L过氧化氢和6 mol/L盐酸,加热至尽干,用2mol/L盐酸溶解并转移到分液漏斗中,依次加入磺基水杨酸、溴甲酚绿,再用氨水和盐酸调节溶液至黄色。再加入偶氮氯膦-PN(CPA-PN)进行显色并与钪形成α型络合物,用20 mL0.02mol/L 1-苯基-3-甲基-4-苯甲酰基-5-吡唑啉酮(PMBP)-苯萃取该络合物。于分光光度计波长680nm处测量其吸光度,从校准曲线上查得钪含量,以此建立了离子交换树脂分离-分光光度法测定赤泥中钪的方法。结果表明,溶液中钪质量浓度在0.01~0.48μg/mL范围内与吸光度呈线性,校准曲线的线性相关系数r=0.998 9,表观摩尔吸光系数为3.86×10~4 L·mol^(-1)·cm^(-1),方法中钪的检出限为0.42μg/g。干扰试验结果表明,溶液中5.0μg Sc(Ⅲ)的测定结果相对误差不超过±5%时,BO33-(600),NH_4(Ⅰ)(200),Ca(Ⅱ)(100),RE_2O_3(60),Be(Ⅱ)(30),Fe(Ⅲ)、Al(Ⅲ)、Mn(Ⅱ)(25),Zr(Ⅳ)(4),Ti(Ⅳ)、Ta(Ⅴ)(2)不干扰测定。按照实验方法测定赤泥实际样品中钪,结果的相对标准偏差(RSD,n=6)为2.5%~3.9%,并与电感耦合等离子体原子发射光谱法测定结果相吻合。 展开更多
关键词 赤泥 Dowex50W-X8(H^+型)分离 偶氮氯膦-pn(CPA-pn)显色 分光光度法
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