云芝NADPH-细胞色素P450还原酶在大肠杆菌中表达及酶学特性分析

云芝Trametes versicolor具有很强的环境有机污染物降解能力,其烟酰胺腺嘌呤二核苷酸-细胞色素P450还原酶(NADPH-cytochrome P450 reductase,CPR)为细胞色素P450酶(Cytochrome P450s,CYPs)提供电子,参与有机污染物的降解过程。序列分析显示,云芝基因组拥有1个潜在CPR序列和多个潜在CYP序列。为深入研究云芝CPR参与细胞降解有机污染物的分子机制,实验进行了云芝CPR在大肠杆菌中异源表达和酶学特性分析。结果表明,经IPTG诱导后,去除预测的N端膜锚定区域(氨基酸残基1–24)的CPR蛋白(CPRΔ24)可在重组菌中实现可溶性表达,且表达蛋白的分子量与理论值(78 kDa)一致。镍离子亲和层析和分子筛层析纯化后测得其比活性为5.82 U/mg。酶学性质分析显示,重组CPRΔ24的最适温度和pH分别为35℃和8.0,并对一些金属离子及有机溶剂具有不同程度的耐受性。酶在35℃、pH 8.0反应条件下对NADPH的动力学参数K_m和k_(cat)分别为19.7μmol/L、3.31/s;对底物细胞色素c的动力学参数K_m和k_(cat)分别为25.9μmol/L、10.2/s。以上研究为探究云芝CPR在环境有机污染物降解途径中的功能机制奠定基础。     

P450酶在植物三萜化合物生物合成中的催化与调控

植物三萜化合物是一类具有6个C₅异戊二烯单元的高附加值天然化合物,具有抗炎、护肝、抗肿瘤、抗氧化和降血压等重要药理活性。在三萜化合物生物合成过程中,细胞色素P450酶通过引入羟基、羧基、羰基以及环氧基等官能团,为丰富三萜结构的多样性起到了重要的作用。然而,目前P450酶底物催化特异性机制仍不清晰,异源底盘细胞中表达率低、与细胞色素氧化还原酶(CPR)的适配性差限制了其在植物三萜化合物微生物异源合成中的应用。本文系统地介绍了植物三萜化合物的合成途径、P450酶的催化系统组成和催化机制。通过P450酶的理性与非理性的分子改造,P450酶及其CPR的适应性匹配以及关键代谢途径的区室化研究,以期为P450酶在高效合成三萜化合物的应用提供研究思路。     

CPR和P450基因在昆虫抗药性中的作用研究进展

P450酶系在昆虫代谢农药中有重要作用,NADPH-细胞色素P450还原酶（NADPH-cytochrome P450 reductase,CPR）和细胞色素P450（P450）在该酶系起核心作用。昆虫具有P450超基因家族,但只有一个单一的CPR基因,CPR是昆虫所有参与农药代谢的P450酶的唯一电子供体,其影响P450活性。P450基因的高水平表达在害虫抗药性中具有重要作用,P450基因介导的昆虫抗药性是最重要的代谢抗性类型。不同P450基因的高表达的调控机制不同,引起P450基因过量表达的原因可能有P450基因的编码区突变、顺式作用元件和反式作用因子变化、基因扩增等。细胞色素P450介导的抗药性存在一定程度的进化可塑性,即同种昆虫不同种群对相同的农药产生抗药性时,导致抗性产生的P450基因不同;同一昆虫品系在某种农药的抗性选择压力下,影响抗性的P450基因的种类和表达特性会随着持续的农药选择而发生变化。最近的研究显示,CPR的变异和昆虫抗药性相关,但是昆虫CPR基因介导抗药性的机制还缺乏深入研究。全面阐释P450酶系介导昆虫抗药性的机制、建立基于P450基因表达量变化与CPR突变的抗性分子标记,对于害虫抗药性治理具有重要意义。     

利用正交法确定测定意大利蜜蜂幼虫细胞色素P4500-脱乙基活性最佳反应条件

荧光分光光度法检测意大利蜜蜂Apis mellifera ligustica Spinal细胞色素P450 O-脱乙基活性方便快捷、较灵敏,以7-乙氧基香豆素为底物.通过测定其产物7-羟基香豆素的荧光变化来确定细胞色素P450 O-脱乙基的活性.在利用荧光分光光度法检测意大利蜜蜂幼虫细胞色素P450 O-脱乙基活性过程...     

吡虫啉胁迫对意大利蜜蜂哺育蜂免疫解毒相关基因表达及酶活力的影响

蜜蜂作为世界上最重要的授粉性昆虫,在采集过程中易接触到杀虫剂,前人研究表明新烟碱类杀虫剂吡虫啉（imidaclorprid）影响意大利蜜蜂Apis mellifera ligustica（简称“意蜂”）的存活和舞蹈、采集等行为。本研究旨在探究亚致死剂量吡虫啉胁迫对意大利蜜蜂哺育蜂（8日龄成年工蜂）免疫解毒相关基因表达、免疫解毒酶系活力及存活率的影响。结果显示哺育蜂连续取食3 d和9 d含0.1 ng/μL吡虫啉的蔗糖液后,其存活率与对照组（饲喂含等量丙酮的蔗糖溶液）无显著差异;连续饲喂11 d含0.1 ng/μL吡虫啉的50%蔗糖溶液后,其存活率与对照组有显著差异。荧光定量PCR检测及双抗体一步夹心法酶联免疫吸附试验结果显示哺育蜂取食吡虫啉3 d后,蜜蜂体内免疫基因多酚氧化酶基因（PPOA3,GB43738）,Abaecin类抗菌肽基因（ABA,GB18323）,葡萄糖脱氢酶基因（GLD, GB43007）和解毒基因细胞色素P450基因（CYP450 6a2,GB49876）,细胞色素B561基因（CYB561 2-like,GB40148）,葡萄糖醛酸转移酶（UDP-glucuronosyltransferase,GB52179）的表达及蜂体内体内细胞色素P450酶（cytochrome P450,CYP450）含量均有上调趋势,超氧化物岐化酶（superoxide dismutase,SOD）和过氧化氢酶（catalase,CAT）均有显著下调趋势;哺育蜂取食吡虫啉9 d后,PPOA3,ABA,GLD,CYP450 6a2,CYB561 2-like,UDP-glucuronosyltransferase的表达及蜂体内体内细胞色素P450酶含量均有下调趋势,多酚氧化酶（polyphenol oxidase,PPO）,超氧化物歧化酶和过氧化氢酶酶活力均有显著下调趋势。本研究在分子水平上提供了亚致死剂量吡虫啉是通过扰乱蜜蜂正常的免疫系统进而影响蜜蜂行为的证据,以期为维护蜜蜂健康提供一定的理论依据。     

构建酿酒酵母细胞工厂高效合成摩尔酸

[目的] 摩尔酸作为齐墩果烷型三萜化合物具有抗HIV、抗炎等多种生物学活性,其前体物质是计曼尼醇,本研究基于合成生物学策略构建酿酒酵母细胞工厂高效合成摩尔酸。[方法] 运用CRISPR/Cas9技术,首先分别整合不同来源的氧化鲨烯环化酶（OSCs）,筛选高产计曼尼醇底盘细胞;进一步异源表达长春花来源的细胞色素P450氧化酶（CYP716AL1）和麻风树来源的细胞色素P450还原酶（JcCPR）,构建摩尔酸生物合成途径;并通过CYP716AL1和不同来源的CPR适配研究以及过表达甲羟戊酸（MVA）代谢途径中关键酶的方式提高摩尔酸的产量。[结果] 整合苹果来源的氧化鲨烯环化酶MdOSC获得的重组菌株计曼尼醇产量最高,达68.3 mg/L;以此为底盘细胞进一步整合CYP716AL1和JcCPR实现了摩尔酸的生物合成,产量为15.0 mg/L;共表达CYP716AL1和拟南芥来源的CPR获得的重组菌株摩尔酸产量最高,达到24.3 mg/L;最后过表达MVA代谢途径中的关键酶法呢基焦磷酸合酶（ERG20）和鲨烯环氧酶（ERG1）,获得的重组菌株摩尔酸产量高达34.1 mg/L。[结论] 本研究实现了摩尔酸的高效生物合成,为构建高产齐墩果烷型三萜酿酒酵母细胞工厂提供了理论和技术依据。     

中国红豆杉细胞色素P450还原酶的基因克隆、表达与活性分析

细胞色素P450还原酶(Cytochrome P450 reductase,CPR)是细胞色素P450羟基化酶电子传递链的组成部分,在生物体内起着重要的电子传递作用。文章从中国红豆杉(Taxuswallichiana var. Chinensis)愈伤组织细胞中克隆CPR基因(TchCPR),TchCPR含有一个2154bp碱基的阅读框,编码717个氨基酸残基;在氨基酸水平上它与裸子植物细胞色素P450还原酶的同源性(82%)高于其他被子植物的细胞色素P450还原酶(74%)。在大肠杆菌BL21(DE3)中诱导表达了全长和从N-端截短不同数目氨基酸残基的6个融合肽段,经亲和层析纯化,分析了表达的不同长度融合蛋白的电子传递效率。结果表明截短长度大于61个氨基酸残基肽段的胞色素P450还原酶都能够诱导表达,在表达水平上无显著差异,而截短61个氨基酸的CPR融合蛋白电子传递的催化活性(1.6057nmol Cyt Cred/min/μg TchCPR融合蛋白)高于其他4个融合蛋白。     

灰飞虱唾液腺三大解毒酶家族的转录组分析

灰飞虱Laodelphax striatellus （Fallén）是危害多种禾本科经济作物的重要刺吸式害虫。唾液腺对刺吸式口器昆虫取食植物尤其重要, 其分泌的唾液可以帮助刺吸式口器昆虫刺穿植物、 消化食物、 解毒植物的次生物质。细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）、 谷胱甘肽S 转移酶（glutathione S transferase, GST）和羧酸酯酶（carboxylesterase, CarE）是昆虫主要的解毒酶系。为了分析解毒酶基因在灰飞虱唾液腺中的表达谱, 本研究对灰飞虱成虫唾液腺进行转录组测序、 重头组装和注释, 并与豌豆蚜Acyrthosiphon pisum和西方蜜蜂Apis mellifera的同源蛋白进行系统发育分析。发现有9个谷胱甘肽S 转移酶（glutathione S transferase, GST）基因、 22个羧酸酯酶（carboxylesterase, CarE）基因和39个细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）基因在灰飞虱唾液腺中表达。通过对同源蛋白进行系统发育分析, 发现灰飞虱唾液腺大部分的CarE是参与消化/解毒和激素/信息素的加工, 而参与神经/发育的CarE很少; 灰飞虱唾液腺表达的P450基因远远少于豌豆蚜和西方蜜蜂基因组的P450基因数, 且只有CYP6和CYP4家族的成员; GST家族在3种昆虫的保守性最高。研究结果为灰飞虱对寄主植物和杀虫剂的适应性研究奠定了基础。     

利用杆状病毒Bac-to-Bac系统在Sf9细胞中表达真菌细胞色素P450nor

利用BactoBac杆状病毒载体表达系统将真菌细胞色素P450nor基因克隆至转移载体pFastBac1中, 得到重组质粒pFastBacP450nor, 再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中发生转座作用, 得到含P450nor基因的重组穿梭载体rBacmid pAcP450nor。分离提取重组Bacmid DNA, 并转染培养的昆虫细胞Sf9, 得到重组病毒rAcp450nor。经酶切和PCR 鉴定, 细胞色素P450nor基因正确地插入到病毒基因组的多角体蛋白基因启动子下, SDSPAGE分析证明：表达蛋白的分子量为43kD左右。Western blotting分析结果表明：有一条特定的杂交带存在, 且分子量相同（约43kD）。进一步证明了含有真菌细胞色素P450nor基因的重组表达载体和重组病毒构建成功,并在昆虫细胞Sf9中实现了高效表达, 经MTT法测定表达的细胞色素P450nor具有还原NO的生物学活性。     

RNA干扰沉默KAT基因对蜜蜂合成10-HDA的影响

合成KAT基因的双链RNA,在蜜蜂体内进行RNA干扰,利用半定量RT-PCR技术和高效液相色谱法,分别检测RNA干扰后KAT基因的表达量和蜜蜂10-HDA的合成量,初步探讨沉默KAT基因与蜜蜂合成10-HDA的关系。结果显示,在蜜蜂体内注射2μl的dsKAT其成活率最高,且注射5天后,与空白组相比,KAT基因的表达量最低,蜜蜂头部10-HDA的合成量降低效果显著(P<0.05),蜜蜂上颚腺表面腺体相对干瘪、疏松。成功实现了蜜蜂体内KAT基因的沉默,且初步推测出KAT基因参与10-HDA的合成途径,阐明了KAT基因与蜜蜂合成10-HDA的关系。这为利用RNA干扰技术分析其他蜜蜂基因功能提供了借鉴,为进一步完善10-HDA的生物合成途径奠定了基础,为通过提高基因表达量来提高10-HDA的产量提供了依据。     

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions.

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met¹), Ad(Met⁻¹), Ad(Met⁻¹²), and Ad(Met⁶). The shoulder numbers represent this site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met⁻¹), were purified from the cell lysate and the ratios of A₄₁₄-to-A₂₇₆ of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450_SCC reduction catalyzed by Ad(Met⁰) was about 60% or that by Ad(Met¹), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450_SCC would need to be considered.     

南海深海新颖低温脂肪酶的克隆、表达及酶学性质鉴定

从南海深海放线菌Pseudonocardia antitumoralis SCSIO 01299中克隆一个脂肪酶基因 lipaseB5。lipaseB5 基因片段大小为972bp,编码的蛋白质具有323个氨基酸残基并与Pseudonocardia sp. HH130629-09中假定脂肪酶有98%的同源性。以pET28a(+)为载体,将重组质粒转化到E. coli BL21(DE3)中,实现lipaseB5高效表达,并利用Ni-NTA 亲和层析纯化lipaseB5。LipaseB5最适反应底物为对硝基苯酚癸酸酯(p-NPD),最适温度为30℃,最适反应pH为7.5。LipaseB5催化p-NPD水解反应的活性达到140.14U/mg,V_max和K_m分别为109.8μmol/min、0.976mmol/L,催化橄榄油的活力为32.019 7U/mg。LipaseB5在pH7.5~8.5保持良好的pH稳定性;在10~20℃低温下保持较高的酶活力,在10~40℃内具有温度稳定性。LipaseB5对大部分金属离子有很好的耐受性,低浓度的Li⁺、Mg²⁺对该酶活性有促进作用。LipaseB5对高浓度的NaCl有很好的耐受性,在100mmol/L的NaCl存在下,酶活性大约保持在103%。LipaseB5对多种短链醇类有机溶剂和表面活性剂有很好的耐受性,TritonX-100、Tween-80和Tween-20对lipaseB5酶有激活作用。     

新颖微生物低温酯酶EstP8的酶学性质研究与在手性催化中的应用

从假单胞菌Pseudonocardia antitumoralis HUP007基因组中克隆了一个1 041bp的酯酶基因EstP8,编码的蛋白具有377个氨基酸残基。在E.coli BL21（DE3）中实现酯酶EstP8的高效异源表达和纯化。EstP8为脂肪酶家族Ⅳ中的一员,具有HGGG保守序列。EstP8最适底物为对硝基苯酚乙酸酯（p-NPO）,最适温度和pH分别为50℃和8.0。EstP8催化p-NPO水解反应的活性、V_max和K_m分别达到105.19U/mg、89.4μM/min、1.144mM。EstP8在pH7.0~8.0范围内具有良好的pH稳定性;在4℃时,酯酶相对活力为41.78%,在10~40℃内具有很好温度稳定性。EstP8对大部分金属离子有很好的耐受性,低浓度的Cu²⁺、Mn²⁺、Zn²⁺对该酶的活性有激活作用。辛烷、庚烷、甲苯、丙酮、DMF等有机溶剂对EstP8的活性同样具有激活作用。酯酶EstP8还可以通过水解拆分高效地制备手性（R）-1-苯基乙醇;添加有机溶剂可以很好地促进该酯酶的光学选择性和产率,在共溶剂甲苯的存在下,所制备的（R）-1-苯基乙醇的e.e.和产率可达91%和18%;在共溶剂DMSO的存在下,所制备的（S）-乙酸苏合香酯的e.e.和产率可达98%和60%。酯酶EstP8在手性生物催化等诸多工业领域有很好的应用潜力。     

Purification and characterization of a soluble form of rat liver NADPH-cytochrome P-450 reductase highly expressed in Escherichia coli

A recombinant cDNA of rat liver NADPH-cytochrome P-450 reductase (CPR), which lacks the N-terminal hydrophobic region, was amplified by PCR and cloned. The N-truncated cDNA named tCPR was ligated into a pBAce vector and expressed. The tCPR protein expressed in Escherichia coli was recovered into the soluble fraction of the cell lysate and purified to homogeneity by three sequential purification procedures; (I) anion-exchange chromatography on a DEAE-cellulose (DE-52) column, (II) affinity chromatography on 2('),5(')-ADP Sepharose 4B, and (III) chromatography on a hydroxyapatite column. The average yield was 47mg per liter of culture medium. The absorption spectrum of the purified tCPR protein was identical to that of a native full-length CPR purified from rat liver, indicating that tCPR also possesses one molecule each of FAD and FMN. The tCPR protein was able to reduce cytochrome c and was also able to assist heme degradation by a soluble form of rat heme oxygenase-1. However, it failed to support the O-deethylation of 7-ethoxycoumarin by cytochrome P-450 1A1, indicating that the presence of the N-terminal hydrophobic domain is necessary for CPR to interact with cytochrome P-450. Previously, to prepare a soluble form of CPR, full-length CPR was treated with proteinases that selectively removed the N-terminal domain. With the expression system established in this study, however, the soluble and biologically active tCPR protein can be readily prepared in large amounts. This expression system will be useful for mechanistic as well as structural studies of CPR.     

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.     

盐度和CO2倍增环境下碱蓬幼苗呼吸酶活性的变化

研究了生长在正常大气CO₂和CO₂倍增环境中的盐生植物碱蓬(Suaedasalsa)幼苗呼吸酶活性对KCl和NaCl的反应。结果表明,在CO₂倍增(700μl·L^-1)和正常大气CO₂(350μl·L^-1)下,300mmol·L^-1KCl和NaCl均能抑制琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)活性,而异柠檬酸脱氢酶(IDH)活性为NaCl抑制、KCl促进; NaCl和KCl明显抑制细胞色素氧化酶(CO)和光呼吸中乙醇酸氧化酶(GO)、羟基丙酮酸还原酶(HPR)活性; 并指出在KCl胁迫下,CO₂使三羧酸循环(TCAC)的运行变慢,NaCl胁迫下使其加快,TCAC运行限速步骤与MDH无关,CO为盐对呼吸代谢影响的重要位点。另外,K⁺、Na⁺对蛋白表达的影响有差异,CO₂可使盐胁迫下的碱蓬幼苗蛋白表达降低。     

Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450

The steroid 11ß-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4–5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011ß system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011 from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11ß-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M_r 60 kD) and a NADPH-cytochrome P450 reductase (M_r 79 kD).     

含硒抗病毒多肽的半胱氨酸缺陷型表达及鉴定

目的：在大肠杆菌中进行含硒抗病毒多肽的可溶性半胱氨酸缺陷型融合蛋白的表达,并对其纯化蛋白进行生物学活性的初步鉴定。方法：将含硒抗病毒多肽Se-GBVA10基因克隆到GST融合表达载体pGEX-2T中,转化至半胱氨酸缺陷型大肠杆菌BL21 cysE51 后进行半胱氨酸缺陷型表达,通过谷胱甘肽Sepharose 4B 亲和柱纯化目的蛋白质,并对其含硒量、抗病毒活力等性质进行初步鉴定。结果：利用半胱氨酸缺陷型表达法成功地可溶性表达了sjGST-Se-GBVA10和sjGST-GBVA10融合蛋白,并经凝血酶切后获得了纯化的Se-GBVA10和GBVA10抗病毒多肽。测定Se-GBVA10的含硒量为0.974 mol/mol peptide,GPX活力为47.52 U/μmol,其EC₅₀为21.73μmol/L,CC₅₀为849.41μmol/L。结论：Se-GBVA10具有与GBVA10相同的抗病毒活性,并具有一定的抗氧化活力。