灰盖鬼伞过氧化物酶功能表达及部分酶学特性

摘要:【目的】旨在用毕赤酵母高效表达灰盖鬼伞过氧化物酶。【方法】借助DNAworks 3.1软件设计、优化引物,用自己构建的基因合成、定点突变平台合成了毕赤酵母密码子偏好性的灰盖鬼伞过氧化物酶基因,测序后构建在表达载体pPICZαA上,整合于巴斯德毕赤酵母GS115染色体,来自酿酒酵母的α因子作为信号肽序列指导重组蛋白的分泌表达。从82个PCR检测为阳性的酵母转化子中筛选出6株高Zeocin抗性的菌进行表达,选表达酶活性最高的作为实验菌株命名为CIP/GS115。【结果】以ABTS为底物时,CIP/GS115 在甲醇诱导第4天酶活最高达到487.5 U/mL,是目前摇瓶培养诱导表达灰盖鬼伞过氧化物酶活性最高报道。纯化后的酶最适反应温度为25℃,45℃酶反应速度是最适温度时的61.5%,在低于40℃时比较稳定,超过45℃稳定性迅速下降。最适反应pH 为5.0,在pH 4.5－6.5之间比较稳定。以不同的底物研究纯酶底物特异性发现最适底物的顺序是:ABTS ＞ 愈创木酚＞ 2,6-二甲氧苯酚＞ 2,4-二氯苯酚＞ 苯酚。【结论】灰盖鬼伞过氧化物酶在毕赤酵母中的高效分泌表达和高的特殊活性为该酶在废水处理、染料脱色等方面的工业化应用奠定了一定基础。     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

理性设计提高酯合成催化反应脂肪酶的热稳定性

【背景】南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)具有优异的酯合成活性,是在非水相催化中应用极为广泛的工业用酶。【目的】在保留CALB优秀催化性能的基础上,提高CALB的热稳定性。【方法】采用预测软件PoPMuSiC和FoldX计算CALB潜在热稳定性突变位点,并根据氨基酸残基的空间位置进一步筛选。利用重叠延伸PCR技术在基因calb中引入10个单点突变,于毕赤酵母GS115中表达。【结果】点突变A146G、A151P、L278M均能有效提高CALB的热稳定性。在单点突变的基础上,组合突变体A146G-L278M和A146G-L278M-A151P的热稳定性得到进一步提高。与野生型相比,突变体A146G-L278M和A146G-L278M-A151P的最适反应温度均提高了5°C,T_m值分别提高了3.3°C和4.2°C。此外,合成己酸乙酯的酶促反应动力学分析表明,相比于野生型,突变体A146G-L278M和A146G-L278M-A151P对己酸和乙醇均具有更高的亲和力,且对己酸的催化效率k_(catA)/K_(m A)是野生型的4.1倍。通过分子动力学模拟,从分子水平阐明了突变体A146G-L278M和A146G-L278M-A151P热稳定性提高的机制。【结论】本研究采用的理性设计策略对提高CALB的热稳定性是行之有效的,该策略可作为其他工业用酶提高热稳定性的参考。     

定向引入N-糖基化位点改进黑曲霉β-1,4-内切木聚糖酶热稳定性

【背景】木聚糖是生物圈中仅次于纤维素的第二大多糖,其结构复杂,完全降解需要多种木聚糖酶协同作用。β-1,4-内切木聚糖酶是木聚糖主链水解过程中最关键的酶,已广泛应用于饲料、造纸、能源、食品和医药等行业。但在实际应用中,由于真菌木聚糖酶的热稳定性较差,限制了其在工业中的应用。【目的】提高来源于黑曲霉(Aspergillusniger)的β-1,4-内切木聚糖酶(xynB)热稳定性。【方法】采用氨基酸虚拟突变技术对xynB定向引入一个N-糖基化位点,将虚拟突变后筛选获得的候选突变体和野生型在毕赤酵母SMD1168中表达,并对纯化后的野生型和突变体酶进行酶学性质和稳定性分析。【结果】经虚拟突变和筛选获得5个候选突变体,在毕赤酵母SMD1168中成功表达了4个突变体,其中3个突变体发生了糖基化。突变体和野生型酶均表现出宽范围的酸碱耐受性,且突变体xynB~(A92N/D94T)在pH4.0–11.0条件下的稳定性明显优于野生型;糖基化突变体xynB~(A92N/D94T)、xynB~(G66N/A68T)和xynB~(G66F/D67N/G69T)在温度为60–80°C时热稳定性明显高于野生型,xynB~(G66N/A68T)在80°C保温30 min后的残留酶活比野生型提高了约30%。【结论】本研究方法可为其他来源木聚糖酶和其他工业酶的热稳定分子改造提供参考。     

宏基因组来源耐Mn2+、热稳定细菌漆酶的分子克隆及酶学特性

【背景】漆酶和锰过氧化物酶(Manganese peroxidase,Mnp)是木质素降解的主要酶,二者有协同效应。Mnp活性依赖于Mn~(2+),而Mn~(2+)是大多数漆酶的抑制剂。【目的】获得耐Mn~(2+)的细菌漆酶用于木质素降解。【方法】构建许昌市某污水河污泥宏基因组文库,通过活性筛选获得细菌漆酶基因lac1542。使用大肠杆菌异源表达Lac1542,研究纯化后的重组蛋白酶学性质并进一步检测了含Lac1542复合酶系降解木质素能力。【结果】测序结果显示lac1542编码一个含513个氨基酸的蛋白。以ABTS为底物Lac1542最适反应pH为4.0,在pH 3.0-6.5范围内酶活性稳定。最适反应温度是75°C,在70°C以下酶活性稳定;100 mmol/L的Mn~(2+)仍能提高酶的活性。动力学参数研究发现,该酶的最适底物顺序为:ABTS丁香醛联氮儿茶酚2,6-DMP愈创木酚。Lac1542/Mnp复合酶系对木质素降解率为47.8%,比单独使用Mnp木质素降解率(22.4%)提高25.4%。Lac1542/Mnp/灰盖鬼伞过氧化物酶(Coprinus cinereus Peroxidase,CIP)复合酶系木质素降解高达71.5%,比Mnp/CIP酶系木质素降解率(48.9%)提高22.6%,加入Lac1542后的复合酶系能明显提高木质素的降解率。【结论】Lac1542的可溶性表达、耐受高浓度Mn~(2+)、热稳定性使得Lac1542可以替代一些经典的真菌漆酶应用于制浆、造纸、纤维素乙醇生产、染料脱色等工业。     

茅草菇菌丝球对铬黑T的脱色降解性能及毒性评价

【目的】利用真菌茅草菇菌丝球对染料铬黑T (EBT)进行脱色和降解,探究在不同环境条件下对染料脱色性能的影响及作用机制。【方法】采用单因素分析探究真菌的最佳脱色能力,分光光度法测定真菌酶活,小麦种子萌发、大肠杆菌接触抑制试验及秀丽隐杆线虫毒性试验测定脱色前后废水的毒性。【结果】茅草菇菌丝球受摇床温度和转速影响较小,在pH 5、28℃、120 r/min下,400 mg/L的EBT溶液脱色率为97.14%。研究表明,茅草菇菌丝球在脱色过程中主要分泌3种木质素酶,即木质素过氧化物酶、锰过氧化物酶和漆酶,其最大酶活分别为(134.15±9.93)、(64.1±2.98)和(12.43±0.34) U/L。推断了染料降解的潜在路径,证实EBT的去除是通过生物吸附与降解的协同作用实现的。最后对脱色后的染料废水进行了多级毒性评价,包括植物毒性、微生物毒性和动物毒性,结果表明,脱色后的染料废水毒性显著降低。【结论】该研究对探讨生物法处理工业染料废水具有重要参考价值。     

北京棒杆菌天冬氨酸激酶突变体R169H的构建及酶学性质表征

【目的】提高北京棒杆菌(Corynebacterium pekinense)中天冬氨酸激酶(aspartokinase,AK)活力。【方法】利用定点突变技术对AK基因进行突变,并将突变体转入大肠杆菌(Escherichia coli)BL21中异源表达。重组菌经超声破碎后、利用镍柱对AK进行纯化,并经SDS-PAGE和Western blot验证。通过检测酶活力比较突变体和野生型动力学变化并研究突变体和野生型的部分酶学性质。【结果】成功构建突变体R169H。经验证知,AK分子量为48kDa。突变体R169H的Vmax为226.3 U/mg·s-1,较野生型提高2.3倍。最适反应温度为26℃,与野生型经验值相同;最适反应pH为9.0,较野生型经验值8.0有所提高;在最适温度和pH值下的半衰期为5.5 h,比野生型的4h稳定性要好;代谢产物赖氨酸、苏氨酸和蛋氨酸在低浓度时对AK均具有激活作用。【结论】突变体中R169与E92间氢键消失,能够影响亚基间聚合度,降低酶对底物的亲和力,减弱代谢产物对AK的反馈抑制作用,从而使R169H中AK的Vmax提高2.3倍。     

基于结构基础的嗜热细菌贝斯其热解纤维素菌木聚糖酶CbXyn10C的热稳定性分子改良

【背景】作为降解木聚糖的核心酶种,木聚糖酶可以有效促进木质纤维素的消化水解,在动物养殖领域应用广泛。来源于嗜热细菌贝斯其热解纤维素菌(Caldicellulosiruptorbescii)的GH10家族木聚糖酶Cb Xyn10C最适温度为85℃,在80℃条件下具有良好的热稳定性,具有饲料工业应用潜力。【目的】为满足饲料制粒尤其是水产饲料加工过程的工艺要求,进一步提高木聚糖酶Cb Xyn10C的热稳定性并阐明其耐热机理。【方法】以Cb Xyn10C晶体结构为基础,采用刚性氨基酸引入、疏水作用网络重排2种策略对其热稳定性进行理性设计,获得在100℃条件下比活提高的单点突变体后,通过有益突变位点叠加策略进一步提升酶的热稳定性,最后采用分子动力学模拟技术分析其热稳定性提高的分子机制。【结果】共获得了4个稳定性提高的单点突变体A45P、T69P、F309V和A325P,其中突变体A45P效果最优。随着在A45P基础上另外3个突变位点的叠加,酶的热稳定性在不损失酶活的前提下得到了逐步提升。获得的四点突变体A45P/F309V/A325P/T69P的耐热性最好,其最适反应温度和熔解温度Tm值较野生型...     

北京棒杆菌AS1.299高丝氨酸脱氢酶突变体L200F/D215K的异源表达及酶学性质

【目的】对北京棒杆菌Corynebacterium pekinense高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)进行空间结构改造从而获得优良性能新酶。【方法】利用定点突变技术构建HSD双突变体L200F/D215A、L200F/D215E、L200F/D215G和L200F/D215K,并将其转入大肠杆菌E.coli BL21中进行高效表达,选取催化效率最高的双突变体L200F/D215K与双突变前的L200F进行动力学和酶学性质比较。【结果】HSD双突变体L200F/D215K的Vmax为36.92 U/mg,较L200F提高1.24倍;最适反应温度为37℃,较L200F提高2℃;最适反应pH为7.5,与L200F经验值相同;最适温度下的半衰期为4.16 h,较L200F提高1.12倍;L200F/D215K和L200F对有机溶剂和金属离子均表现出较好的抗性。【结论】HSD通过空间结构改造活力得到提高,并且其酶学性质得到优化。本研究有助于认识HSD突变体的酶学性质,为其新酶的研发利用提供有力依据。     

腾冲嗜热厌氧菌丙氨酸消旋酶底物通道氨基酸位点的功能

【目的】通过定点突变探究腾冲嗜热厌氧菌MB4中生物合成型丙氨酸消旋酶Tt Alr底物通道内氨基酸位点A172和S173的功能。【方法】利用定点突变PCR技术构建突变体,通过亲和层析法纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测各突变蛋白的活性及其稳定性。【结果】通过定点突变PCR成功得到8个突变体,酶学特性分析发现,A172位点突变为丝氨酸(S)后酶蛋白的相对活性有所提升,但含有该位点突变的酶蛋白稳定性均大幅下降;S173位点突变为天门冬氨酸(D)后导致突变体蛋白的最适反应温度提升了15°C,半衰期大幅延长,但相对活性明显下降。【结论】丙氨酸消旋酶Tt Alr底物通道内A172和S173位点均是影响酶蛋白催化活性和稳定性的关键位点。     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.     

（鱼句）亚科花（鱼骨）型鱼类骨骼系统的比较

对我国花型Hemibarbuspattern鱼类作了骨骼系统比较,结果表明,此类型鱼类脑颅较长,副蝶骨平直或稍弯曲,眶蝶骨腹纵嵴发达(铜鱼Coreius septentrionalis例外),下颞窝和咽突中等大,基枕骨后突发达;脑颅中的上筛骨的后突、侧筛骨的外筛突,蝶耳骨的外突、上耳骨的后突、围眶骨和后颞窝等均有明显的差异;咽颅中的舌颌骨、尾舌骨、鳃盖骨和下咽齿的列数等又有显著的区别;附肢骨骼中的腰带骨、脊椎骨中的复合神经骨和第4椎骨腹侧的悬器等也有不同之处。据此,这些差异和区别可作为属间或种间的分类依据。     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.