深海沉积物微生物元基因组文库来源的新的酯酶基因的克隆、表达及酶学性质

【目的】从深海沉积物微生物元基因组文库中克隆新的酯酶基因,并进行酶学性质研究。【方法】利用含有三丁酸甘油酯的酯酶选择性筛选培养基,从深海沉积物微生物元基因组文库中筛选得到酯酶阳性Fosmid克隆。对筛选得到的fosmid FL10进行部分酶切构建亚克隆文库,筛选得到酯酶阳性亚克隆pFLS10。PCR扩增目的片段后与pET28a连接构建酯酶基因原核表达质粒,转化大肠杆菌(Escherichia coli)BL21。纯化表达产物并对其进行活性测定及酶学性质研究。【结果】序列分析显示该pFLS10亚克隆质粒含有一段924bp的ORF(Open Reading Frame),与一海洋元基因组文库中筛选出的酯酶ADA70030序列一致性为71%。该酶为一新的低温酯酶,对C4底物(对硝基苯丁酸酯)水解能力最强。该酶最适作用温度为20℃,最适作用pH为7.5,20℃时较为稳定,pH8-10的范围内有良好的pH稳定性,K+、Mg2+对该酶具有一定的激活作用,Mn2+等对其具有不同程度的抑制作用。【结论】应用元基因组技术筛选到了新的酯酶基因fls10并进行了克隆表达,该酶在低温及碱性条件下较为稳定且活力较高,对于工业化生产具有一定的应用潜力。关键词:深海沉积物;元基因组文库;低温酯酶;酶学特征     

烟曲霉脯氨酰内肽酶在巴斯德毕赤酵母中的分泌表达与重组酶性质

【目的】研究烟曲霉脯氨酰内肽酶cDNA基因的异源表达及重组酶性质。【方法】以烟曲霉CICIM F0044总RNA为模板,反转录合成cDNA;再以cDNA为模板,通过PCR扩增去除自身信号肽的脯氨酰内肽酶基因,构建表达载体pPIC9K-PEP;电转化酵母宿主菌Pichia pastoris GS115,获得重组菌PEP-09;纯化并分析重组酶性质。【结果】重组菌摇瓶发酵酶活力最高可达647.3 U/L。表达产物纯化后的分子量为63 kD左右。重组酶最适反应温度为65°C,有较好的温度稳定性,在55°C保温8 h能保留90%以上的酶活力。该酶最适pH为5.5,在pH 3.0 9.0范围内有很好稳定性,在pH 6.0 8.0的缓冲液中37°C保温10 d酶活没有明显变化。【结论】烟曲霉脯氨酰内肽酶cDNA基因在巴斯德毕赤酵母中实现了分泌表达,重组酶活性稳定,有一定的应用潜力。     

来源于海洋细菌Altererythrobacter luteolus SW109T的新型酯酶E29的克隆表达及其酶学性质

【目的】克隆表达一个来源于海洋细菌的酯酶E29,并研究其酶学性质。【方法】从细菌Altererythrobacter luteolus SW109T中筛选并扩增出一个酯酶基因,将其克隆至p SMT3载体上,并将重组质粒转化至大肠杆菌BL21(DE3)中进行异源表达,分析表达产物的酶学性质。【结果】氨基酸序列分析结果表明,酯酶E29属于脂类水解酶第二家族(Family II)。酶学性质分析结果显示,酯酶E29的最适反应底物为对硝基苯酚丁酸酯,最适反应温度为45°C,最适反应pH为8.5;10 mmol/L的Co~(2+)和Mn~(2+)及15%的异丙醇和乙腈能强烈抑制酯酶E29的活性,1%的SDS能使酶失活,甘油的存在能促进酶活性。【结论】酯酶E29是一个海洋来源的新型酯酶,其具有较高的酶活力值、较宽的底物谱以及对部分有机溶剂和金属离子较好的耐受性,在工业方面具有潜在的应用价值。     

黑曲霉阿魏酸酯酶基因密码子优化及在毕赤酵母中的高效表达

【目的】对黑曲霉(Aspergillus niger)阿魏酸酯酶基因进行克隆和密码子优化,使其在毕赤酵母(Pichia pastoris X-33)中高效表达。【方法】以黑曲霉基因组为模板,经重叠延伸PCR扩增得到阿魏酸酯酶基因(Anfae A),并对Anfae A基因进行毕赤酵母密码子偏好性"随机优化"和"一对一优化",全基因合成后分别与表达载体pPICZαA连接,构建表达载体pPICZαA-Anfae A、pPICZαA-op Anfae A I和pPICZαA-op Anfae A II。经Sac I线性化后电转化至P.pastoris X-33中,筛选阳性转化子。摇瓶发酵4.5 d后,测定并比较重组阿魏酸酯酶(re Anfae A)酶活。【结果】密码子优化前阿魏酸酯酶酶活为6.8±0.1 U/m L,基因"一对一优化"和"随机优化"后的重组酶酶活分别为5.2±0.1 U/m L和39.9±0.1 U/m L,"随机优化"后酶活比优化前提高了近6倍,而"一对一优化"后酶活仅为优化前酶活的76.5%。重组阿魏酸酯酶的最适p H为5.5,且在pH 4.5-7.0稳定性较好;最适反应温度50°C,在45-50°C较稳定。【结论】阿魏酸酯酶基因经密码子"随机优化"后进行重组表达,酶活显著提高,对研究阿魏酸酯酶在毕赤酵母及其它宿主中的高效表达具有一定的借鉴意义,也为大规模工业化应用奠定了基础。     

来源于海洋细菌Altererythrobacter epoxidivorans CGMCC 1.7731T的新型碱性酯酶E22的酶学性质

【目的】克隆表达海洋细菌Altererythrobacter epoxidivorans CGMCC 1.7731~T中的酯酶基因e22,并研究其酶学性质。【方法】分析菌株的全基因组序列,筛选获得一个酯酶基因e22,将其克隆至p ET-28a载体上,并转化至大肠杆菌BL21(DE3)细胞中表达,研究纯化后表达产物的酶学性质。【结果】通过氨基酸序列分析,确定酯酶E22属于脂类水解酶第二家族(Family Ⅱ)。酶学性质研究结果表明,该酶最适反应底物为对硝基苯酚丁酸酯(C4);最适反应pH 10.5,为碱性酯酶;最适反应温度为55°C,并在60°C孵育2 h后仍保留超过50%的活性,显示了良好的热稳定性;1%甲醇、1%Triton X-100或0.1%SDS对酯酶E22的活性无显著影响,而10 mmol/L的Ba~(2+)或Ca~(2+)则对其活性有抑制作用。【结论】E22是一个新型海洋来源酯酶,具有耐碱性、热稳定性、有机溶剂和去垢剂耐受性等优良特性,在工业生产中具有较好的应用潜力。     

蜡样芽胞杆菌磷脂酶C在乳酸克鲁维酵母中重组表达、纯化及酶学性质分析

【目的】构建蜡样芽胞杆菌(Bacillus cereus)磷脂酶C(Phospholipase C,PLC)的重组乳酸克鲁维酵母(Kluyveromyces lactis)菌株、纯化重组蛋白并对其进行酶学性质分析。【方法】以B.cereus基因组DNA为模板,PCR扩增得到磷脂酶C基因(bcplc),构建重组乳酸克鲁维酵母表达质粒并转化到乳酸克鲁维酵母中,实现bcplc基因的表达。利用镍柱亲和层析纯化和脱盐柱得到电泳纯的重组磷脂酶C(rbcPLC)。【结果】成功构建产磷脂酶C的重组乳酸克鲁维酵母并纯化了重组磷脂酶C,纯化后rbcPLC经SDS-PAGE分析在40 kDa附近出现显性条带。NPPC法测得rbcPLC酶活为19251 U/mg,最适反应温度为80°C,最适pH为9.0。在低于40°C时,pH 7.0-8.0时,rbcPLC重组酶较稳定。Cu~(2+)和Co~(2+)对其有明显的抑制作用;Zn~(2+)、Mn~(2+)、Ca~(2+)、Mg~(2+)对其有明显的促进作用。【结论】首次实现了对蜡样芽胞杆菌来源的磷脂酶C在乳酸克鲁维酵母中的重组表达、纯化及其酶学性质分析,为其它食品安全性微生物来源的磷脂酶C的研究提供了借鉴意义。     

特异腐质霉内切葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质

【目的】在毕赤酵母中表达特异腐质霉Humicola insolens的中性内切葡聚糖酶Ⅱ,并对其性质加以研究。【方法】利用RT-PCR的方法,以特异腐质霉(Humicola insolens)NC3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因(egⅡ)的cDNA。将其插入表达载体pPIC9K,重组质粒经线性化后电击转化毕赤酵母(Pichia pastoris)菌株GS115。【结果】SDS-PAGE和酶活的检测结果均表明:egⅡ基因在毕赤酵母中成功表达。重组酶的部分酶学性质研究表明,该酶的最适反应温度为70°C,且在65°C以下具有较好的热稳定性。最适反应pH为6.5,在pH 6.0?7.0之间有较好的稳定性。【结论】用重组毕赤酵母可高效表达外源中性内切葡聚糖酶,为其今后在工业应用奠定了基础。     

芽孢杆菌N1碱性蛋白酶的克隆表达及酶学性质

【背景】蛋白酶广泛应用于制革行业中,酶法脱毛对环境污染较小,但蛋白酶对化学试剂的不稳定性及胶原降解活性限制了其工业应用。【目的】克隆芽孢杆菌(Bacillussp.)N1基因组的碱性蛋白酶基因,实现其在大肠杆菌中的异源表达,并对重组酶酶学性质及脱毛作用进行研究。【方法】利用基因组文库法克隆获得蛋白酶基因aprG,构建重组大肠杆菌(Escherichiacoli)BL21(DE3)pLysS/pET-28a-aprG。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达该重组酶,以福林酚显色法对其酶学性质进行研究,并将AprG作用于羊皮、兔皮和羽毛。【结果】克隆得到蛋白酶基因aprG,并实现其在大肠杆菌中的表达。重组酶AprG最适反应温度为50°C,最适反应pH为10.0。各种金属离子对AprG活性影响较小,且AprG对表面活性剂和氧化剂、还原剂的耐受性较强。底物特异性分析表明,该酶胶原活性较低。AprG对羊皮和兔皮作用显著,且降解羽毛效果明显。【结论】蛋白酶AprG在制革行业中具有良好的应用前景。     

耐碱性木聚糖酶基因在巨大芽孢杆菌中的表达及其酶学性质

摘要：【目的】从耐碱性木聚糖酶高产短小芽孢杆菌中克隆得到带有自身启动子的木聚糖酶基因,将其在巨大芽孢杆菌中进行表达,并对表达产物进行性质分析。【方法】将克隆得到的木聚糖酶基因xynA以及带有自身启动子序列的结构基因, 构建在芽孢杆菌表达载体pWH1520和改造后的载体pWG03中,得到重组质粒pWTEJX和pWGXYN,分别转化到巨大芽孢杆菌BM70中,获得重组巨大芽孢杆菌BMJXH9和BMGpp12;经过诱导产酶培养,均得到分泌表达。【结论】重组巨大芽孢杆菌BMGpp12比BMJXH9产酶活力提高了三倍     

细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达

【目的】实现地衣芽孢杆菌麦芽糖淀粉酶在枯草芽孢杆菌中的高效异源表达,并研究该重组酶的酶学性质。【方法】克隆巨大芽孢杆菌木糖异构酶基因的启动子区域及其调控蛋白,构建一个大肠杆菌/芽孢杆菌穿梭型诱导表达质粒,使用该诱导型启动子介导麦芽糖淀粉酶编码基因,实现其在枯草芽孢杆菌中的功能表达。对重组枯草芽孢杆菌的诱导条件进行优化,提高麦芽糖淀粉酶的产量。【结果】获得了诱导表达麦芽糖淀粉酶基因的重组枯草芽孢杆菌菌株。最适诱导温度为45°C,最适诱导剂添加浓度为1%,最适添加诱导剂时间为接种培养9 h后。重组酶蛋白分子量大小为67 k D,对该酶的酶学性质研究发现,以可溶性淀粉为底物,反应生成麦芽糖和葡萄糖,其中麦芽糖含量为60.42%。重组酶最适作用温度为45°C,最适作用p H为6.5,Ca2+、Co2+、EDTA对该重组麦芽糖淀粉酶具有激活作用。【结论】通过木糖诱导表达系统可以实现麦芽糖淀粉酶在枯草芽孢杆菌中的高效诱导型表达,酶活最高可达296.64 U/m L发酵液,在工业上有着较好的应用前景。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV