多聚磷酸盐激酶双底物通道腔的理性设计及其在无细胞催化生产谷胱甘肽中的应用

三磷酸腺苷(adenosine triphosphate,ATP)是一种重要的辅助因子,参与许多需能的生物催化反应。多聚磷酸盐激酶(polyphosphate kinases,PPK)由于其底物聚磷酸盐廉价易得,可以为消耗ATP的反应提供能量。本研究选择哈氏噬纤维菌(Cytophaga hutchinsonii)来源的ChPPK,进行了底物谱和耐受性分析,通过分子对接和定点突变,理性改造多聚磷酸盐激酶的双底物通道腔来提高PPK酶的催化活性。与野生型相比,筛选得到突变体ChPPK_K81H-K103V的相对酶活提高了326.7%,同时,双突变扩大了ChPPK的底物利用范围与耐受性,提高了该酶的耐热性与耐碱性。基于该ATP再生系统,本研究偶联谷胱甘肽双功能酶GshAB和ChPPK_K81H-K103V,破细胞后采用无细胞催化生产谷胱甘肽,加入5 mmol/L ATP后,该体系6 h可以生产(25.4±1.9) mmol/L的谷胱甘肽,比突变前的催化体系提高了41.9%。优化无细胞催化体系的缓冲液、裂解液菌体量、补料时间后,无细胞体系可产生(45.2±1.8) mmol/L谷胱甘肽,底物l-半胱氨酸的转化率达到90.4%。提高ChPPK生产ATP的能力,可有效增强底物的转化率,降低催化成本,实现了无细胞催化生产谷胱甘肽的高产量、高转化率与高经济价值的统一。本研究提供了一种绿色高效的ATP再生系统,可为消耗ATP的生物催化反应平台提供可持续动力。     

多聚磷酸激酶及其在ATP再生体系构建中的进展

构建高效的腺嘌呤核苷三磷酸(adenosinetriphosphate,ATP)再生体系可显著提高生物催化磷酸基团转移反应的效率。多聚磷酸激酶(poly phosphate kinase, PPK)能利用来源广、廉价且稳定的多聚磷酸(polyphosphate, Poly P)盐作为磷酸基供体，能够实现单磷酸腺苷(adenosine monophosphate,AMP)、二磷酸腺苷(adenosinediphosphate,ADP)、ATP、PolyP之间磷酸基的高效定向转移，已成为构建ATP再生体系的首选。本文介绍了不同类型PPK的结构特征、相关催化机制以及不同来源的PPK在酶活、催化效率、稳定性和底物偏好性的特征差异；归纳和列举了针对野生PPK酶学性质不足进行分子改造的实例，并对PPK在ATP再生体系构建的研究进展进行了总结。     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。     

细菌3-脱氧葡糖醛酮代谢酶的纯化及性质研究

细菌Bacillus sp.2粗酶液通过(NH₄)₂SO₄分级分离、Q Sepharose FF、Sephadex G-100(Ⅰ)、Hydroxyapatite和Sephadex G-100(Ⅱ)柱层析分离,纯化了一种以NADPH为辅酶的3-脱氧葡糖醛酮(3-DG)代谢酶,定性为2-羰基醛还原酶.纯化酶的比活力为63.75 U/mg,在SDS-聚丙烯酰胺凝胶上显示一条蛋白质带.该酶分子质量约为32 ku,酶反应最适pH约为6.2, 在pH 5～8, 温度25～30℃之间酶保持稳定;该酶对3-DG的K_m为2.3 mmol/L.添加适量的EDTA、巯基乙醇或二硫苏糖醇能明显提高酶的活性;而碘乙酸、N-乙基顺丁烯二酰亚胺抑制酶的活性.     

多聚磷酸相关蛋白结构及生物学功能

多聚磷酸（polyphosphate,polyP）是由几个到数百个磷酸基通过高能磷酸酐键连接而成的链状多聚体,存在于所有细胞生物中.多聚磷酸相关蛋白包括多聚磷酸相关酶和多聚磷酸结合蛋白.多聚磷酸相关酶如多聚磷酸激酶（polyphosphate kinase,PPK）催化polyPn生成polyPn+1的可逆反应;外切聚磷酸酶（exopolyphosphatase,PPX）、内切聚磷酸酶（endopolyphosphatase,PPN）能将polyP水解成磷酸残基;多聚磷酸依赖的激酶将polyP的磷转移到生物小分子上,如葡萄糖和烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD）,使其分别磷酸化为6 磷酸葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate,NADP）.多聚磷酸结合蛋白可与多聚磷酸结合,发挥各种生物学功能.本文将简要介绍多聚磷酸相关蛋白的结构与主要生物学功能,以阐述多聚磷酸参与的细胞内生化过程.     

过氧化氢酶-无机杂化纳米花的制备及催化特性

过氧化氢酶(catalase,CAT)是一种在食品、医疗、纺织等领域广泛应用的工业酶,具有催化效率高、专一性强、绿色环保等突出特点。工业中游离过氧化氢酶无法回收再利用,导致以其为核心的工业生物转化过程成本较高。开发一种简单、温和、低成本并且体现绿色化学理念的方法对过氧化氢酶进行固定化有望提高其利用率并且强化酶学性能,具有迫切的现实需求。本研究将源自枯草芽孢杆菌(Bacillus subtilis)168的过氧化氢酶KatA在大肠杆菌中进行重组表达,之后将分离纯化得到的纯酶以酶-无机杂化纳米花形式制备成固定化酶并进行酶学性质研究。结果显示,利用乙醇沉淀、DEAE阴离子交换层析、疏水层析3步纯化,最终获得电泳纯的重组KatA,之后通过优化制备条件获得了一种新型KatA/Ca₃(PO₄)₂杂化纳米花固定化酶。酶学性质研究结果显示,游离酶KatA的最适反应温度为35℃,KatA/Ca₃(PO₄)₂杂化纳米花的最适反应温度为30−35℃,二者最适反应pH值均为11.0。游离酶KatA和KatA/Ca₃(PO₄)₂杂化纳米花在pH4.0−11.0和25−50℃条件下均表现出较好的稳定性。KatA/Ca₃(PO₄)₂杂化纳米花显示出比游离酶KatA更好的储存稳定性,在4℃储存14d后仍保留82%的酶活力,而游离酶仅具有50%的酶活力。此外,纳米花在进行5次催化反应后仍具有55%的酶活力,表明其具有较好的操作稳定性。动力学研究结果显示,游离酶KatA对底物过氧化氢的K_m为(8.80±0.42)mmol/L,k_cat/K_m为(13151.53±299.19)L/(mmol·s);而KatA/Ca₃(PO₄)₂杂化纳米花的K_m为(32.75±2.96)mmol/L,k_cat/K_m为(4550.67±107.51)L/(mmol·s)。与游离酶KatA相比,KatA/Ca₃(PO₄)₂杂化纳米花对底物过氧化氢的亲和力下降,同时其催化效率也有所降低。综上所述,本研究以Ca²⁺作为自组装诱导剂,成功将KatA以酶-无机杂化纳米花形式制备成固定化酶,不仅对部分酶学性能实现了强化,而且为固定化过氧化氢酶的绿色制备和规模化应用奠定了基础。     

pH-比色法测定CaATPase活力

基于ATP被酶水解后H⁺生成的速度作为酶活力的指标,在560um波长和pH7.4,以酚红作指示剂,建立了pH-比色测定肌浆网CaATPase(Ca²⁺,Mg²⁺-ATPase)活力的分光光度法。方法简易,可观察反应进行的过程。SR CaATPase对底物ATP的K_m=97.4μmol/L;在22—42℃间求得活化能(E_Ca)=20.40千卡。用本法初步观察了汉防己甲素(简称汉甲)对酶的抑制作用。     

固定化尿苷-胞苷激酶和聚磷酸激酶偶联催化制备5′-胞苷酸

尿苷-胞苷激酶作为生物体核苷酸代谢补偿途径中的重要催化剂，可以催化胞苷的磷酸化反应合成5′-胞苷酸 (简称胞苷酸)，但需要NTP作为磷酸供体。为了提高胞苷酸的生产效率，文中首先使用大肠杆菌分别异源表达来源于嗜热栖热菌Thermus thermophiles HB8的尿苷-胞苷激酶和来源于类球红细菌Rhodobacter sphaeroides的聚磷酸激酶，其中尿苷-胞苷激酶用于催化胞苷和ATP形成胞苷酸，聚磷酸激酶则用于ATP的循环再生。然后，使用D403金属螯合树脂吸附Ni2+形成固定化载体，再利用固定化载体特异性吸附重组酶形成固定化酶。最后，单因素优化实验确定固定化酶的催化反应条件，在30 ℃、pH 8.0的条件下，以60 mmol/L胞苷和0.5 mmol/L ATP为底物，可实现5批次的高效连续催化反应，胞苷酸平均摩尔得率达到91.2%。上述制备方法反应成本低，产物得率高，酶利用率高，在工业生产中具有较好的应用潜力。     

牛樟芝非核糖体多肽合成酶基因(ＡcNRPS)的克隆与鉴定

胶霉毒素属于真菌天然次生代谢产物epipolythiodioxopiperazine (ETP)家族,具有免疫抑制剂、抗真菌等多种生物活性,是由非核糖体多肽合成酶(NRPSs)催化合成。从牛樟芝(Antrodia camphorata)基因组中挖掘出非核糖体多肽合成酶基因(AcNRPS,NCBI登录号为KX430967),克隆获取其全长cDNA,并对其进行生物信息学分析和表达谱分析。结果显示AcNRPS基因cDNA全长6 687 bp;与其DNA序列比对发现AcNRPS基因含有12 个内含子;其开放阅读框编码2 229 个氨基酸残基,BLAST比对发现其含有2 个A-C-T结构域,底物需2个氨基酸;系统发育树结果显示AcNRPS与其他合成产物为胶霉毒素的NRPS基因聚为一类,其可合成胶霉毒素类化合物;表达谱分析显示,以葡萄糖和土豆蛋白胨作为碳、氮源的培养基能够有效促进牛樟芝NRPS基因的表达。     

植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产g-氨基丁酸

γ-氨基丁酸可由谷氨酸脱羧酶(glutamate decarboxylase, GAD)催化谷氨酸一步合成,反应体系成分简单、环境友好。然而,绝大多数GAD酶催化pH偏酸性且反应范围狭小,需要加入无机盐维持最适催化环境,增加了生产附加成分。此外,随着产物γ-氨基丁酸的生成,溶液pH会逐渐上升,不利于GAD酶的持续转化。本研究首先从实验室保藏的一株高产γ-氨基丁酸的植物乳杆菌(Lactobacillus plantarum)中克隆得到谷氨酸脱羧酶LpGAD,基于酶蛋白表面电荷修饰,选择9个位点进行定点突变及组合突变,酶学性质表征结果显示三突变体LpGAD^{S24R/D88R/Y309K}在催化pH区间内酶活力整体提高,尤其拓宽了在偏中性pH 6.0下的酶活,为野生酶的1.68倍。接下来,通过分子动力学模拟解析了酶活提高的机理。此外,将Lpgad与Lpgad^{S24R/D88R/Y309K}突变基因分别在谷氨酸棒杆菌(Corynebacterium glutamicum) E01中过表达,通过优化确定了摇瓶最适转化条件为反应温度40 ℃,菌体量OD₆₀₀=20,底物L-谷氨酸100.0 g/L,5-磷酸吡哆醛添加量为100 μmol/L。5 L发酵罐中,不调节pH,通过分批投料底物L-谷氨酸,γ-氨基丁酸产量高达402.8 g/L,较对照菌株提高了1.63倍。本研究成功拓宽了LpGAD的pH催化范围及酶活,提高了γ氨基丁酸的转化效率,为实现其规模化工业生产奠定了基础。     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Biological control of three floating water weeds,<Emphasis Type="Italic">Eichhornia crassipes,Pistia stratiotes</Emphasis>, and <Emphasis Type="Italic">Salviniamolesta</Emphasis> in the Republic of Congo

Since 1999, four specific weevils (Coleoptera, Curculionidae) were released in the Republic of Congo against three exotic floating water weeds: Neochetina eichhorniae Warner and N. bruchi Hustache against water hyacinth, Neohydronomus affinis Hustache against water lettuce, and Cyrtobagous salviniae Calder and Sands against water fern. Recoveries of exotic weevils were made from all 24 release sites except one, and all four species have established and spread (up to 800 km for water hyacinth weevils). Within a few years of releases, control of water fern and water lettuce was such that fishing and navigation could be resumed, while reductions of water hyacinth populations were only beginning.     

New species and new combinations in Peruvian Orchidaceae

seventeen new species and combinations are proposed in the generaChondrorhyncha, Cischweinfia, Cochlioda, Eloyella, Encyclia, Kefersteinia, Koellensteinia, Macroclinium, Rodriguezia, Solenidiopsis, andStenia. All new species are illustrated. A key is provided for 2-flowered species ofMacroclinium, PeruvianSigmatostalix, and PeruvianStenia. Solenidium (Solenidiopsis) peruvianum Schltr. is lectotypified.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.     

Die GattungKarschia — Bindeglied zwischen bitunicaten Ascomyceten und lecanoralen Flechtenpilzen?

The genusKarschia, in the earlier sense, including saprophytes and parasites on lichens, has been thought to be a non-lichenized parallel genus of the lichen genusBuellia. Modern workers included it on the one hand inBuellia, on the other hand combined it with bitunicate ascomycetes. It is now proved thatKarschia is heterogeneous and contains but superficially similar members both of the genusBuellia of theLecanorales and of typical or masked bitunicateAscomycetes. Therefore, it can not be regarded as a link betweenLecanorales andDothideales. The type species ofKarschia belongs to theDothideales.

     

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.