破囊壶菌脂肪酶基因的克隆、双温控自诱导表达及酶学性质研究

【目的】克隆破囊壶菌Aurantiochytrium sp.PKU#SW7的脂肪酶基因,实现其在大肠杆菌细胞中异源高效表达,并进行初步酶学性质研究。【方法】基于转录组数据注释,获得脂肪酶基因lip,构建重组基因工程菌Rosetta(DE3)/p ET30-lip,利用双温控自诱导方法高效表达蛋白,表达产物(LIP)经Ni-Agarose His亲和层析柱纯化后进行酶学性质研究。【结果】从Aurantiochytrium sp.PKU#SW7中克隆得到一个大小为873 bp的脂肪酶基因(Gen Bank登录号为KT305964),该酶对p-NPB最适反应温度和pH分别为40°C和8.0。以不同浓度的金属离子Ca~(2+)和Co~(2+)溶液分别保温处理酶液30 min可使酶活提高1.3倍左右;甲醇对脂肪酶的抑制作用不明显。在最适反应条件下对p-NPP与p-NPB的酶活力分别为70.0±3.1 U/mg和102.5±2.6 U/mg。【结论】Aurantiochytrium sp.PKU#SW7脂肪酶具有良好的特性,符合生物柴油生物催化剂基本要求。     

高分子量腈水合酶在大肠杆菌中的表达策略及重组菌的细胞催化

【目的】实现红球菌Rhodococcus rhodochrous J1来源的高分子量腈水合酶H-NHase在Escherichia coli BL21(DE3)中过量表达,以提高菌体总酶活,缩短菌体发酵周期,提高生产效率。【方法】将H-NHase中编码α亚基的基因nhh A和调控基因nhh G上游的核糖体结合位点(Shine-Dalgarno sequence,SD)替换成翻译起始强度更高的异源SD,同时优化各亚基之间间隔序列的长度,并根据大肠杆菌密码子偏好性对目的基因进行优化。通过E.coli重组表达系统过表达优化后的腈水合酶基因。采用离子交换层析对H-NHase进行纯化,并通过凝胶过滤层析确定重组酶的相对分子量。优化了全细胞催化条件,并建立底物恒速流加的细胞催化工艺,模拟了烟酰胺的生产工艺。【结果】H-NHase在E.coli中实现了过量表达。重组蛋白粗酶液的活性为85.5±4.3 U/mg,纯酶比活为234.0±11.7 U/mg,H-NHase相对分子质量为504.5±9.8 k D。细胞催化最适p H为7.5,最适温度为25°C,底物浓度为400 mmol/L。在此条件下,重组菌细胞酶活为256.0±10.4 U/m L,流加工艺最终的产物转化率可达99.9%。【结论】重组H-NHase大肠杆菌细胞生长迅速,发酵周期短,应用此重组菌进行细胞催化可以提高酰胺类物质的生产效率,具有潜在的工业价值。     

牦牛瘤胃中产脂肪酶微生物的分离与鉴定

【背景】脂肪酶是一类特殊的酯键水解酶,广泛应用于工业化生产中,微生物是工业脂肪酶的主要来源。瘤胃中微生物种类繁多、数量庞大,已有关于瘤胃微生物产纤维素酶的报道,尚无产脂肪酶瘤胃微生物的分离筛选报道。【目的】从牦牛瘤胃中分离筛选出能够产脂肪酶的微生物,并进行菌株鉴定及其酶学性质的研究。【方法】以橄榄油为唯一碳源,通过中性红油脂平板进行初步筛选后,用改进铜皂-分光光度法测定酶活力进行复筛;再经形态学观察、生理生化实验和16S rRNA基因序列分析进行菌种鉴定;研究3种脂肪酶的最适作用温度、pH值及金属离子、有机溶剂和表面活性剂对酶活力的影响。【结果】筛选出6株酶活力较高的菌株,其中3株为液化沙雷氏菌,2株为白地霉,1株为卷枝毛霉。脂肪酶的酶学性质研究表明:液化沙雷氏菌、白地霉和卷枝毛霉所产脂肪酶的最适作用温度为45、35和40°C;最适pH为8.0、7.0和7.0;Ca2+和Mg2+对3种脂肪酶均有激活作用;Zn2+对3种脂肪酶有不同程度的抑制作用,EDTA、SDS可使3种脂肪酶失活;3种脂肪酶对丙三醇的耐受力较高,卷枝毛霉脂肪酶对甲醇、乙醇、丙酮的耐受力较高。【结论】从牦牛瘤胃中分离出3种产脂肪酶的微生物,且证实瘤胃微生物在脂肪酶研究方面具有较高的价值。     

火球菌Pyrococcus furious瓣状核酸内切酶1的表达纯化及酶学特征

【目的】克隆表达和纯化火球菌Pyrococcus furious来源的瓣状核酸内切酶1基因pFEN1(PF1414),对该蛋白的活性和酶学特征进行鉴定和分析。【方法】将pFEN1在大肠杆菌中进行重组表达,经亲和层析纯化得到电泳纯蛋白;利用人工合成的荧光标记的寡核苷酸片段作为底物,用变性聚丙烯酰胺凝胶电泳鉴定pFEN1在体外的酶学特性以及与其他蛋白的相互作用。【结果】pFEN1重组蛋白能在大肠杆菌中进行高效表达;高于100 mmol/L的NaCl会抑制pFEN1的活性;pFEN1的核酸酶活性依赖于金属离子Mg~(2+)或Mn~(2+),且Mn~(2+)的催化效率优于Mg~(2+);来自嗜热古菌的pFEN1是一种耐高温蛋白,最适反应温度为60–65°C;增殖细胞核抗原(PCNA)能促进pFEN1的内切酶活性。【结论】本研究证实pFEN1是一种Mg~(2+)或Mn~(2+)依赖的核酸内切酶,且PCNA能促进该酶的活性。     

对SDS稳定的V8(V125T)蛋白酶突变体的高效表达及性质研究

谷氨酰内切酶能特异性切割谷氨酸、天冬氨酸残基羧基端结合的肽键。将含有V8蛋白酶突变体(V125T)基因的表达质粒的重组大肠杆菌BL21(DE3),在50L发酵罐中发酵,融合蛋白为可溶性表达,可得菌湿重50g/L,相对蛋白表达量为33%。融合蛋白采用GST亲和纯化、肠激酶激活、DEAE-FF阴离子交换层析,得到纯的V8(V125T)突变体,经纯化后可获得0.998mg蛋白/g菌(湿重),比活为13.47 U/mg pro.纯化过程的酶活回收率达到了97.9%。对纯酶进行酶学性质分析,以Z-Phe-Leu-Glu-p NA作为底物测得V8(V125T)蛋白酶的Km为0.339mmol/L,Vmax为16.642μmol/min。其最适pH为8.0,在pH4.0~10.0之间较稳定;最适反应温度在45℃,12h内在4~35℃有很好的温度稳定性;25℃条件下1mmol/L的金属离子对酶具有不同程度的影响,其中Fe~(3+)的抑制作用最强;2mol/L尿素及1mmol/L EDTA对酶活性无影响,在0.1%SDS中保存12h、在0.5%SDS中4h和在1%SDS中1h,活性均能维持90%以上,在0.5%,0.1%SDS保存12h,仍能保持80%和64%的活性,与未突变的重组V8蛋白酶相比,该突变体对SDS的耐受性得到极大提高。     

褐藻胶裂解酶基因的克隆表达及重组酶酶学性质

【目的】克隆交替假单胞菌(Pseudoalteromonas sp.)BYS-2的褐藻胶裂解酶基因,实现其在大肠杆菌细胞中异源表达,对分离纯化的重组酶进行酶学性质研究。【方法】以交替假单胞菌BYS-2菌株基因组DNA为模板,克隆得到褐藻胶裂解酶基因alg738,构建重组基因工程菌BL21(DE3)/p ET22b-alg738,诱导表达,表达产物通过Ni-NTA树脂纯化后进行酶学性质研究。【结果】重组酶的最适反应p H为8.0,在p H 6.0-9.0范围内37°C保温1 h仍能保持84%以上的相对酶活力,具有较好的p H稳定性;最适反应温度为45°C,热稳定性实验显示在37°C下保温60 min其残余酶活力仍达66.6%;在5 mmol/L浓度下,Na~+、Mg~(2+)、Mn~(2+)对该酶具有明显的促进作用,Ni~(2+)、Co~(2+)、Cu~(2+)、Hg~(2+)、Zn~(2+)、EDTA、β-巯基乙醇、SDS具有明显的抑制作用。动力学参数Km、Vmax分别为1.11 g/L和0.011 g/(L·min),底物特异性分析表明该重组酶为偏好聚甘露糖醛酸钠(Poly M)裂解作用的双功能酶。【结论】重组褐藻胶裂解酶具有良好的酶学特性,为褐藻胶裂解酶的开发应用打下基础。     

乙酸钙不动杆菌磷脂酶C在大肠杆菌中重组表达、纯化及酶学性质分析

【目的】构建乙酸钙不动杆菌(Acinetobacter calcoaceticus)ATCC17902磷脂酶C(Phospholipase C,PLC)的重组大肠杆菌菌株、纯化重组酶并进行酶学性质分析比较。【方法】以A.calcoaceticus ATCC17902基因组DNA为模板,PCR扩增得到两段磷脂酶C基因(PLC1、PLC2),构建重组大肠杆菌表达质粒并转化大肠杆菌BL21(DE3)中,实现PLC1、PLC2基因的表达。IPTG诱导表达后,经镍柱亲和层析纯化重组蛋白。【结果】成功构建两株产磷脂酶C的重组大肠杆菌并纯化,样品经SDS-PAGE分析在80 kDa附近均出现显著的特异性条带。NPPC法测得PLC1、PLC2酶活分别为31160±418 U/mg、13640±354 U/mg,最适反应温度分别为65、50℃,最适pH值分别为8、7.5。在低于30℃时,pH值7-8时,PLC1、PLC2重组酶较稳定,40℃处理30min,PLC1酶活稳定而PLC2残余酶活低于25%。Mg2+、Ca2+增强PLC1、PLC2的活性,Zn2+增强PLC1酶活性却抑制PLC2酶活。底物特异性分析表明PLC1、PLC2均水解磷脂酰肌醇(Phosphatidylinositol,PI),对其他种类磷脂不能水解或水解程度很低。【结论】本文首次实现了A.calcoaceticus ATCC17902来源的磷脂酶C的重组表达与功能验证,为其它食品安全性微生物来源的磷脂酶C的研究提供了一定的借鉴意义。     

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。     

流感嗜血杆菌ATCC49247来源的IgA酶理化性质及其对低糖基化IgA1的分解作用

【背景】近年来,能够特异性切割人IgA1分子的IgA蛋白酶(IgA酶)被认为是治疗IgA肾病(IgAN)的潜在药物,但各种物理化学因素均可能影响其生物学活性。【目的】研究流感嗜血杆菌(Haemophilus influenzae) ATCC49247 IgA酶的理化性质,确定IgA酶最适的作用条件后观察IgA酶对低糖基化IgA1的作用。【方法】从细菌培养液中分离纯化IgA酶,SDS-PAGE电泳后银染法分别检测多种理化条件下IgA酶的水解活性及其对低糖基化IgA1的消化作用。【结果】H. influenzae ATCC49247IgA酶可耐受的反应温度较广,最适温度为50°C;在高于60°C的环境中,IgA酶便不可逆地丧失了稳定性;IgA酶在pH 6.0-9.0的环境下能够保持完整催化活性;1 mmol/L的PMSF和高于10 mmol/L的SDS能够强烈地抑制IgA酶的活性,DTT和EDTA对IgA酶活性无明显影响;所有浓度的Al~(3+)、Fe~(3+)和高浓度的Cu~(2+)、Zn~(2+)、Fe~(2+)对IgA酶均表现出强烈的抑制作用,同时Co~(2+)、Mn~(2+)、Ca~(2+)、Ni~(2+)和Mg~(2+)都对IgA酶活性没有明显影响。选择了最适宜的IgA酶作用条件,发现IgA酶能够有效降解低糖基化IgA1底物。【结论】确定IgA酶最适宜的作用条件能保持良好的酶活性,更好地发挥了IgA酶对低糖基化IgA1的降解作用,为IgA酶的临床研究和药用价值的进一步开发提供一定的依据。     

联苯利用节杆菌YC-RL1中2,3-二羟基联苯-1,2-双加氧酶BphC功能验证

【目的】通过节杆菌(Arthrobacter sp.)YC-RL1对多氯联苯降解过程中关键基因bph C的克隆与原核表达,鉴定其编码的2,3-二羟基联苯-1,2-双加氧酶Bph C的酶活特性与功能。【方法】以菌株YC-RL1全基因组为模板进行PCR扩增获得bph C基因,将该基因转入Escherichia coli BL21(DE3)感受态细胞后进行原核表达;利用镍柱亲和层析法对Bph C酶进行纯化并分别测定该酶在不同条件下对底物2,3-DHBP的催化特性,确定其最适反应pH、温度及不同金属离子对酶活特性的影响;进一步根据米氏方程对该酶的动力学参数进行测定与分析。【结果】通过PCR扩增获得了bphC基因,其大小为930 bp;对该基因进行原核表达,所得重组蛋白BphC携带有6个组氨酸标签,经纯化后体外仍具有活性,该酶作用于2,3-DHBP时的最适pH与温度分别为pH 7.4和30°C,且在最适条件下,Fe~(2+)、Cu~(2+)及Cd~(2+)等金属离子可明显促进其酶活作用,但多数金属离子对该酶有不同程度的抑制作用;该酶在与底物2,3-DHBP作用过程中,酶促动力学常数分别为K_m:8.67 mmol/L,V_(max):27.32μmol/s,k_(cat):15.55 s~(–1),k_(cat)/K_m:1.79 L/(mmol·s),其催化效率同有关报道中同类酶的动力学特性比较均有所提高。【结论】菌株YC-RL中的bphC基因对于多氯联苯的生物降解具有至关重要的作用,其编码的BphC是重要的芳香环裂解酶,该酶对其底物具有较高的亲和性,可在体外环境中发挥高效的酶促作用,具有良好的应用价值。     

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.     

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.     

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.     

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.     

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.

     

Demographic characteristics of an intertidal bay goby (Lepidogobius lepidus)

Synopsis In a fourteen month study (May 1976 – June 1977) I examined the following characteristics of an intertidal bay goby (Lepidogobius lepidus) in Morro Bay, California, U.S.A.: annual and seasonal patterns of abundance, age composition and growth rates, survivorship and mortality patterns, and the reproductive cycle for female gobies. Fishes were collected with the aid of quinaldine and otoliths and ovaries removed. Age and growth rates were estimated from otolith annuli using a back calculation formula and a Brody-Bertalanffy growth curve. Mortality rates were derived using the methods of Heincke (1913), Robson & Chapman (1960), mean age, and a catch curve (Ricker 1975). A gonad index was used to describe the annual reproductive cycle. Results indicated that abundance fluctuated seasonally and that these fluctuations appeared to be caused by reproductive emigrations. Bay gobies reached an age of 7+ and a standard length of 87 mm. Growth was relatively constant (6 mm yr⁻¹) until age 5, at which point it began to decline. The mean rates of survivorship, mortality, and instantaneous mortality were 0.75, 0.25, and 0.29 respectively. Mortality rates for individual age classes ranged from 0.13 to 0.51 and increased with age. This stock appears to reproduce mainly during the winter.     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.