灵芝UDP-葡萄糖4-差向异构酶酶学性质研究及其应用

【背景】灵芝多糖是灵芝的重要活性物质之一。UDP-葡萄糖4-差向异构酶（UDP-glucose 4-epimerase,UGE,EC 5.1.3.2）是灵芝多糖合成途径中糖供体生成的重要酶,其参与了UDP-葡萄糖与UDP-半乳糖的相互转化,与多糖中半乳糖残基含量密切相关。【目的】通过对来源于灵芝的UGE基因进行异源表达,丰富灵芝多糖糖供体合成途径重要酶的酶学特性信息,深入了解灵芝多糖代谢合成途径。【方法】以灵芝菌株（Ganoderma lingzhi） CGMCC 5.26的cDNA为模板,克隆得到UGE基因GL30389,并在Escherichia coli BL21（DE3）中诱导表达,产物纯化后进行酶学性质、酶动力学、底物专一性及转化率的研究。【结果】灵芝UGE的分子量为45 kDa。最适反应pH值为6.0,在pH 7.0—9.0范围内有较好的稳定性;最适反应温度为30℃,温度在40℃时稳定性最好。Fe²⁺和Mg²⁺对UGE有激活作用。以UDP-葡萄糖为底物时,K_m为0.824 mmol/L,V_max为769.230 μmol/（L·min）,k_cat为1.333 s^—1,k_cat/K_m为1.618 L/（mmol·s）。灵芝UGE对D-葡萄糖、半乳糖醛酸及N-乙酰葡萄糖胺有催化活性。通过优化pH、温度、底物与酶的配比、添加金属离子将转化率从16.0%提升至39.4%。【结论】灵芝UGE与植物来源的UGE酶学性质较为相似,其催化效率优于大部分细菌来源的UGE。本研究丰富了灵芝多糖糖供体合成途径重要酶的酶学特性信息,有利于深入了解灵芝多糖代谢合成途径。     

一株海洋来源铜绿假单胞菌Gxun-7角蛋白酶基因的克隆、表达及重组酶酶学性质

【背景】角蛋白酶是一类特异性降解角蛋白的水解酶,在动物饲料、生物肥料、医学、洗涤、制革及环境治理等方面具有重要的应用潜力。【目的】对前期从海洋环境筛选出的一株铜绿假单胞菌Gxun-7的角蛋白酶基因进行克隆、表达,并探究重组酶酶学性质,为角蛋白酶在工业生产中的应用奠定基础。【方法】以铜绿假单胞菌Gxun-7基因组推定的角蛋白酶基因为基础,设计引物克隆获得角蛋白酶基因kp2,构建重组表达质粒pET22b-kp2,并转化到E. coliRosettagamiB (DE3)中进行诱导表达,同时对重组表达菌株的表达条件进行优化。利用镍柱分离纯化重组角蛋白酶并研究其酶学性质。【结果】重组角蛋白酶的分子量约为33 kDa,最适温度和pH值分别为40 ℃和8.0,在温度30-60 ℃和pH 6.5-8.0具有较好的稳定性。金属离子Co²⁺、Cu²⁺和化学试剂十二烷基磺酸钠(sodium dodecyl sulfonate,SDS)、乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)、苯甲基磺酰氟(phenylmethylsulfonyl fluoride,PMSF)对酶活力有抑制作用,而Mg²⁺、K⁺、巯基乙醇和二硫苏糖醇(dithiothreitol,DTT)对酶活力有促进作用。重组角蛋白酶具有良好的耐盐性,在12.5%的NaCl作用下相对酶活为87.55%。以酪蛋白为底物时,酶的K_m值为60.92 mg/mL、V_max值为9.70 U/mL。【结论】海洋来源铜绿假单胞菌Gxun-7的重组角蛋白酶具有良好的温度、碱、盐稳定性,可应用于工业生产中。     

栓菌漆酶在毕赤酵母中高效表达及重组酶的性质

栓菌420（Trametes sp. 420）漆酶基因lacD以两种方式在巴斯德毕赤酵母（Pichia pastoris）进行异源表达,产生两种重组漆酶：rLacDx（具有天然N-末端）和rLacDe（N-末端带有8个额外的氨基酸残基）。摇瓶发酵18d,rLacDx和rLacDe的产量分别为1.21×10⁵u/L、7.38×10⁴u/L ［以2,2′-连氮-3-乙苯-二噻唑-6磺酸（ABTS）为底物］。在高密度发酵条件下,rLacDx的产量增加到2.39×10⁵u/L,同时其生产周期降至7.5 d。两种重组酶对愈创木酚底物的氧化特性相似,且在50℃和pH3～10的范围内均稳定。然而,rLacDx对底物ABTS的比活力（1761u/mg）高于rLacDe （1122u/mg）,其表观K_m值(427μmol/L)低于rLacDe (604μmol/L)。     

杀鱼假交替单胞菌C923几丁质酶基因PpchiC的克隆表达与酶学性质

【背景】某些假交替单胞菌可分泌几丁质酶,在降解利用几丁质为水产动物提供营养、免疫、抗病等方面有着重要潜力。【目的】克隆杀鱼假交替单胞菌(Pseudoalteromonas piscicida)C923的一个几丁质酶基因,实现其在大肠杆菌中的异源表达,并对重组几丁质酶的酶学性质进行研究。【方法】从菌株C923测序的基因组中注释到一个几丁质酶家族基因PpchiC,设计引物克隆该基因后进行生物信息学分析;构建载体进行异源表达并从温度、时间与诱导剂浓度进行表达优化;对表达蛋白进行最适温度与pH等酶学性质研究,同时比较了重组菌破碎后上清与沉淀及纯化的酶蛋白对几丁质的降解效应。【结果】基因PpchiC长1350bp,编码450个氨基酸,PpchiC蛋白理论分子量为48.76kDa,等电点为4.78,不稳定系数为29.08。结构域分析发现该蛋白含有一个类型Ⅲ几丁质结合域和一个糖苷水解酶18家族(glycosyl hydrolase 18,GH18)的催化域;PpchiC蛋白含有GH18家族几丁质酶的保守催化基序DxxDxDxE、YxR和[E/D]xx[V/I]。16℃、0.25mmol/L IPTG、诱导12h为其最优化表达条件,PpchiC在50℃、pH8.0时表现出最大酶活性;以胶体几丁质为底物时,PpchiC的K_m值为2.58mg/mL、V_max值为5.04mg/(mL·min)。降解结果表明,菌体的沉淀与上清及从上清中纯化的酶蛋白均有着较好的几丁质降解效应。【结论】杀鱼假交替单胞菌C923基因PpchiC编码GH18家族的几丁质酶,能被大肠杆菌高效表达且降解几丁质效应明显,这为PpchiC及菌株C923的应用提供了参考依据。     

来源于超嗜热古菌雅氏火球菌(Pyrococcus yayanosii)CH1耐热耐压脯肽酶的酶学性质研究

【目的】脯肽酶是一种能从二肽(Xaa-Pro)的C末端水解脯氨酸或羟脯氨酸残基的肽酶。对深海来源的雅氏火球菌(Pyrococcus yayanosii) CH1基因组中PYCH_07700基因编码的蛋白Pyprol的体外酶学性质进行研究,以期发现新型脯肽酶。【方法】在小宝岛热球菌(Thermococcus kodakarensis) TS559中异源表达Pyprol。使用二肽Met-Pro作为底物,检测重组蛋白的脯肽酶活性。【结果】Pyprol的最适温度为100 ℃,最适pH为6.0。Pyprol在与Co²⁺结合时活性最高,最适的金属离子浓度为1.2 mmol/L。与P. furiosus来源的脯肽酶Pfprol相比,Pyprol在更宽的pH范围具有活性,并且能够耐受更高浓度的金属离子。Pyprol是耐压蛋白,最适静水压为40 MPa。与常压条件下相比,40 MPa下,Pyprol在40、70和100 ℃均有更高的活性。【结论】来源于深海热液喷口的严格嗜压的超嗜热古菌P. yayanosii CH1的新型脯肽酶Pyprol具有热稳定和耐压特性。     

Nonomuraea candida HMC10T中新结构套索肽noncaromin生物合成基因簇的克隆及异源表达

【目的】本研究旨在通过定向克隆菌株Nonomuraea candida HMC10^T中一个新的Ⅱ型套索肽类生物合成基因簇,通过在放线菌底盘宿主中的异源表达,获得新结构套索肽noncaromin,并完成其抑菌活性分析。【方法】通过antiSMASH软件分析菌株N.candida HMC10^T全基因组序列,确定新的Ⅱ型套索肽noncaromin的生物合成基因簇(biosynthetic gene cluster of noncaromin,nonc-BGC)。然后,利用ExoCET重组技术(exonuclease combined with RecET recombination)获得完整的nonc-BGC,得到重组质粒pJQK652,并通过λ-Red重组技术改造得到整合型质粒pJQK653。采用接合转移方法,将该质粒分别导入白色链霉菌、2株变铅青链霉菌、2株天蓝色链霉菌和红色糖多孢菌宿主中进行异源表达,再通过发酵和分离纯化获得目标套索肽noncaromin。最后,利用QTOF-ESI-MS²完成套索肽noncaromin的结构鉴定,并通过抗菌活性检测确定该化合物的生物活性。【结果】本研究利用ExoCET技术成功获得了完整的nonc-BGC,在6种放线菌宿主中成功异源表达,完成了noncaromin的结构鉴定,确定了其具有微弱的抗枯草芽孢杆菌活性。【结论】本研究在克隆得到新结构套索肽nonc-BGC的基础上,实现了该基因簇在6个放线菌底盘宿主中的成功表达,获得了1个具有微弱抑制枯草芽孢杆菌活性的新结构Ⅱ型套索肽noncaromin。本研究结果为发掘菌株N.candida HMC10^T及其他放线菌中的新结构化合物提供了借鉴。     

嗜热古菌Infirmifilum uzonense来源的双功能高温β-葡萄糖苷酶IuBgl3的原核表达及酶学性质分析

β-葡萄糖苷酶在食品、医药、生物质转化等领域具有重要的应用价值,因此发掘适应性强、性质优良的β-葡萄糖苷酶是国内外研究热点。本研究从嗜热古菌Infirmifilum uzonense中成功克隆出一个GH3家族的β-葡萄糖苷酶基因,命名为Iubgl3。基因序列分析显示Iubgl3全长为2109bp,编码702个氨基酸,理论分子量为77.0kDa。将该基因在大肠杆菌中进行克隆表达并对纯化后的IuBgl3进行酶学性质研究。结果显示,重组酶IuBgl3最适pH5.0,最适温度85℃。该酶具有良好的热稳定性,80℃处理2h后仍能保持85%以上的酶活力。其具有优良的pH稳定性,在pH4.0−11.0范围内处理1h,仍维持85%以上的酶活力。通过底物特异性测定发现,该酶对对硝基苯-β-d-吡喃葡萄糖苷(p-nitrophenylβ-d-glucoside,pNPG)和对硝基苯-β-d-吡喃木糖苷(p-nitrophenyl β-d-xylopyranoside,pNPX)均有很高的水解能力,是典型的双功能酶。以pNPG为底物时的动力学参数K_m和V_max分别为0.38mmol和248.55μmol/(mg·min),催化效率k_cat/K_m=6149.20s⁻¹mmol⁻¹。大多数金属离子对IuBgl3的酶活力没有显著影响,SDS可导致酶完全失活,而EDTA却能提高30%的酶活力。本研究丰富了高温古菌GH3家族的β-葡萄糖苷酶基因,获得了一个稳定性优良的高温酸性双功能酶,具有良好的工业应用前景。     

巴斯德毕赤酵母不同生长阶段内参基因的筛选与验证

【背景】巴斯德毕赤酵母(Komagataella phaffii)是一种甲基营养型酵母,近年来作为生产重组蛋白和构建生物合成途径的细胞工厂受到广泛关注。实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)是巴斯德毕赤酵母表达系统研究中一种快速、高效的基因表达水平检测技术,但需要进行归一化处理才能保证所得结果的可靠性。【目的】筛选并验证巴斯德毕赤酵母在不同生长阶段最稳定的内参基因用于精准归一化RT-qPCR的结果。【方法】通过转录组数据分析初步筛选出16个候选内参基因(rps8b、rpl35a、rpl10、eif5a、rpl19a、por1、rpl23b、0887、tif1、ole1、rpl14b、gs、sun、sdh2、trx1和ccp1)。通过RT-qPCR技术得到候选内参基因的C_t值,利用qBASE软件中的geNorm程序综合NormFinder算法评估内参基因的表达稳定性。【结果】通过geNorm分析得出精准归一化所需的最佳内参基因个数为2,最稳定的基因是rpl19a和tif1,NormFinder分析得到稳定性最高的内参基因为tif1。此外,利用甲酸脱氢酶编码基因fdh和乙醇脱氢酶甲醛脱氢酶双功能酶的编码基因afdh对候选内参基因进行验证。【结论】巴斯德毕赤酵母不同生长阶段的RT-qPCR进行精准归一化需要tif1和rpl19a这2个内参基因,为相关功能基因的表达定量提供了可靠的分析依据,补充了RT-qPCR分析中的内参基因,为巴斯德毕赤酵母不同生长阶段的基因表达调控及其应用研究提供了新的参考。     

西黑冠长臂猿粪便微生物宏基因组来源的高比活α-l-阿拉伯呋喃糖苷酶的重组表达及酶学性质

【背景】α-l-阿拉伯呋喃糖苷酶是一类重要的半纤维素酶,能协同其他半纤维素酶降解木聚糖,在食品、医药、生物质能转化中具有应用价值。【目的】挖掘新型α-l-阿拉伯呋喃糖苷酶基因,对其进行异源表达、纯化并研究其酶学性质。【方法】从西黑冠长臂猿粪便微生物宏基因组中扩增α-l-阿拉伯呋喃糖苷酶基因,在大肠杆菌BL21(DE3)中进行异源表达,并进行酶学性质研究。【结果】从粪便微生物宏基因组中扩增得到α-l-阿拉伯呋喃糖苷酶基因AbfNC2b_38,并获得重组α-l-阿拉伯呋喃糖苷酶AbfNC2b_38,其分子量为57.04 kDa。AbfNC2b_38的最适作用条件为55 ℃、pH 6.0,K_m和V_max分别为(6.48±0.73) mmol/L和(1 248.0±114.6) U/mg,与其他宏基因组来源的α-l-阿拉伯呋喃糖苷酶相比具有最高比活300.81 U/mg。AbfNC2b_38具有较好的乙醇和NaCl耐受性,30%乙醇下耐受1 h保持68%的活性;25% NaCl中耐受1 h,相对酶活仍保持在约70%。与木聚糖酶协同降解山毛榉木聚糖时,协同率最高为1.21。【结论】从西黑冠长臂猿粪便微生物宏基因组中获得新型α-l-阿拉伯呋喃糖苷酶基因AbfNC2b_38并成功异源表达。AbfNC2b_38具有较好的乙醇和NaCl耐受性,能与木聚糖酶协同作用提高木聚糖的降解效率,在饲料、食品加工等领域具有潜在的应用价值。     

木聚糖酶生产菌Saccharothrix variisporea YJ的筛选、发酵条件及酶学性质研究

以Azo-xylan为底物,利用双层平板法从堆肥中筛选到可降解木聚糖的菌株,16S rRNA测序分析显示该菌株与糖丝菌属（Saccharothrix variisporea）的同源性最高（99.33%）,命名为S. variisporea YJ。研究发现以酵母提取物或(NH₄)₂SO₄作为氮源、甘蔗叶作为碳源、初始pH值 7.0、发酵温度40 ℃、发酵时间5 d时,发酵液中木聚糖酶的酶活性最高。酶学性质研究表明该木聚糖酶的最适反应温度及pH值分别为55 ℃和8.0,在55 ℃以下及pH值 4.0~10.0的范围内保持较高稳定性。Na⁺能有效提高木聚糖酶活性,Mg²⁺和Mn²⁺没有明显影响,Cu²⁺则严重抑制木聚糖酶活性。此外,发酵液还可以直接对天然底物玉米芯进行降解。     

Coccystes undoxylophus

Ohne Zusammenfassung     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Aquila pennata undminuta

Ohne Zusammenfassung     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

UeberAquila pennata undminuta

Ohne Zusammenfassung     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.     

Biogeography of Hysterangiales (Phallomycetidae, Basidiomycota)

To understand the biogeography of truffle-like fungi, DNA sequences were analysed from representative taxa of Hysterangiales. Multigene phylogenies and the results of ancestral area reconstructions are consistent with the hypothesis of an Australian, or eastern Gondwanan, origin of Hysterangiales with subsequent range expansions to the Northern Hemisphere. However, neither Northern Hemisphere nor Southern Hemisphere taxa formed a monophyletic group, which is in conflict with a strictly vicariant scenario. Therefore, the occurrence and importance of long-distance dispersal could not be rejected. Although a pre-Gondwanan origin of Hysterangiales remains as a possibility, this hypothesis requires that Hysterangiales exist prior to the origin of the currently recognized ectomycorrhizal plants, as well as the arrival of mycophagous animals in Australia. This also requires that a basal paraphyletic assemblage represents parallel evolution of the ectomycorrhizal symbiosis, or that Hysterangiales was mycorrhizal with members of the extinct flora of Gondwana. Regardless, models for both ancient and more recent origins of Hysterangiales are consistent with truffle-like fungi being capable of transoceanic dispersal.