腾冲嗜热厌氧杆菌Cas2原核表达及生物信息学分析

为研究CRISPR/Cas系统及其相关蛋白Cas2(TTE2657)在腾冲嗜热厌氧杆菌热适应中的作用,应用PCR技术构建了原核重组质粒pET-28a::cas2,并在大肠埃希菌BL21表达Cas2蛋白;结合生物信息学软件对cas2编码蛋白的基本理化性质、氨基酸同源性、空间结构及蛋白质相互作用网络进行预测和分析。结果显示,成功构建了原核表达载体pET-28a::cas2并在大肠埃希菌BL21中得到表达,Cas2分子质量大小为9.9 ku,主要以可溶性形式存在;qRT-PCR显示cas2 mRNA在60℃和75℃高表达;生物信息学分析显示cas2基因其完整的ORF全长264 bp,编码88个氨基酸,其中Ile(14)、Ser(14)、Phe (12)含量较高,等电点为9.31,不存在跨膜结构。其蛋白质二级空间结构以α-螺旋、无规则卷曲、β-折叠为主,蛋白互作预测网络显示Cas2与Cas3、Cas5、Cas7等其家族大部分蛋白存在相互作用。进化树分析显示腾冲嗜热厌氧杆菌cas2基因与厌氧菌芽胞杆菌B7M1同源性最高(39.5%)。腾冲嗜热厌氧杆菌cas2编码蛋白是一种亲水性蛋白,在原核系统能高效表达。本研究为嗜热蛋白质的热稳定性机制的研究提供参考。     

嗜热微生物酶的嗜热机制及应用研究进展

从细胞膜组分、蛋白质结构、遗传物质、钨元素等方面阐述了嗜热微生物酶的嗜热机制 ,并简要介绍了其应用的研究进展。     

诱导嗜热四膜虫(Tetrahymena thermophila)细胞凋亡样变化的研究

本文以单细胞真核生物嗜热四膜虫(Te-trahymena thermophila)作为实验材料以抗肿瘤药物高三尖杉脂碱(Homoharringtonine,HHT)、糖皮质激素类药物地塞米松(9α-Fluo-ro-16α-methylprednisolone,Dex)和抗生素类药物放线菌素D(Actinomycin D)诱导嗜热四膜虫凋亡并研究其细胞凋亡过程的生物化学特性。结果表明抗肿瘤药物及抗生素类药物均不能明显地诱导嗜热四膜虫细胞凋亡。但糖皮质激素类药物在含一定量的Ca~(2 )、Mg~(2 )离子时能诱导嗜热四膜虫发生凋亡。作者认为诱导嗜热四膜虫凋亡过程可能与糖皮质激素类药物诱导鼠胸腺细胞凋亡的机制是类似的,嗜热四膜虫与胸腺细胞的凋亡过程可能同样被Ca~(2 )、Mg~(2 )离子依赖性的核酸内切酶的活化机制所控制着。     

嗜热真菌的分离鉴定与系统发育分析

目的:研究广西热带地区嗜热真菌的多样性.方法:从广西热带地区各地采集的土壤样品,将土样撒在PDA平板上,50℃高温培养,挑取真菌菌丝进一步划线分离纯化,20℃低温培养验证获得嗜热真菌,对其进行形态观察和ITS基因序列分析.结果:共分离到33株嗜热真菌,形态和分子生物学鉴定结果显示分离到的菌株中有20株属于Thermomyces lanuginosus,4株属于Thermoascus aurantiacus,1株属于Chaetomium thermophilum,l株属于Talaromyces emersonii,1株属于Myceliophthora thermophila,还有6株的ITS序列与已知真菌的同源性很低,尚无法鉴定到种属.结论:广西热带地区的嗜热真菌存在多样性,Thermomyces lanuginosus为该地区主要嗜热真菌种.     

极端嗜热古菌的热休克蛋白

随着生物工程产业对于耐高温酶和菌体的需求, 极端嗜热古菌热休克蛋白(heat shock proteins, HSPs)的研究更受重视, 其热休克蛋白体系非常简洁, 不含HSP100s和HSP90s, 就是HSP70(DnaK)、HSP40、(DnaJ)和GrpE等嗜温古菌可能含有的在极端嗜热古菌中几乎不含有, 即仅包括HSP60, sHSP, prefoldin和AAA+蛋白四大类, 因此对其结构、功能和作用机制的研究在理论和实践上都特别有意义。系统地介绍了这四大类组分的结构、功能和作用机制和协同作用的研究进展, 论述了极端嗜热古菌热休克蛋白的系列研究难点和困惑, 展望了进一步的研究方向和重点。     

嗜酸乳杆菌与嗜热链球菌共发酵互生机理的研究

目的:对嗜酸乳杆菌和嗜热链球菌共发酵时两者的互生作用机理进行研究。方法:以质量浓度100gL脱脂乳为培养基,将嗜酸乳杆菌主要代谢产物—氨基酸,如赖氨酸、精氨酸、缬氨酸分别以0.0083mgml、0.0036mgml、0.0053mgml的量加入含嗜热链球菌脱脂乳培养基中,40℃培养,测定凝乳时间;将嗜热链球菌的代谢产物-乳酸、甲酸分别为0.1332mgml和0.075mgml的量加入含嗜酸乳杆菌脱脂乳培养基中,37℃培养,测定其发酵乳的凝乳时间。结果:嗜热链球菌发酵乳凝乳时间由12h缩短到4h,pH为4.52～4.62,吉尔涅尔度为54.23～64.74°T;嗜酸乳杆菌发酵乳的凝乳时间由16h缩短为5h,pH为4.61～4.65;且嗜酸乳杆菌在CO2环境中发酵时,发酵时间明显缩短。结论:嗜酸乳杆菌和嗜热链球菌共发酵时具有互生关系。     

同源过表达BglR对嗜热毁丝霉β-葡萄糖苷酶活性的影响

目的:克隆嗜热毁丝霉(Myceliophthora thermophila ATCC42464)bglr基因,构建Mtbglr过表达载体,研究同源过表达bglr对嗜热毁丝霉β-葡萄糖苷酶活性及总纤维素酶活性的影响。方法:利用SLIC技术构建Mtbglr过表达载体;使用MtPpdc启动子及MtTpdc终止子将该基因进行同源过表达;利用原生质体转化、实时荧光定量PCR和酶活测定等技术实现bglr基因在嗜热毁丝霉中表达及酶活水平的鉴定。结果:成功构建Mtbglr过表达载体,并转化嗜热毁丝霉,结果表明,诱导培养条件下,转化子Mt8菌株的β-葡萄糖苷酶活性和胞外蛋白质浓度分别是WT菌株的1.7倍和1.9倍。结论:bglr基因对嗜热毁丝霉β-葡萄糖苷酶活性具有增强作用,为嗜热真菌β-葡萄糖苷酶基因调控奠定了理论基础。     

嗜热和嗜碱木聚糖酶研究进展

木聚糖酶是降解半纤维素主要成分木聚糖的关键酶,广泛应用在食品、饲料、制浆造纸、生物脱胶等行业。特别是在造纸工业中,木聚糖酶显示出巨大的应用潜力,已成为国内外研究的热点。纸浆漂白工艺中需要酶在高温碱性条件下发挥作用。目前,主要通过筛选野生型木聚糖酶资源和对现有中性中温木聚糖酶分子改造的方法获得嗜热碱木聚糖酶。文中就嗜热嗜碱木聚糖酶的筛选、嗜热嗜碱机制研究及分子改造进展进行了综述,并对其前景进行了展望。     

嗜热酶的结构特征与热稳定性

相对于嗜温酶,嗜热酶作为生物催化剂有许多优势而受到人们的广泛重视。嗜热酶的热稳定性机制历来是生物化学家们研究的重点。本文综述了嗜热酶的结构特征及其与热稳定性的关系。相对于嗜温酶,嗜热酶的结构比较僵硬,折叠紧密,廿螺旋更长,构象张力低,大多数为多聚体。了解嗜热酶的结构特征与热稳定性的关系,对于采用酶工程改造嗜温酶从而提高其热稳定性具有重要的指导意义。     

嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a( )上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。     

Characterization of native glutamate dehydrogenase from an aerobic hyperthermophilic archaeon Aeropyrum pernix K1

Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285 +/- 15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 degrees C). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7-15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.     

Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1.

We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV, which was slightly higher than that of Trx from E. coli (-270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.     

Bifunctional phosphoglucose/phosphomannose isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum constitute a novel enzyme family within the phosphoglucose isomerase superfamily

The hyperthermophilic crenarchaeon Aeropyrum pernix contains phosphoglucose isomerase (PGI) activity. However, obvious homologs with significant identity to known PGIs could not be identified in the sequenced genome of this organism. The PGI activity from A. pernix was purified and characterized. Kinetic analysis revealed that, unlike all known PGIs, the enzyme catalyzed reversible isomerization not only of glucose 6-phosphate but also of epimeric mannose 6-phosphate at similar catalytic efficiency, thus defining the protein as bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). The gene pgi/pmi encoding PGI/PMI (open reading frame APE0768) was identified by matrix-assisted laser desorption ionization time-of-flight analyses; the gene was overexpressed in Escherichia coli as functional PGI/PMI. Putative PGI/PMI homologs were identified in several (hyper)thermophilic archaea and two bacteria. The homolog from Thermoplasma acidophilum (Ta1419) was overexpressed in E. coli, and the recombinant enzyme was characterized as bifunctional PGI/PMI. PGI/PMIs showed low sequence identity to the PGI superfamily and formed a distinct phylogenetic cluster. However, secondary structure predictions and the presence of several conserved amino acids potentially involved in catalysis indicate some structural and functional similarity to the PGI superfamily. Thus, we propose that bifunctional PGI/PMI constitutes a novel protein family within the PGI superfamily.     

Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.     

A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1

An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was purified by Ni-chelation affinity chromatography. It showed a single band with a molecular mass of 18kDa in SDS-PAGE. The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C. The enzyme activity was Ca(2+)-independent. Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.     

A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix.

The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400 mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe.     

Characterization of novel hexadecameric thioredoxin peroxidase from Aeropyrum pernix K1

A gene (APE2278) encoding the peroxiredoxin (Prx) homologous protein of yeast and human was identified in the genome data base of the aerobic hyperthermophilic archaeon Aeropyrum pernix. We cloned the gene and produced the encoded protein in Escherichia coli cells. The isolated recombinant protein showed peroxidase activity in vitro and used the thioredoxin system of A. pernix as an electron donor. These results indicate that the recombinant protein is in fact thioredoxin peroxidase (ApTPx) of A. pernix. Immunoblot analysis revealed that the expression of ApTPx was induced as a cellular adaptation in response to the addition of exogenous H2O2 and may exert an antioxidant activity in vivo. An analysis of the ApTPx oligomers by high pressure liquid chromatography and electron microscopic studies showed that ApTPx exhibited the hexadecameric protein forming 2-fold toroid-shaped structure with outer and inner diameters of 14 and 6 nm, respectively. These results indicated that ApTPx is a novel hexadecameric protein composed of two identical octamers. Although oligomerization of individual subunits does not take place through an intersubunit-disulfide linkage involving Cys50 and Cys213, Cys50 is essential for the formation of the hexadecamer. Mutagenesis studies suggest that the sulfhydryl group of Cys50 is the site of oxidation by peroxide and that oxidized Cys50 reacts with the sulfhydryl group of Cys213 of another subunit to form an intermolecular disulfide bond. The resulting disulfide can then be reduced by thioredoxin. In support of this hypothesis, ApTPx mutants lacking either Cys50 or Cys213 showed no TPx activity, whereas the mutant lacking Cys207 had a TPx activity. This is the first report on the biochemical and structural features of a novel hexadecameric thioredoxin peroxidase from the archaea.     

江浙蝮蛇磷脂酶A2基因的多样性研究

我们利用简并引物从江浙蝮蛇腺总ＲＮＡ经ＲＰ－ＰＣＲ扩增磷脂酶Ａ２（简称ＰＬＡ２）基因,并以碱性ＰＬＡ２（Ｂ－ＰＬＡ２）基因为探针,分离出了酸性ＰＬＡ２（Ａ－ＰＬＡ２）和两个未见报道的特征结构类同的基因,分别命名为Ａｓｎ＾４８－ＰＡＬ２和ＢＡ－ＰＡＬ２。双向测序测定了这组ＰＬＡ２同工酶（除信号肽外）基因的全序列,并由此推导编码的氨基酸序列。其中Ａ－ＰＬＡ２基因编码的氨基酸序列与较早报道的由蛇毒中分离     

嗜热泉生古细菌及其他泉古菌同义密码子使用偏向性分析

比较分析了嗜热泉生古细菌(Aeropyrum pernix K1)和其他两种系统发育相关的泉古菌[嗜气菌(Pyrobaculum aerophi-lumstr.IM2)和嗜硫菌(Sulfolobus acidocaldarius DSM 639)]的同义密码子使用偏向性。结果表明嗜热泉生古细菌(Aeropyrum pernix K1)的密码子偏向性很小,并且与GC3S成高度的相关性。这3种泉古菌的密码子使用模式在进化上很保守。与基因的功能对密码子使用的影响相比,这些泉古菌密码子的使用偏向性更是由其物种所决定的。嗜热泉生古细菌(A.pernix K1),嗜气菌(P.aerophilum str.IM2)和嗜硫菌(S.acidocaldarius DSM 639)生存在不同的极限环境中。推测正是这些极限环境决定了这些泉古菌的密码子使用偏向性模式。此外在这些泉古菌的基因组中并没有发现其正义链和反义链的密码子使用偏向性差别。嗜热泉生古细菌(A.pernix K1)和嗜硫菌(S.acidocaldarius DSM 639)的密码子偏向性程度与基因表达水平有高度的相关性,而嗜气菌(P.aerophilum str.IM2)的基因组并没有发现这种规律。