假单胞菌ND6菌株水杨酸羟化酶基因(nahG)的核苷酸序列分析和酶鉴定

水杨酸羟化酶是细菌萘降解途径中的关键酶，它能催化水杨酸脱羟和羟化，生成儿茶酚．假单胞菌(Pseudomonas)DN6菌株的萘降解基因位于102kb的质粒pND6-1上，以pUC18质粒为载体，制备了含有1～3kb pND6-1 DNA随机片段的基因库，通过DNA测序和DNA序列同源性分析，从基因库中筛选出含有水杨酸羟化酶基因nahG的克隆．nahG基因的大小为1305bp，编码的水杨酸羟化酶(NahG)由434个氨基酸组成，与Pseudomonas putida NCIB 9816-4菌株的水杨酸羟化酶基因相比，核苷酸序列的同源性为100％，与其它10种细菌的水杨酸羟化酶基因相比，核苷酸序列的同源性为32％～99％．酶学实验表明，ND6菌株的水杨酸羟化酶具有广泛的底物特异性，它不仅能代谢水杨酸，还能代谢多种水杨酸的衍生物，如乙酰水杨酸、黄基水杨酸、3-甲基水杨酸、5-甲基水杨酸和5-氯水杨酸等．     

生物滴滤塔处理甲醛和三苯混合气体的实验研究

对生物滴滤塔处理甲醛和三苯混合气体的影响因素及性能进行了研究,在室温、pH值为6-7的条件下，当进气流量为600L／h，表面液体速度为3．14-3．93m／h时，生物滴滤塔对低浓度甲醛、苯、甲苯和二甲苯的混合气体有很好的去除效果，其相应的去除率分别在99％、73％、91％、88％以上．生物滴滤塔中的微生物对去除甲醛起到了重要作用．通过富集实验，经单菌对比验证，有4株菌降解效果显著．经16SrDNA测序和生理生化鉴定，可基本确定菌株J1和B4为Pseudomonas sp．，菌株JB2为Bacillus sp．，菌株E5为Arthrobacter sp．     

膜生物反应器异养硝化菌的筛选与硝化特性研究

从膜生物反应器中分离出3株异养硝化细菌,初始菌体浓度为105个/m L时,菌株X1、X2和X3经过12 h的硝化作用后,分别可去除50.7%、63.4%和46.7%的氨氮。菌株X2还表现出反硝化能力,12 h可去除44.5%的总氮。经16Sr DNA序列的测序和比对,菌株X1、X2和X3分别与假单孢菌(Pseudomonas sp.)、蜡状芽孢杆菌(Bacillus cereus)和鞘脂杆菌目(Sphingobacteriales)的同源性最高。未来异养硝化-反硝化菌株X2的应用对实现同步硝化反硝化具有重要的意义。     

阿特拉津氯水解酶基因的定点诱变和酶活力检测

阿特拉津氯水解酶(AtzA)是一种对除草剂的生物降解和环境净化有重要意义的酶．采用定点诱变方法将假单胞菌ADP株的atzA基因第832位碱基(鸟嘌呤)诱变成腺嘌吟，然后将其插入表达载体pET21b( )，并在大肠杆菌中表达．表达的蛋白特性研究表明：AtzA或AtzA—NK融合蛋白系水溶性蛋白，很容易通过Ni—NTA Magnetic Agarose Beads分离纯化．采用阿特拉津脱氯反应产生HCl而引起pH指示剂颜色改变的测定方法能方便地对其酶活力进行定量．酶活力结果表明，突变酶的比活力与假单胞菌ADP菌株的AtzA相比没有明显改变，暗示突变位点(第278位缬氨酸突变成甲硫氨酸)不是酶的活性中心或底物结合部位。     

假单胞杆菌BC001对吡啶和喹啉的生物去除

从首钢焦化厂废水处理系统的活性污泥中分离出1株能在高浓度的吡啶（约400mg/L）和喹啉（约500mg/L）双基质条件下良好生长的细菌,经16S rDNA及生理形态特征鉴定为假单胞杆菌（Pseudomonas sp. BC001）,它对吡啶的去除主要通过生物吸附,而对喹啉的去除包括生物吸附和降解两个阶段。该菌能利用喹啉作为唯一的碳源和氮源代谢生长,适量的外加碳源对喹啉降解具有促进作用,经检测喹啉降解的中间产物主要为2-羟基喹啉和8-羟基香豆素, 氮的主要代谢终产物为NH₄⁺。     

The secretion of lecithinase of Pseudomonas alcaligenes S2 was via type II secretion pathway

Phosphorus is one of the major essential macronutrients for virtually metabolic processes in plant growth and de-velopment[1]. This creates a paradox with major agro-nomic implications since the phosphate form of phospho-rus is one of the least soluble mineral nutrient ions in the soil. The concentration of soluble phosphorus in soil is usually very low, normally at levels of 1 ppm or less (10 mol/L H2PO4?). Mineral forms of phosphorus are repre-sented in soil by primary minerals, such as ap…     

溴氰菊酯降解菌Pseudomonas sp.P1-1-B3产酶条件的优化

农药降解酶对去除农药残留污染具有良好的应用前景.从海洋沉积物中筛选到一株高产溴氰菊酯降解酶的菌株Pseudomonas sp.P1-1-B3,研究了碳源、氮源、pH值、培养温度、培养时间、接种量及溴氰菊酯对菌株产酶的影响.结果表明,降解菌产酶的最适条件为:以可溶性淀粉作为碳源,m(蛋白胨):m(酵母浸膏)=2:1为氮源,pH 7.5,温度30℃,培养时间2 d,接种量3%,溴氰菊酯含量15μmol/L.在此条件下,菌株产酶的比活力最大为113.3 U/mg.该降解酶对溴氰菊酯的降解,在12 h内降解率达54.6%.     

β-甘露聚糖酶产生菌的鉴定及其粗酶性质的研究

从土壤中分离得到一株产β-甘露聚糖酶菌株.16SrDNA序列分析结合常规细菌形态学分析表明,该细菌属于芽孢杆菌菌(Bacillus)属,并命名为Bacillus sp.102.该菌株产酶高及迅速,且粗酶液中甘露聚糖酶的量占绝对优势.薄板层析显示粗酶水解魔芋甘露聚糖和角豆胶产物以甘露寡糖为主.对酶性质的研究发现,酶的最适反应温度为50℃,最适pH值为9.0,在pH值为中性时能保持很好的稳定性,有在碱性条件下的工业应用潜力.     

纤维素降解菌的分离及单菌株与菌群纤维素酶活性质

从实验室保存的5号菌群中，分离筛选出四株具有纤维素酶活力的细菌F1、F2、F3和F4．经形态学观察并进行16SrDNA序列分析，鉴定F1、F2、F3、F4分别是苏云金芽孢杆菌、生淀粉糖化酶产生菌、粪产碱菌和蜡样芽孢杆菌．通过分别测定四株细菌、5号菌群以及四株菌混合菌群的CMC酶活．进行比较分析，说明分离筛选单菌株，了解了菌群主要成分，同时有利于克服5号菌群活性不稳定，无法保存等缺点．混合单菌株形成新的菌群，利于后续对菌群内部各组分之间相互作用的研究．     

Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase

Disulphide bonds in secreted proteins are considered to be inert because of the oxidizing nature of the extracellular milieu. An exception to this rule is a reductase secreted by tumour cells that reduces disulphide bonds in the serine proteinase plasmin. Reduction of plasmin initiates proteolytic cleavage in the kringle 5 domain and release of the tumour blood vessel inhibitor angiostatin. New blood vessel formation or angiogenesis is critical for tumour expansion and metastasis. Here we show that the plasmin reductase isolated from conditioned medium of fibrosarcoma cells is the glycolytic enzyme phosphoglycerate kinase. Recombinant phosphoglycerate kinase had the same specific activity as the fibrosarcoma-derived protein. Plasma of mice bearing fibrosarcoma tumours contained several-fold more phosphoglycerate kinase, as compared with mice without tumours. Administration of phosphoglycerate kinase to tumour-bearing mice caused an increase in plasma levels of angiostatin, and a decrease in tumour vascularity and rate of tumour growth. Our findings indicate that phosphoglycerate kinase not only functions in glycolysis but is secreted by tumour cells and participates in the angiogenic process as a disulphide reductase.     

Production of Medium Chain Length Polyhydroxyalkanoates by Pseudomonas mendocina 0806 from Related and Unrelated Carbon Sources

ＩｎｔｒｏｄｕｃｔｉｏｎＰｏｌｙｈｙｄｒｏｘｙａｌｋａｎｏａｔｅｓ (ＰＨＡｓ)ａｒｅａｆａｍｉｌｙｏｆｉｎｔｒａｃｅｌｌｕｌａｒｂｉｏｐｏｌｙｍｅｒｓｓｙｎｔｈｅｓｉｚｅｄｂｙｍａｎｙｂａｃｔｅｒｉａａｓｃａｒｂｏｎａｎｄｅｎｅｒｇｙｓｔｏｒａｇｅｃｏｍｐｏｕｎｄｓ[1,2 ] .ＡｔｔｅｍｐｔｓｈａｖｅｂｅｅｎｍａｄｅｔｏｐｒｏｄｕｃｅｓｏｍｅｏｆｔｈｅＰＨＡｓｆｏｒａｐｐｌｉｃａｔｉｏｎａｓｂｉｏｄｅｇｒａｄａｂｌｅｐｌａｓｔｉｃｓ[3 5] .ＰＨＡｓｃａｎｂｅｂｒｏａｄｌｙｄｉｖｉｄｅｄｉｎｔｏｔｗｏｇ…     

一株高效高温菌的筛选初步鉴定和性质研究

从高温堆肥物料中通过平板培养法分离、纯化并筛选出一株高效高温菌YB16.YB16具有同时分解淀粉、蛋白质、油脂和纤维素等大分子有机物的能力.经鉴定YB16为革兰氏阳性,杆状,有芽孢,好氧, G+Cmol%为50.66%,初步鉴定YB16属于芽孢杆菌属.pH生长范围为6.0～9.0,最适生长温度为65 ℃左右.     

假单胞杆菌BC001对吡啶和喹啉的生物去除

从首钢焦化厂废水处理系统的活性污泥中分离出1株能在高浓度的吡啶(约400mg/L)和喹啉(约500mg/L)双基质条件下良好生长的细菌,经16SrDNA及生理形态特征鉴定为假单胞杆菌(Pseudomonassp.BC001),它对吡啶的去除主要通过生物吸附,而对喹啉的去除包括生物吸附和降解两个阶段。该菌能利用喹啉作为唯一的碳源和氮源代谢生长,适量的外加碳源对喹啉降解具有促进作用,经检测喹啉降解的中间产物主要为2-羟基喹啉和8-羟基香豆素,氮的主要代谢终产物为NH4 。     

耐热碱性磷酸酯酶基因的DNA序列分析

从栖热菌中克隆到产耐热碱性磷酸酯酶（FD－TAP）基因并进行了DNA序列分析,结果表明此2．0kb的片段含有一个1056bp的开放阅读框,编码501个氨基酸的蛋白质,其N端有一26个氨基酸的信号肽．在起始密码子的上游5bp处有一个5＇－GGAGGT－3＇的SD序列．基因编码区的（G＋C）％为68．7％,第3位密码子（G＋C）％为92．7％．FD－TAP的氨基酸序列与大肠杆菌等生物的碱性磷酸酯酶氨基酸序列比较,相同性为27％,相似性为38％．中央β－折叠区及与活性中心相关的氨基酸残基高度保守．表明FD－TAP具有与大肠杆菌碱性磷酸酯酶相似的结构和作用机制．在相当于大肠杆菌碱性磷酸酯酶的His370至His412两个金属离子结合部位之间,FD－TAP有一72个氨基酸的插入片段,提示该插入片段与FD－TAP的高耐热性相关．     

嗜碱芽孢杆菌NTT33β-甘露聚糖酶的特性研究

本文研究了嗜碱芽孢杆菌（ Ｂａｃｉｌｌｕｓ ａｌｋａｌｏｐｈｉｌｉｃ） Ｎ Ｔ Ｔ３３β－ 甘露聚糖酶的产生条件和酶学性质．其产酶的最佳碳源为 １％ 槐豆角,最佳氮源为 １％ 蛋白胨＋ ０．２％ 酵母膏,发酵培养３６ｈ 产酶量最高（达 ６１．３ｕ／ｍ ｌ）．甘油、葡萄糖、甘露醇等对产酶有强的阻遏作用．经分离纯化后测得纯酶的分子量为３８．９ Ｋ Ｄ,等电点 ｐ Ｉ为３．０,酶反应最适 ｐ Ｈ 为 ９．０～１０．０,最适温度为８０℃,稳定 ｐ Ｈ 为６．０～９．０,稳定温度为６０℃以下,金属离子中 Ｃｕ２＋ , Ｂａ２＋ , Ｍ ｇ２＋ , Ａｌ３＋ , Ｃｏ２＋ 对该酶有一定的激活作用,而 Ａｇ＋ , Ｈｇ２＋ , Ｍｎ２＋ 对该酶有明显抑制作用     

绿色荧光蛋白基因转化内生解淀粉芽孢杆菌的研究

通过重叠延申法将枯草芽孢杆菌的启动子PSJ2与绿色荧光蛋白基因（gfp）的ORF连接起来,构建绿色荧光蛋白表达盒,再通过Rco R I和Pxt I双酶切将表达盒连接到pUS186载体上,转化解淀粉芽孢杆菌TB2菌株,得到可发出绿色荧光工程菌,工程菌对黄瓜枯萎病菌的拮抗作用与野生菌株相当。     

Substantial increase of protein stability by multiple disulphide bonds

Disulphide bonds can significantly stabilize the native structures of proteins. The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide. In phage T4 lysozyme, a disulphide-free enzyme, engineered disulphide mutants that crosslink residues 3-97, 9-164 and 21-142 are significantly more stable than the wild-type protein. To investigate the effect of multiple-disulphide bonds on protein stability, mutants were constructed in which two or three stabilizing disulphide bridges were combined in the same protein. Reversible thermal denaturation shows that the increase in melting temperature resulting from the individual disulphide bonds is approximately additive. The triple-disulphide variant unfolds at a temperature 23.4 degrees C higher than wild-type lysozyme. The results demonstrate that a combination of disulphide bonds, each of which contributes to stability, can achieve substantial overall improvement in the stability of a protein.     

Convergent evolution of similar function in two structurally divergent enzymes

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.     

Structural snapshots along the reaction pathway of ferredoxin-thioredoxin reductase

Oxygen-evolving photosynthetic organisms regulate carbon metabolism through a light-dependent redox signalling pathway. Electrons are shuttled from photosystem I by means of ferredoxin (Fdx) to ferredoxin-thioredoxin reductase (FTR), which catalyses the two-electron-reduction of chloroplast thioredoxins (Trxs). These modify target enzyme activities by reduction, regulating carbon flow. FTR is unique in its use of a [4Fe-4S] cluster and a proximal disulphide bridge in the conversion of a light signal into a thiol signal. We determined the structures of FTR in both its one- and its two-electron-reduced intermediate states and of four complexes in the pathway, including the ternary Fdx-FTR-Trx complex. Here we show that, in the first complex (Fdx-FTR) of the pathway, the Fdx [2Fe-2S] cluster is positioned suitably for electron transfer to the FTR [4Fe-4S] centre. After the transfer of one electron, an intermediate is formed in which one sulphur atom of the FTR active site is free to attack a disulphide bridge in Trx and the other sulphur atom forms a fifth ligand for an iron atom in the FTR [4Fe-4S] centre--a unique structure in biology. Fdx then delivers a second electron that cleaves the FTR-Trx heterodisulphide bond, which occurs in the Fdx-FTR-Trx complex. In this structure, the redox centres of the three proteins are aligned to maximize the efficiency of electron transfer from the Fdx [2Fe-2S] cluster to the active-site disulphide of Trxs. These results provide a structural framework for understanding the mechanism of disulphide reduction by an iron-sulphur enzyme and describe previously unknown interaction networks for both Fdx and Trx (refs 4-6).     

耐热α-淀粉酶的筛选、基因克隆和酶学性质分析

【目的】筛选耐热α-淀粉酶并实现异源表达,同时分析其酶学性质特征。【方法】以M9培养基加上可溶性淀粉作为选择性分离培养基,从腾冲火山温泉土壤中筛选到产淀粉酶的菌株Anoxybacillus sp.GXS-3。根据淀粉酶氨基酸保守序列,设计引物进行PCR扩增,然后对目标序列进行步移扩增,获得淀粉酶基因AmyGX。将AmyGX与表达载体pQE30连接,导入大肠杆菌M15中表达,对重组酶进行分离纯化和酶学性质分析。【结果】AmyGX基因长1 515 bp,编码505个氨基酸残基,前23个氨基酸残基为信号肽序列;重组质粒pQE30-AmyGX编码的蛋白分子量为58.04kDa,对可溶性淀粉催化水解反应的最适温度为60℃,最适pH值为8.0,V_(max)、K_m值分别为0.19U/mg、3.14mg/mL,热半失活温度T_(50)~(30)值为65.2℃;Zn~(2+)、Cu~(2+)、Co~(2+)、Fe~(3+)、Ba~(2+)对该酶具有明显的抑制作用,Na~+、K~+对该酶有激活作用,Mg~(2+)、Ca~(2+)的影响则不明显。【结论】AmyGX是一种中等耐温碱性酶,在造纸、洗涤剂生产和有毒废弃物去除等方面具有潜在的应用前景。