Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Abstract Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (p I = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.     

Purification and Characterization of an Esterase Involved in Poly(vinyl alcohol) Degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45°C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K _m and V _max of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 μM, and 6.52 and 12.6 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

Cloning and Sequence Analysis of the Gene (alyII) Coding for an Alginate Lyase of Pseudomonas sp. OS-ALG-9

Pseudomonas sp. OS-ALG-9 produces several kinds of alginate-degrading enzymes both intra- and extracellularly. As a second alginate lyase of this bacterium, the gene encoding alyII has been cloned in Escherichia coli JM109 by shotgun techniques and then sequenced. The alyII gene has an open reading frame of 2141 bp encoding 713 amino acid residues with a calculated molecular mass of 79,803 Da. The deduced amino acid sequence did not show any extensive similarity with those of other known alginate lyases, however, hydrophobic cluster analysis showed that alyII belonged to class 3 of alginate lyases. The alginate lyase from E. coli harboring the alyII gene showed a single active band, which coincided with one of four major alginate lyases from the crude cell extracts of Pseudomonas sp. OS-ALG-9 on a zymogram.     

交替假单胞菌(Pseudoalteromonas sp.)DY3菌株产内切葡聚糖酶的性质研究及基因克隆

从深海样品ESO109中分离到一株具有高内切葡聚糖酶活力的细菌DY3,16SrDNA序列分析表明该菌与交替假单胞菌属(Pseudoalteromonas sp．)的Pseudoalteromonas citrea和Pseudoalteromonas elyakovii的同源性为99％。PCR扩增DY3的内切葡聚糖酶基因celX全长1479bp,编码一个492AA的蛋白质。酶的氨基酸序列分析表明CelX与Rseudoalteromonas haloplanktis的内切葡聚糖酶CelG有95％的相似性,包括一个糖基水解酶家族5的催化结构域,一个连接序列和位于C端的的CBM5结构域。对酶性质的初步研究发现,CelX的最适温度为40℃,酶的最适pH在6～7之间。     

荧光假单胞菌香兰素脱氢酶基因的克隆及表达

对荧光假单胞菌(Pseudomonas fluorescens ATCC13525)香兰素脱氢酶基因vdh进行了克隆、序列分析以及表达。PCR扩增获得了长度为1 449 bp的核苷酸序列,该序列编码含438个氨基酸,分子量约为50 ku的多肽。序列分析表明该基因与GenBank提供的部分已知vdh基因具有高度的同源性。该基因在大肠杆菌DH5α中能高效表达,而在野生型P.fluorescens ATCC13525中本身并不表达出功能。Vdh基因表达产物香兰素脱氢酶(Vdh)在细胞中主要以可溶性蛋白的形式存在。同时研究表明诱导剂IPTG对vdh基因在大肠杆菌中的表达并不起作用。     

Characterization of Two Isozymes of Coniferyl Alcohol Dehydrogenase from Streptomyces sp. NL15-2K

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.     

Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

L—乳酸脱氢酶基因克隆及功能分析

构建了一株产D,L-乳酸的乳杆菌(Lactobaeillus sp．)MD—1的基因库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJ144作为宿主,通过互补筛选分离克隆到乳酸脱氢酶基因(ldhL)。核酸序列分析表明,该基因以ATG为起始密码子编码316个氨基酸残基组成的蛋白质,预测的分子量为33．84kD;5′端存在典型的启动子结构,3′端的终止子是不依赖于ρ因子的转录终止子。ldhL编码的蛋白质有3个保守区域,其中Gly13～Asp50保守区域是NADH的结合位点,Asp73～Ile100和Asn123～Arg154保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低,核苷酸序列相似性最高仅为64．1％,氨基酸序列相似性最高仅为68．9％,是新的L—乳酸脱氢酶基因。     

耐热芽孢杆菌E2菌株纤维素酶基因克隆的研究

用质粒ｐＢＲ３２２作载体,大肠杆菌Ｅ．ｃｏｌｉ ＤＨ５αＦ’作受体,克隆到耐热孢杆菌Ｅ２菌株的羧甲基纤维素酶基因。重组质粒ＰＢＧ３从产生ＣＭＣａｓｅ的转化子中分离得到。克隆到的ＣＭＣａｓｅ基因位于４．０ｋｂＨｉｎｄⅠⅠⅠ片段上。功能状态ＣＭＣａｓｅ基因被亚克隆到２．４ｋｂＤＮＡ片段上。     

南极嗜冷假单胞菌7197 S- 腺苷同型半胱氨酸水解酶 ( SAHH) 基因的克隆与序列分析

从南极普利兹湾深海沉积物中筛选到一株耐冷菌株7197,其16S rDNA序列分析表明该菌株属于假单胞菌属（Pseudomonas）。作者通过设计引物,从该菌的全基因组DNA中克隆到编码S-腺苷-L-高半胱氨酸（SAHH）的完整ORF,全长为1424bp。使用DNAMAN（5,1）软件对全长ORF为1424bp的SAHH基因进行分析,SAHH基因编码一个由474AA残基组成、分子量预计为52523Da的SAHH蛋白质,与Psychrobacter sp．273—4的SAHH有96．84％的相似性;与Acinetobacter sp．ADP1的SAHH有79％的相似性;与Pseudomonas fluorescens Pf-5的SAHH有75％的相似性。     

重组酵母菌乙醇脱氢酶基因的克隆及序列分析

目的:克隆产麻黄碱重组酵母菌乙醇脱氢酶基因片段,并对其进行序列分析,为研究该基因在重组酵母中与麻黄碱生物合成途径的关系提供参考.方法:根据一段利用抑制差减杂交技术获得的来源于重组酵母乙醇脱氢酶基因片段,采用RACE的方法扩增Adh基因,使用分子生物学软件对该基因进行生物信息学分析.结果:获得一段大小为1 245 bp的基因片段,编码375个氨基酸,含有两个催化域和两个锌结合域,与来源于Gandida boidinii ADH3基因的同源性为85％.结论:克隆的基因为乙醇脱氢酶基因,并在GenBank注册,登录号为JF293468.     

D-乳酸脱氢酶基因克隆及其表达

构建了一株产D ,L 乳酸的乳杆菌 (Lactobacillussp .)MD 1的基因文库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的EscherichiacoliFMJ1 4 4作为宿主 ,在厌氧条件下通过互补筛选获得乳酸脱氢酶基因 (ldh) ,非变性聚丙烯酰胺凝胶电泳 (Native PAGE)检测证明其阳性克隆表现出D 乳酸脱氢酶 (D LDH)活性。核酸序列分析表明 ,ldhD的ORF编码 331个氨基酸残基组成的蛋白质有两个保守区域 ,其中V1 47～D1 76 区是NADH的结合位点 ,R77～E1 0 7区据报道是酶的活性部位。该菌株D LDH和D羟基异己酸脱氢酶 (D HicDH)属于NADH依赖性脱氢酶家族 ,ldhD和其他乳杆菌属的ldhD及D HicDH基因和编码的氨基酸序列相似性较低 ,核酸序列相似性最高达 4 9 33% ,氨基酸序列相同性最高为 4 2 % ,是一个新的D 乳酸脱氢酶基因     

Immobilization of hog pancreas lipase in macroporous poly(vinyl alcohol)-cryogel carrier for the biocatalysis in water-poor media

Hog pancreas lipase was covalently attached to the beads of poly(vinyl alcohol)-cryogel – a macroporous hydrogel prepared by means of freeze-thaw technique. The immobilized biocatalyst thus obtained was examined in the reaction of enantioselective hydrolysis of the ethyl ester of N-benzylidene derivative of DL-phehylalanine in the medium of acetonitrile (contained 5 vol.% of water without any buffers). Eighty-three %-enantiomeric excess of the l-amino acid was reached after 144 h. Virtually the same result was obtained in the repeated use of the same immobilized biocatalyst after its 6-months-storing in a refrigerator.     

Research on the Long Time Swelling Properties of Poly (vinyl alcohol)/Hydroxylapatite Composite Hydrogel

Poly(vinyl alcohol)/hydroxylapatite(PVA/HA)composite hydrogel was prepared by repeated freezing and thawing.Thewater loss properties of the resultant hydrogel were investigated by using optical microscope.Long time immersion tests ofPVA/HA composite hydrogel were carried out in the diluted calf serum solution to study the change laws of swelling propertieswith the freezing-thawing cycles and HA content.The micro-morphologies of PVA/HA composite hydrogel after long timeimmersion were observed by means of the high-accuracy 3D profiler.The results show that the swelling process of PVA/HAcomposite hydrogel is the converse process of its water loss.Long time swelling ratio curves of PVA/HA composite hydrogel inthe calf serum solution are manifested as four stages of quick increase,decrease,slow decrease and stable balance,and itsequilibrium swelling ratio decreases with the increase of freezing-thawing cycles and HA content.It is revealed that the networkstructure of the composite hydrogel immersed for a long period is significantly improved with the increase of HA content.Perfect network structures of PVA/HA composite hydrogel as well as full and equilibrium tissues after swelling equilibrium areobtained when the HA content is 3% and the number of freezing-thawing cycles is 7.     

盐穗木甜菜碱醛脱氢酶基因（BADH）的克隆及其在盐胁迫下的表达分析

根据我们实验室已发表的植物甜菜碱醛脱氢酶基因（BADH）的同源保守区设计引物,通过RT—PCR扩增获得了由1503个核苷酸组成的盐穗木BADH基因开放阅读框,推测该基因编码500个氨基酸,分子量约为54．49kDa的多肽。推测的盐穗木BADH氨基酸序列中包含一段甜菜碱醛脱氢酶中高度保守的十肽序列（VTLELGGKSP）以及与酶功能有关的半胱氨酸残基（Cys）。序列比对结果显示,盐穗木BADH与盐地碱蓬、中亚滨藜、盐爪爪以及菠菜等的核苷酸序列同源性在81％以上,与水稻的同源性也达到68％。半定量RT—PCR分析结果表明,盐穗木BADH基因的表达受盐胁迫诱导,推测BADH可能与盐穗木具有较强的耐盐能力有关。     

假单胞菌Na+/H+逆向转运蛋白基因nhaA的克隆与鉴定

根据3种生物的Na^ ／H^ 逆向转运蛋白基因(nhaA)的两端序列设计引物,利用PCR从假单胞菌(Pseudomonas sp．cn4902)中克隆得到一结构基因。该基因长1089bp,编码362个氨基酸,与E．coil K12的nhaA基因的同源性高达97．0％。将该结构基因与pBV220构建成重组载体pBVA。SDS—PAGE电泳表明：含pBVA的转化子产生较高浓度的分子量约为41kD的蛋白,与预期相符。在含NaCl 1．0mol／L的培养基中生长达到平衡期时,转化子的菌浓度约是对照的2．3倍。经原子吸收光谱测定,转化子细胞质中Na^ 浓度仅为对照菌的60．4％。SDS—PAGE电泳表明该基因的表达蛋白位于细胞膜(壁)上。提纯外源基因表达蛋白并对其N端8个氨基酸进行测序,与nhaA基因推测的氨基酸序列完全相符。这些实验证实,克隆得到的基因是假单胞菌的nhaA基因。该基因已经在GenBank登记,收录号为AY643494。     

链霉菌UDP-葡萄糖脱氢酸编码基因Ste6的克隆表达和性质研究

链霉菌139能够产生一种全新的胞外多糖——依博素（139A）,该多糖体内具有显著抗类风湿性关节炎活性。其生物合成基因簇（GenBank Accession Number：AYl31229）已被鉴定约31．3kb,包含22个开放阅读框（ste1—ste22）。以pET-30a为载体,克隆并在大肠杆菌BL21（DE3）中进行了ste6基因的表达,对该基因的克隆、表达与性质进行了研究。亲和层析法证实,纯化后重组蛋白具有催化UDP-葡萄糖脱氢变成UDP-葡萄糖醛酸的活性。这表明ste6编码产物是葡萄糖脱氢酶。为了证实ste6基因与依博素生物合成的关系,采用单交换基因破坏策略构建了ste6基因阻断突变株。结果初步显示ste6和依博素生物合成相关。     

酿酒酵母by1.1b产D-(-)-扁桃酸脱氢酶的发酵条件优化

对一株产D-(-)-扁桃酸对映选择性脱氢酶的酿酒酵母菌(Saccharomyces cerevisiae sp. strain by1.1b)发酵产酶条件进行了优化。研究各种碳源、氮源及无机盐对产酶的影响, 应用正交试验优化发酵培养基组成, 结果为: 蛋白胨 60 g/L, 麦芽糖 30 g/L, MgSO4 0.5 g/L, ZnSO4 0.01 g/L, KCl 1.0 g/L。优化后酶产量提高了7.9倍(由2.56 U/mL增至20.21 U/mL)。摇瓶培养最佳条件为: 装液量40 %, 发酵pH 6.5, 接种量10 %, 发酵温度30 ℃。考察了细胞生长及产酶的时间进程, 最佳培养时间为25 h。     

酿酒酵母by1.1b产D-(-)-扁桃酸脱氢酶的发酵条件优化

对一株产D-(-)-扁桃酸对映选择性脱氢酶的酿酒酵母菌(Saccharomyces cerevisiae sp.strain by1.1b)发酵产酶条件进行了优化.研究各种碳源、氮源及无机盐对产酶的影响,应用正交试验优化发酵培养基组成,结果为:蛋白胨60 g/L,麦芽糖30 g/L,MgSO4 0.5 g/L,ZnSO4 0.01 g/L,KCl 1.0 g/L.优化后酶产量提高了7.9倍(由2.56 U/mL增至20.21 U/mL).摇瓶培养最佳条件为:装液量40%,发酵pH 6.5,接种量10%,发酵温度30℃.考察了细胞生长及产酶的时间进程,最佳培养时间为25 h.     

牛肝辅酶Ⅱ依赖性视黄醇脱氢酶cDNA的克隆及组织表达

迄今为止的研究证明 ,维生素A亦称视黄醇(retinol)的生理功能是通过其两步氧化代谢产物视黄醛与视黄酸 (亦称维甲酸 )来完成的 .视黄醛通过其光学异构体 1 1 顺式视黄醛与视觉细胞内的视蛋白 (opsin)结合组成视色素 .感光时 ,1 1 顺式视黄醛转变成全反式视黄醛从视蛋白脱落 ,这一过程同时传导到大脑产生视觉[1 ] .全反式维甲酸 (all transretinoicacid)则通过与其在核内受体 (RARα ,β ,γ)结合调节基因的转录来发挥其许多重要的生理功能 ,包括正常胚胎的发育 ,形态、神经系统的形成 ,成体动物的生长、发育、繁殖等 ,并通过调解组织及…