摩氏摩根菌产酯酶条件优化及在普瑞巴林手性中间体合成中的应用

为建立廉价、高效的普瑞巴林关键手性中间体生产工艺,经单因素优化和正交试验,确定了摩氏摩根菌CCTCC M 2011175最佳产酯酶培养基,组成(g/L)为葡萄糖15.0,牛肉膏7.0,Na2HPO41.0,Fe2(SO4)30.1,吐温-8010.0。优化后酯酶比酶活达到1 071.0 U/L,为出发培养基的2.5倍。以培养基优化后获得的摩氏摩根菌菌体为催化剂,立体选择性拆分外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯水解,转化率达到45%,对映体过量值(e.e.)大于94%,为酶法生产普瑞巴林关键手性中间体奠定了良好基础。     

黄喉拟水龟摩氏摩根菌的分离鉴定及药敏试验

<正>黄喉拟水龟(Mauremys mutica),俗称石龟、石金钱龟、黄板龟等,为龟科拟水龟属,主要分布于中国江苏、安徽、福建以及越南、日本等。黄喉拟水龟具有食用、药用和观赏价值,近年来野生资源日趋减少,人工养殖发展迅速。细菌性病原感染是黄喉拟水龟养殖过程中常见疾病,目前已报道黏质沙雷氏菌(Serratia marcescens)[1]、松鼠葡萄球菌(Staphylococcus sciuri)[2]、摩氏摩根菌(Morganella morganii)[3]、嗜麦芽窄食单胞菌(Stenotrophmomas     

卡瑞苯西思伯克霍尔德氏菌卤乙酸脱卤酶的分离纯化及性质

从云南西北部土样中分离到一株卡瑞苯西思伯克霍尔德氏菌(Burkholderia caribensis),它在氯乙酸培养基中能产生较高活性的卤乙酸脱卤酶。经硫酸铵盐析、DEAE SephadexA50柱层析、羟基磷灰石柱层析、Sephadex G200 凝胶过滤后,获得电泳纯酶。用SDSPAGE测定酶分子量为46kD。水解氯乙酸的Km值为3.7×10-3mol/L。酶反应的最适温度为40℃,最适pH值为9.5。金属离子及CN-、EDTA对该酶有不同程度的影响,Hg2+和CN-则对该酶有强烈的抑制作用。     

酯酶产生菌的分离与酶学性质研究

从餐馆附近下水道收集到的土壤中分离获得6株酯酶产生菌,其中S2菌株活性最高,从其形态特征、生理生化试验以及16S rRNA序列分析等方面,初步鉴定S2菌株为假单胞属(Pseudomonas sp.)。对该菌株的部分酶学性质进行了研究,发现该菌株所产的酯酶最适反应温度为40℃,最适反应pH为8.0;且该酯酶在温度为60℃以下和pH 7.0～10.0具有良好的稳定性。     

粗毛栓菌木聚糖酶的纯化及性质

以麦草粉为基质培养粗毛栓菌Trametes gallica,浸提固态培养物得浸提液,后经超滤浓缩、硫酸铵盐析、Phenyl-Sepharose CL-4B疏水层析、DEAE Sepharose fast flow阴离子交换层析和Sephadex G-150分子筛层析等分离与纯化步骤,获得部分纯化的木聚糖酶,其回收率和纯化倍数分别为1.45%和15.6。进一步经活性-PAGE回收,获得三种SDS-PAGE电泳纯级的木聚糖酶同工酶组分:XⅠ、XⅡ和XⅢ(按等电点从大到小排列)。三种组分分子量均约为19.0kDa;等电点分别为:5.6、4.7和4.0;含糖量分别为:0.25%、0.63%和3.4%;XⅠ既能降解木聚糖,又能降解纤维素;XⅡ的最适作用pH值为5.0,最适作用温度45℃;Mg2+、Fe2+对XⅡ有激活作用;Mn2+和Co2+有抑制作用;测得XⅡ的Km值为0.75mg/mL,Vmax为5,000mmoL/min·mg。     

摩氏摩根菌耐药性和超广谱β-内酰胺酶检测

目的了解摩根摩根菌临床分离株对抗菌药物的体外敏感性和临床分布,研究摩氏摩根菌超广谱β-内酰胺酶基因类型分布。方法用K—B法对67株摩氏摩根菌进行药物敏感性检测;PCR法检测超广谱β-内酰胺酶基因,并对PCR结果进行测序分析。结果摩氏摩根菌株对氨苄西林、头孢唑林、头孢呋辛、氨苄西林／舒巴坦、庆大霉素、呋喃妥因均已产生较高的耐药率;而对环丙沙星、左旋氧氟沙星和第三代头孢菌素的耐药率保持在较低水平;对亚胺培南、美洛培南、头孢西丁100％敏感。基因型分析显示有5株携带CTX-M基因,其中3株摩根摩根菌基因型为CTX—M-14型,2株为CTX—M-15型。结论摩氏摩根菌ESBLs检出率仍较低;5株产超广谱β-内酰胺酶摩氏摩根菌对多种抗菌药物耐药,其基因型为CTX—M-15或者CTX—M-14;需加强对不常见肠杆菌科细菌的耐药监测。     

毛栓菌漆酶的分离纯化及酶学性质研究

对毛栓菌产漆酶的分离、纯化及酶学性质进行研究。粗酶液经硫酸铵盐析、透析、DEAE-Sepharose柱层析,得到2种漆酶同工酶LacA和LacB。LacA和LacB回收率分别为17.1%和2.74%。SDS-PAGE电泳测得2漆酶的分子量分别为54.6 ku和7.7 ku;LacA和LacB最适作用温度分别为50℃和60℃;最适反应pH值分别为4.5和4.0;Cu2＋、Mg2＋对LacA有激活作用,对LacB影响不大;Ag＋对LacA和LacB表现为完全抑制;Fe3＋对LacA和LacB有一定的抑制作用;Ca2＋、Mn2＋、K＋、Na＋、Zn2＋对LacA和LacB影响不大。DTT、EDTA、DMSO、SDS对酶均有不同程度的抑制作用,且随其浓度的升高抑制作用增强。     

克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究

以克雷伯氏菌基因组DNA为模板,扩增得到编码甘油脱氢酶（GDH）的基因dhaD,将其克隆到大肠杆菌表达载体pET-28a(+)上,在E.coliBL21（DE3）中诱导表达,利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni柱亲和层析法纯化表达具有活性的甘油脱氢酶(GDH),纯化后比酶活达到156U/mg,纯化倍数达4.6倍,回收率为67.4%。并初步研究了该酶的酶学性质,酶反应的最适pH为11.0,在pH7.0～12.0范围内稳定;酶反应的最适温度为30℃,稳定范围为25～45℃; 酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49 μmol/(mL·min)。     

昆虫来源的几丁质酶的分离纯化及酶学性质

几丁质酶在真菌和昆虫的生理和发育过程中起着关键作用,该酶本身及其酶抑制剂是获取生物农药的重要途径。本研究从蚕蛹体内提取几丁质粗酶,经硫酸铵分级沉淀和Sephadex G-150分离得到几丁质酶。用SDS-PAGE测得该酶的分子量为88kDa。水解胶体几丁质的Km值为22.3μmol/L。酶反应的最适温度为45℃,最适pH值为6.0,金属离子和有机试剂对几丁质酶活性都有影响,其中高浓度的Mn2+对酶有较强的激活作用,而Cu2+、SDS则有较强的抑制作用。研究结果为基于几丁质酶的生物农药筛选研究奠定了基础。     

M-26碱性木聚糖酶的分离纯化及酶学性质研究

从Bacillus pumilus M-26发酵液中分离纯化碱性木聚糖酶,进行酶学性质研究,同时制备工业用碱性木聚糖酶制剂。首先将M-26发酵液进行硫酸铵盐析,制备工业用碱性木聚糖酶干品;然后进行sephadexG-25层析脱盐和cellulose DE-52层析得以纯化。硫酸铵的饱和度50%,酶制剂的酶活可达9 000 IU/g,收率为85%;分离纯化使酶的比活为126.32 IU/mg蛋白,纯化倍数为19.89,酶的回收率12.83%;分子量约为20 ku;M-26碱性木聚糖酶的最适温度和pH分别是55℃和pH 8.0,具有一定的耐碱性;该酶无纤维素酶活性,Fe2＋对其有激活作用;Mn2＋、Zn2＋、Fe3＋、Cu2＋对其具有抑制作用。短小芽胞杆菌M-26碱性木聚糖酶具有纸浆生物漂白应用前景。     

(3R)-2-羧乙基-3-氰基-5-甲基己酸乙酯水解酶产生菌的筛选与产酶条件优化

以2-羧乙基-3-氰基-5-甲基己酸乙酯为唯一碳源,采用手性气相法检测,筛选到一株能产对映选择性水解酶的假单胞菌Pseudomonas CGMCC No.4184.该菌产的水解酶能优先水解R型底物产生(3R)-2-羧乙基-3-氰基-5-甲基己酸,产物对映体过量值达到90%以上.对菌株Pseudomonas CGMCC ...     

来自桔青霉的阿魏酸酯酶的分离纯化、理化性质

【目的】从桔青霉的发酵液中分离纯化了胞外阿魏酸酯酶(PcFAE)并进行了酶学性质的研究,初步探讨了PcFAE对麦糟的酶解作用。【方法】利用(NH4)2SO4沉淀、DEAE-Sepharose Fast Flow离子交换层析、Phenyl Sepharose6Fast Flow疏水层析纯化得到电泳纯的阿魏酸酯酶。【结果】从该菌株的发酵液中获得一阿魏酸酯酶,该酶亚基分子量约为31kDa,全酶分子量约为58kDa。其最适pH为6.0,最适温度为45℃-65℃,在pH5.0-6.0及25℃-55℃之间,酶保持了较好的稳定性。Mg2+、Fe2+、Mn2+、Ca2+和Na+对酶活有一定的促进作用,Zn2+对PcFAE酶活有一定的抑制作用,而Cu2+、亮抑肽素、抑肽酶有显著的抑制作用,Hg2+、苯甲基磺酰氟几乎完全抑制了酶活。EDTA对PcFAE活性无明显影响。PcFAE的kcat/Km对香豆酸甲酯、芥子酸甲酯、阿魏酸甲酯、咖啡酸甲酯的值分别为823、416、103、0,PcFAE对MpCA的催化效率最高。PcFAE作用于麦糟,当5U PcFAE/g麦糟时,其阿魏酸的释放量为7.2%。【结论】获得了一阿魏酸酯酶,其理化性质与至今报道的阿魏酸酯酶有所不同,为阿魏酸酯酶的开发提供了重要的实验依据。     

Purification of a stereospecific 2-ketoreductase from Gluconobacter oxydans

The 2-ketoreductase from Gluconobacter oxydans (SC 13851) catalyzes the reduction of 2-pentanone to (S)-(+)-2-pentanol. The 2-ketoreductase was purified 295-fold to homogeneity from G. oxydans cell extracts. The purified 2-ketoreductase had a molecular mass of 29 kDa with a specific activity of 17.7 U/mg. (S)-(+)-2-pentanol was prepared on a pilot scale (3.2 kg of 2-pentanone input) using Triton X-100-treated G. oxydans cells. After 46 h, 1.06 kg (32.3 M%) of (S)-(+)-2-pentanol of >99% enantiomeric excess (ee) was produced. Journal of Industrial Microbiology & Biotechnology (2000) 25, 171–175. Received 01 May 2000/ Accepted in revised form 28 June 2000     

Morganella morganii J-8羰基不对称还原酶的分离纯化及性质研究

由本实验室筛选得到的摩尔摩根氏菌J-8菌株可将底物1-苯基-2-甲氨基丙酮专一性地转化为d-伪麻黄碱。以M.morganiiJ-8为出发菌株,菌体超声破碎后,经硫酸铵沉淀、Phenyl Superose疏水柱层析、DEAD阴离子柱层析和非变性凝胶电泳四步纯化获得电泳纯羰基不对称还原酶。亚基分子质量为42.5kD,高效液相色谱分析酶的分子质量约为84.1kD,初步认为该酶为二聚体蛋白。对所得到的部分纯化酶的酶学性质做了初步研究,纯酶进行基质辅助激光解析电离-飞行质谱分析,比对结果显示为与亮氨酸脱氢酶蛋白有很高相似性。     

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni²⁺-NTA, and POROS Q columns obtained approximately 100 mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M_r = 144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α (M_r 77,000) and β (M_r 70,000) sGC subunits. It showed an A₄₃₀/A₂₈₀ = 1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k_cat/K_m = 1.7 × 10⁻³ s⁻¹ μM⁻¹ that increased to 5.8 × 10⁻¹ s⁻¹ μM⁻¹ upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.     

Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile

The cellulolytic system of the thermophilic fungus Sporotrichum thermophile contains a recently discovered esterase that may hydrolyze the ester linkage between the 4- O -methyl- d -glucuronic acid of glucuronoxylan and lignin alcohols. The glucuronoyl esterase named St GE1 was purified to homogeneity with a molecular mass of M _r 58 kDa and pI 6.7. The enzyme activity was optimal at pH 6.0 and 60 °C. The esterase displayed a narrow pH range stability at pH 8.0 and retained 50% of its activity after 430 and 286 min at 50 and 55 °C, respectively. The enzyme was active on substrates containing glucuronic acid methyl ester, showing a lower catalytic efficiency on 4-nitrophenyl 2- O -(methyl-4- O -methyl-α- d -glucopyranosyluronate)-β- d -xylopyranoside than its mesophilic counterparts reported in the literature, which is typical of thermophilic enzymes. St GE1 was proved to be a modular enzyme containing a noncatalytic carbohydrate-binding module. LC-MS/MS analysis provided peptide mass and sequence information that facilitated the identification and classification of St GE1 as a family 15 glucuronoyl esterase that showed the highest homology with the hypothetical glucuronoyl esterase CHGG_10774 of Chaetomium globosum CBS 148.51. This work represents the first example of the purification and identification of a thermophilic glucuronoyl esterase from S. thermophile .     

Purification and characterization of 2S albumin from Nelumbo nucifera

The 2S albumins are a group of seed storage proteins that have recently attracted considerable attention in the field of allergen science due to their allergenic potential. A new 2S albumin from seeds of Nelumbo nucifera (Nn-2S alb) was purified to electrophoretic homogeneity by the combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The protein has a molecular mass of about 12 kDa estimated by SDS–PAGE, in good agreement with 12.5 ± 0.01 kDa determined by ESI–MS. Circular dichroism data showed that protein contained about 66% α-helices as estimated by K2D3, indicating that the protein was predominantly helical. The sedimentation coefficient (s°_20,w) of the predicted model was 1.72 ± 0.21 S. The predicted 3-dimensional structure of the Nn-2S alb revealed that the protein has a region of 12 amino acids which largely corresponds to the conserved immuno-dominant epitope of 2S allergens.     

Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis.

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s(-1)), K(M(JH-III)) (84 +/- 12 nM) of nearly-purified enzyme, and k(cat)/K(M) (1.67 x 10(7) s(-1) M(-1)) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifluoro-2-propanone; I(50) = 10(-7) M) and by DFP (diisopropyl fluorophosphate; I(50) = 10(-7) M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed alpha-napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 microM) K(M) for long-chain alpha-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect.