一株产纤溶酶菌株的分离、鉴定及其酶活特征的初步研究

[目的]分离筛选并鉴定产纤溶酶的菌株.[方法]采用血粉培养基富集,琼脂糖-纤维蛋白平板筛选,从自然界中分离筛选出一株产纤溶活性物质的菌株.通过形态学特征、生理生化特征研究,并结合16S rRNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类.[结果]从自然界分离筛到一株产纤溶酶的菌株EF608,经鉴定该菌株为粪肠球菌(Enterococcus faecalis). SDS-PAGE和纤维蛋白自显影表明该纤溶酶的分子量为37 kD,最适反应温度和pH分别为35℃和7.5,EDTA能完全抑制其纤溶活性,而PMSF对其活性无抑制作用.菌株EF608发酵液不仅可以直接水解纤维蛋白,而且具有体外溶栓的作用,对血红细胞没有溶解作用.[结论]筛选到一株具有纤溶活性的粪肠球菌——EF608,为获取新型纤溶酶提供了一种的新的菌源.     

假交替单胞菌LJ1菌株产褐藻胶裂解酶的培养条件优化及酶学性质

[目的]为了优化Lj1菌株的培养条件使之产生高活性的胞外褐藻胶裂解酶.[方法]通过富集培养技术从海带筛选到一株褐藻胶裂解酶产生菌Lj1,依据表型特征、脂肪酸组成分析及16S rRNA基因序列分析对该菌株进行鉴定.通过单因子和正交试验对Lj1菌株产胞外褐藻胶裂解酶的培养条件进行了优化.[结果]Lj1菌株属于假交替单胞菌属(Pseudoalteromonas).该菌株产酶的最佳培养基组成为:褐藻胶3g/L、(NH4)2SO43 g/L、NaCl 20 g/L、KH2PO4 0.1 g/L、CaCl2 0.1 g/L;最佳培养条件为:250 mL三角烧瓶中装液量25 mL、接种量3%、摇瓶转速150 r/min、pH7.5、培养温度为28℃、培养时间为24 h.LJl菌株所产褐藻胶裂解酶的最适温度为40℃,最适pH7.6,最适NaCl浓度为0.3 mol/L.1 mol/1.金属离子Mg2+对酶活力有明显的促进作用,而C02+和Zn2+对酶活力有较强的抑制作用.[结论]LJ1菌株是Pseudoalteromonas新的胞外褐藻胶裂解酶产生菌,在最佳培养条件下,该菌株的酶活力提高了66%.     

一株产耐热右旋糖酐酶真菌的筛选、鉴定及其酶学性质

【目的】从土壤中筛选得到1株产耐热右旋糖酐酶的真菌。【方法】采用营养缺陷型培养基,结合稀释涂布法和平板透明圈法分离筛选出产耐热右旋糖酐酶的菌株。通过观察菌落形态、菌体形态和培养特征,结合ITS r DNA序列分析对菌株进行鉴定。研究菌株所产右旋糖酐酶的酶学性质。【结果】通过筛选得到1株产耐热右旋糖酐酶的菌株DG001,经鉴定为淡紫拟青霉(Paecilomyces lilacinus)。菌株DG001所产右旋糖酐酶的最佳催化条件为55°C,p H 5.0;最适底物为5%Dextran T70。酶在60°C以下和p H 4.0–7.0之间稳定。urea、Mn~(2+)和Mg~(2+)对酶活均有促进作用,低浓度的Mn~(2+)和urea可使酶活分别提高到116.91%和110.14%,而Cu~(2+)则对其有强烈抑制作用。该酶水解右旋糖酐T2000的产物主要是异麦芽糖和异麦芽三糖,被确定为内切右旋糖酐酶。酶对底物的亲和性随底物分子量的增加而增强。【结论】成功筛选获得1株产耐热右旋糖酐酶的菌株DG001,所产酶在较宽温度范围内具有较高活力,热稳定性好。该酶在制糖工业及不同分子量右旋糖酐的制备中具有很好的应用前景。     

一株产褐藻胶裂解酶的需钠弧菌筛选及酶解产物分析

[背景]褐藻胶裂解酶种类丰富、降解机制多样,是高效环保降解褐藻胶、制备褐藻寡糖的工具酶,成为褐藻植物高值化开发利用的研究热点.[目的]从海泥中筛选获得褐藻胶裂解酶高效产酶菌株,确定菌株发酵产酶最优条件,鉴定和分析酶降解产物,进而解析该酶的降解特性.[方法]以褐藻胶为唯一碳源,从海带养殖场附近海泥中筛选菌株,通过形态学观...     

一株产漆酶细菌的分离鉴定及酶学性质研究

[目的]分离获得产漆酶的细菌菌株,研究漆酶的酶学性质并应用于染料脱色.[方法]利用含铜的富集培养基筛选产漆酶细菌;通过形态特征、生理生化试验及16SrDNA序列分析等方法进行鉴定;以丁香醛连氮为底物测定漆酶的酶学性质;通过测定染料在最大吸收波长下吸光值的变化评价漆酶对染料的脱色效果.[结果]从森林土壤中筛选到一株漆酶高产菌株LS05,初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);菌株LS05的芽孢漆酶以丁香醛连氮为底物的最适pH为6.6,最适温度为70℃;该酶具有较好的稳定性,经70℃处理10h或在pH 9.0条件下放置10d后可保留活性.对抑制剂SDS和EDTA具有一定的抗性,在碱性条件下可有效脱色不同的工业染料,RB亮蓝、活性黑和靛红1h内的脱色率达93％以上.[结论]Bacillus amyloliquefaciens LS05的芽孢漆酶在高温和碱性条件下稳定性强,相对于真菌漆酶具有更好的工业应用特性,可有效用于工业染料废水的处理.     

右旋糖酐蔗糖酶工程菌株的构建及其培养条件的研究

[目的]右旋糖酐蔗糖酶是一种以蔗糖为底物,催化转移D-葡萄糖基生成α-葡聚糖或低聚糖的葡萄糖基转移酶.[方法]利用PCR扩增技术,将已获得的右旋糖酐蔗糖酶基因dexYG亚克隆到表达载体PET28a( )上,转化E.coli BL21(DE3),经过卡那霉素抗性筛选和酶切验证后,得到右旋糖酐蔗糖酶工程菌株BL21(DE3)/pET28-dexYG.[结果]经IPTG诱导该基因在E.coli BL21(DE3)中能有效表达,在诱导过程中菌体生长受到抑制.通过对培养时间、IPTG浓度、培养温度、菌浓(OD600)和pH值等产酶因素的优化考察,得到最佳培养条件为:培养时间5h、IPTG浓度0.5mmol/L、25℃、OD600值1.0和pH6.0.酶活力由最初的5.39U/mL提高到35.62U/mL,其中pH值对产酶活力影响最大,在pH6.0时的最高产酶活力是LB原始pH条件下最高酶活的3.5倍,并且pH值也是导致在诱导后期酶活迅速下降的主要原因之一.[结论]酶的表达和酶活的研究结果表明,构建的工程菌株能够异源高效表达右旋糖酐蔗糖酶,并且表现出较高的酶活力.     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

高浓度氯苯优势降解菌的筛选及其降解酶的纯化

[目的]分离纯化出一株高浓度氯苯优势降解菌株,对其所产氯苯降解酶进行分离与纯化,为该菌株及其氯苯降解酶的研究提供理论参考.[方法]利用梯度富集培养技术和无菌滤纸片平板法分离菌株,通过形态特征及16S rRNA基因序列分析初步鉴定菌株,用气相色谱法测定培养液中氯苯浓度,以单位细胞氯苯降解率评价菌株对氯苯的降解能力,以氯苯降解率表示氯苯降解酶的活性.取纯化菌株的发酵酶液制备粗酶液,经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后,进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量.[结果]从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13,该菌株在以2000 mg/L氯苯为唯一碳源的无机盐培养基中仍能正常生长,其单位细胞氯苯降解率可达1.37 ×10-10.扫描电镜观察到该菌株细胞大小约为2.3 ×0.8μm,长有数根端生鞭毛.16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5％.所纯化的氯苯降解酶为胞外酶,带正电荷,其分子大小约为57 kDa.整个纯化过程中酶纯化倍数化达8.0倍,酶活回收率达52.51％,酶量回收率达6.57％.纯化后的氯苯降解酶在30℃-55℃和pH在6.0-8.0之间都保持较高的酶活性,其最适反应温度和pH分别在40℃和pH8.0左右.[结论]所分离的氯苯优势降解菌属于Lysinibacillus属菌株,该菌株能有效降解高浓度(500-2000 mg/L)氯苯废水,通过逐级分离纯化,可获得氯苯降解酶纯酶,纯化指标符合分离纯化基本规律,纯化效果较为理想.     

天山1号冰川底部沉积层产β-半乳糖苷酶低温菌株的系统发育分析及生理多样性

[目的]通过对天山1号冰川底部沉积层冻土中细菌的分离和产β-半乳糖苷酶低温菌株的筛选,了解天山冻土微生物的物种多样性,并对产β-半乳糖苷酶低温菌株的系统发育和生理多样性进行分析.[方法]以乳糖为主要碳源,X-Gal为显色剂,分离筛选出产低温β-半乳糖苷酶菌株.对细菌常规生理生化实验、最适生长温度、耐盐性、药物敏感性进行测定.根据16S rRNA基因序列初步确定产β-半乳糖苷酶低温菌种的系统进化地位,并采用BOX-PCR指纹图谱技术对16S rRNA基因高度同源性的菌株进一步区分.[结果]分离到90株可培养低温菌中25株可产β-半乳糖苷酶,其中76％为革兰氏阳性菌.依据生长温度,产酶菌株80％为嗜冷菌,20％为耐冷菌.在系统发育上,产酶菌株隶属于4个类群,其中肠球菌属(Enterococcus)占26％,短波单胞菌属(Brevundimonas)占22％,假单胞菌属(Pseudomonas)占13％.[结论]天山1号冰川底部沉积层冻土中产β-半乳糖苷酶的低温细菌具有比较丰富的物种和生理多样性.     

重组大肠杆菌右旋糖酐蔗糖酶的纯化及其性质

[目的]研究工程菌E.coli BL21(DE3)/pET28-dexYG产右旋糖酐蔗糖酶的纯化和酶学性质.[方法]工程菌经过IPTG诱导后生产含His-tag融合蛋白的右旋糖酐蔗糖酶,通过硫酸铵沉淀、Ni-NTA亲和层析纯化,得到纯度较高的酶蛋白,并对纯酶进行了酶学性质及动力学研究.[结果]经过SDS-PAGE测得该酶的分子量约为170 kDa,与理论推测值基本相同.以蔗糖为底物,酶促反应的最适温度为25～30℃,最适pH值为5.4,动力学常数Km值为10.43 mmol/L;酶活在pH 5.0～8.0较为稳定,在室温(25 ℃)保藏4天仍有59%的酶活力,4℃保存7周酶活力仅下降一半,但在35℃以上失活很快;Ca2 对催化作用有较大的促进,Mg2 有微弱的促进作用,K 对催化反应无影响,Cu2 的抑制作用最强.其他试剂对重组酶的活性有不同程度的影响,其中SDS抑制作用很强.[结论]研究为重组右旋糖酐蔗糖酶纯酶的获取、得到稳定性好、活性高的酶反应体系及利用该酶进行催化反应和工业化应用提供了重要参数.     

一株产右旋糖苷酶海洋细菌的筛选鉴定

【目的】筛选鉴定产右旋糖苷酶的海洋细菌,并对其所产右旋糖苷酶的酶学性质及在变异链球菌牙菌斑生物膜中的应用进行初步研究。【方法】利用平板透明圈法从海洋环境中筛选产右旋糖苷酶的细菌,根据菌株形态特征、生理特征及16S rDNA序列确定其分类学地位,采用体外生物膜模型研究该酶对变异链球菌牙菌斑生物膜形成的抑制作用。【结果】从海泥中筛选出一株产右旋糖苷酶的细菌KQ11,初步鉴定为节杆菌(Arthrobacter sp.)。该菌株的最适生长温度为30°C,最适生长pH 7.5,最适生长NaCl浓度为0.4%。右旋糖苷酶的最适作用温度为45°C,最适作用pH为5.5。该酶能有效地抑制变异链球菌牙菌斑生物膜的形成。【结论】菌株KQ11右旋糖苷酶能够抑制变异链球菌牙菌斑生物膜的形成,可望用于漱口液等口腔护理产品中。     

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.     

Characterization of an Extracellular Dextranase from Fusarium moniliforme

An extracellular dextranase (EC 3.2.1.11) was purified approximately 75-fold from cell-free culture filtrates of Fusarium moniliforme. The purified dextranase was of the endo type, and isomaltose was identified as the primary end product of dextran hydrolysis. The molecular weight of the dextranase was determined to be 39,000 by gel permeation chromatography. The enzyme was most active at pH 5.5, and the temperature optimum was near 55 C. Activity was not inhibited by either ethylenediaminetetraacetic acid or iodoacetate. The Km for dextran with an average molecular weight of 10,000 was estimated to be 1.1 X 10(-4) M. The electrophoretic mobility of the dextranase was distinctly different from that of a Penicillium-derived commercial dextranase. The F. moniliforme dextranase was also found to differ from the commercial preparation by its greater relative activity against glucans isolated from Streptococcus mutans.     

Studies on Mold Dextranases

Nine strains capable of producing dextranase were isolated from soil. Among them, a strain belonging to the genus Aspergillus was chosen as the best producer of the enzyme. The mold produced greater amounts of dextranase than those found in some strains in the genus Penicillium, when grown aerobically at 28°C for 5 to 6 days in medium containing 1% dextran, 1% NaNO₃ or polypeptone, 0.2% yeast extracts, 0.4% K₂HPO₄ and small amounts of inorganic salts, pH 8.5. From the comparative taxonomic experiments, the mold used here was identified to be a strain belonging to Aspergillus carneus.     

Purification and characterization of extracellular dextranase from a novel producer, Hypocrea lixii F1002, and its use in oligodextran production

Fungi were isolated from natural soil samples and screened for extracellular dextranase synthesis. The strain F1002 was identified as Hypocrea lixii using a standard internal transcribed spacer ribosomal DNA analysis and was selected for extracellular dextranase synthesis. The enzyme was purified via ammonium sulfate precipitation and Sepharose 6B chromatography, which resulted in an 8.3-fold increase in the specific activity and a 10.73% recovery. This enzyme is a monomeric protein with a molecular mass of 62 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme, which was identified as an endodextranase, had an optimum pH of 5.0 and an optimum temperature of 25 °C. The dextranase activity was enhanced by Mg²⁺, Al³⁺, and especially Zn²⁺ at a low concentration, which improved its activity to 124.22%. The enzyme has a very high hydrolytic affinity toward high-molecular weight dextrans. Setting the concentrations of the H. lixii F1002 dextranase (2.31 U/mL) and dextrans (6%), as well as the reaction time (45 min), allowed the dextranase to hydrolyze dextrans of controlled molecular weights (20–70 kDa). Three types of oligodextrans with different molecular weights (namely, 69,376, 38,251, and 21,364 Da) were obtained, with a total yield of 80.32%.     

Some properties of an endodextranase inhibitor from continuous cultures of Streptococcus sobrinus.

Cell-free filtrates of Streptococcus sobrinus, cultured at low growth rate in the chemostat, contain a dextranase inhibitor that can completely inhibit the activity of S. sobrinus endodextranase. The range of conditions under which inhibition occurs, and the situations in which enzyme activity can reappear, have been examined in continuous cultures of strain 6715-13WT and the dextranase-deficient mutant 6715-13-201. A purified preparation of the inhibitor was specific for S. sobrinus dextranase, having no action on dextranases from other oral streptococci. The percentage inhibition of S. sobrinus dextranase varied with the enzyme concentration, and the complete inhibition of low amounts of enzyme indicated a very tight bond between the inhibitor and the enzyme.     

湘江河岸土壤中高产甲壳素脱乙酰酶菌株的筛选及鉴定

【目的】甲壳素脱乙酰酶(CDA)是将天然甲壳素生物转化为可商品化利用的壳聚糖的关键酶。本文旨在从湘江河岸的土壤中筛选可高产CDA的新菌株。【方法】以甲壳素为唯一碳源,利用4'-硝基乙酰苯胺为显色剂,通过变色圈法进行产CDA菌株初筛,产酶活性分析复筛;通过形态学和ITS区序列特征对菌株进行鉴定。【结果】从湘江(长沙段)河岸边的土壤中分离出的117株菌株中筛选到可产CDA的菌株30株,其中4株具有较强产CDA的能力。进一步经发酵产酶分析验证,菌株A1具有较强的产CDA能力,其胞外CDA酶活高达13.21 U/m L。结合形态学和ITS区序列特征,菌株A1初步鉴定为层生镰孢菌。【结论】从湘江河岸边的土壤中筛选到可高产CDA的菌株A1,具有较好的开发应用前景。     

Identification and characterization of a dextranase gene of Streptococcus criceti

The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene-walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200-amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell-surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C-terminus was partially purified and showed dextranase activity on blue-dextran sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDSPAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD-SDS-PAGE, whereas no dextranase activity of the cells was observed on blue-dextran brain heart infusion (BD-BHI) agar plates. Furthermore, PCR-based mutations of dextranase indicated that a deletion mutant of the C-terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C-terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp-453 and Trp-793 residues of S. criceti dextranase are critical to the enzyme's activity.     

Characterization of a yeast D-mannan with an alpha-D-glucosyl phosphate residue as an important immunochemical determinant

Conditions for the reaction of concanavalin A and dextranase with glutaraldehyde have been established to give a soluble, intermolecularly cross-linked conjugate possessing both dextranase and concanavalin activities. Evidence is presented that the dextranase and concanavalin molecules are linked to each other in the conjugate. The conjugate gives a different pattern of hydrolysis products on incubation with dextran than does dextranase.     

Cloning and expression of Penicillium minioluteum dextranase in Saccharomyces cerevisiae and its exploitation as a reporter in the detection of mycotoxins

A dextranase gene from Penicillium minioluteum (strain IMI068219) has been cloned, sequenced and expressed in Saccharomyces cerevisiae via fusion of the DNA segment encoding the mature dextranase protein with α-factor signal sequence, and insertion into the GAL1–controlled expression vector pYES2/CT. Galactose-induced expression yielded extracellular dextranase activity of 0.63 units/ml and cell-associated dextranase activity of 0.48 units/ml, after 24 h incubation. The dextranase construct was introduced into a strain of S. cerevisiae expressing the human cytochrome P450 3A4 (CYP3A4) and the cognate reductase, which was then used to develop a microplate toxicity bioassay. Toxicity was signalled as inhibition of dextranase activity, assayed fluorimetrically. This novel bioassay was assessed using six economically significant mycotoxins.