一株快速转化甜菊苷的细菌鉴定、产酶及转化特性

【目的】本研究鉴定了一株筛选的甜菊苷特异降解菌、优化了该菌产β-葡萄糖苷酶的条件以及研究了该菌对甜菊苷的转化特性。【方法】经16SrRNA基因序列测序和系统发育学分析,结合形态学特征确定该菌株的系统发育地位。用单因素及多因素分析探讨了其对甜菊苷的降解,通过液质联用检测了降解产物。【结果】菌株-J2与巨大芽孢杆菌的16SrRNA基因序列相似性达到100%,结合形态学特征,鉴定该菌为巨大芽孢杆菌(Bacillus megaterium)。在玉米淀粉4%、豆粕粉1%、硫酸镁0.04%、pH7.0、37℃、220r/min、接种量10%、培养36h的条件下,该菌产β-葡萄糖苷酶活力为779.68U/mL。甜菊苷转化的结果表明:3d可将10mg/mL甜菊糖溶液中甜菊苷转化74%,使莱鲍迪甙A和甜菊苷的比例(RA/SS)由转化前的0.38上升至0.99,RA的相对量增加160.5%,5d时转化完全。转化产物经液质联用鉴定为甜菊双糖甙。【结论】确定菌株-J2为巨大芽孢杆菌(Bacillus megaterium),该菌对甜菊苷具有高效、特异的转化能力,为首次报道的新型、安全菌株。     

一株转化琼胶为新琼寡糖菌株的筛选及鉴定

摘要:【目的】筛选一株可以将琼胶转化为新琼寡糖的菌株,并对该菌株进行鉴定。【方法】从紫菜生长区域采集紫菜和该区域海水,用含1‰琼胶的培养基富集培养,逐级稀释涂布、平板划线进行初筛,液体培养进行复筛,DNS法测定琼胶降解产物中还原糖的含量。通过16S rDNA序列分析,结合菌体形态、菌落特征及生理生化特性,确立该菌的系统发育学地位。【结果】从紫菜振荡液中筛选出一株可以产琼胶酶的菌株HJPHYXJ-1,该菌属于革兰氏阴性菌,16S rDNA序列同源性与需钠弧菌(Vibrio natriegens)的达到了99%,结合形态特征和生理生化实验结果鉴定该菌为需钠弧菌。HPLC法测定酶解产物为新琼寡糖。【结论】HJPHYXJ-1被筛选用于转化琼胶,酶解产物的聚合度在2－12 之间,是以二糖为单位的新琼寡糖,该菌产生的酶为β-琼胶酶。     

一株转化大豆苷元为S-雌马酚菌株的筛选和鉴定

【目的】筛选一株可转化大豆苷元为S-雌马酚的微生物菌株,并对该菌株进行鉴定。【方法】在厌氧条件下采用抗生素抑制非目标菌生长并结合稀释涂平板法进行菌株分离,分离可转化大豆苷元生成S-雌马酚的肠道细菌,并对产物进行结构鉴定。之后通过16S rDNA序列分析,构建该菌系统进化树,结合菌体形态及菌落特征,确立该菌系统发育学地位。【结果】从大鼠肠道内筛选分离到一株可以将大豆苷元转化为S-雌马酚的革兰氏阴性兼性厌氧菌株LH-52(JN861767),16S rDNA序列测序结果 BLAST比对表明该菌株与奇异变形杆菌(Proteus mirabilis)相似度达到了99%,结合形态特征和生理生化实验结果鉴定该菌为奇异变形杆菌。根据HPLC保留时间、质谱、核磁共振等波谱数据分析确定产物为S-雌马酚。【结论】菌株P.mirabilis LH-52为首次筛选到的可转化大豆苷元为S-雌马酚的兼性厌氧菌,相对于文献报道的严格厌氧菌更适合于工业化生产。     

棘孢曲霉转化甜菊糖为甜菊醇及纯化莱鲍迪苷A

【目的】本工作对棘孢曲霉固体发酵抽提酶液转化甜菊糖进行了研究,并对转化产物进行鉴定及纯化分析。【方法】用高效液相色谱、液质联用及红外光谱等方法对转化新产物进行鉴定,对上清液中莱鲍迪苷A(RA)成分进行纯化。【结果】棘孢曲霉酶液在10 h内对甜菊糖中的甜菊苷(SS)、莱鲍迪苷C(RC)进行高效特异性转化,以沉淀的形式析出的转化产物经鉴定为甜菊醇,转化率高达98.0%,分离提纯后纯度为95.2%,回收率达84.0%.由于甜菊醇的沉淀分离,留在溶液中的RA更易被纯化。RA通过树脂吸附分离的回收率为80.5%.【结论】棘孢曲霉酶液对甜菊糖的一次转化可以同时得到甜菊醇和莱鲍迪苷A两种产品,是一种经济高效的工艺。     

不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响

摘要: 【目的】探讨不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响。【方法】有氧条件下,采用稀释涂布法分别从ICR 小鼠、芦花鸡、长白猪和獭兔等4 种健康动物肠道中分离优势需氧菌,将不同动物的优势需氧菌分别与不同类型黄豆苷原转化菌株进行厌氧混合培养,高效液相色谱检测培养液中黄豆苷原的转化情况。【结果】16S rRNA 基因序列分析,结合形态学及相关理化特性分析表明,分离的22 株优势需氧菌分属埃希氏菌属(10 株) 、变形菌属(5 株) 、肠球菌属(4 株) 、芽胞杆菌属(2 株) 和假单胞菌属(     

人肠道产柚苷酶菌株分离、鉴定及其对柚皮苷的转化特性

【目的】从健康人肠道微生物菌群中分离能将柚皮苷高效转化为柚皮素的特定细菌菌株,对分离得到的菌株进行菌种鉴定并研究菌株对柚皮苷转化特性,目的是为柚皮素的高效生物合成提供新细菌资源。【方法】取健康人新鲜粪样,在厌氧工作站内培养24 h后进行梯度稀释并涂板,从板上挑取单菌落与底物柚皮苷进行厌氧培养,用高效液相色谱检测底物被转化情况。根据16S rDNA序列及相关生理生化特性分析,对所分离的柚皮苷转化菌进行菌种鉴定,并测定转化菌株对底物柚皮苷的转化动态和转化能力。【结果】从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,分别命名为AUH-JLD3、AUH-JLD7、AUHJLD104和AUH-JLD109。根据16S rDNA序列分析,结合形态学及相关生理生化特性等,将其分别鉴定为布劳特氏菌(Blautia sp.AUH-JLD3)、肠球菌(Enterococcus sp.AUH-JLD7)、拟杆菌(Bacteroides sp.AUHJLD104)和巴氏链球菌(Streptococcus pasteurianus subsp.AUH-JLD109)。转化动态研究结果表明,所分离的4株细菌菌株均能在12 h内将0.2 mmol/L底物柚皮苷转化为柚皮素;转化能力测定结果显示,菌株AUHJLD3、AUH-JLD7、AUH-JLD104及AUH-JLD109能够高效转化底物柚皮苷的最大浓度分别为0.2 mmol/L(平均转化率66.67%)、0.8 mmol/L(平均转化率86.49%)、0.2 mmol/L(平均转化率73.68%)以及1.6 mmol/L(平均转化率93.20%)。【结论】本研究首次从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,其中巴氏链球菌AUH-JLD109对底物柚皮苷转化能力最强。     

微囊藻毒素降解菌的筛选、鉴定及其胞内粗酶液对藻毒素MCLR的降解

【目的】从巢湖底泥中分离筛选高效的藻毒素降解菌,并初步研究其胞内粗酶液降解藻毒素-LR(MC-LR)的特性,为水体中藻毒素污染的微生物治理提供有效的菌源与理论依据。【方法】利用富集驯化培养技术,以MC-LR为唯一碳源,分离筛选MC-LR降解菌,通过形态观察、生理生化实验及16S rRNA序列分析鉴定菌株,并考察其胞内粗酶液在不同条件下对MC-LR的降解特性。【结果】分离得到1株能高效降解MC-LR的菌株M6。分子鉴定结果表明,该菌株为蜡状芽胞杆菌(Bacillus cereus)。其降解MC-LR的活性物质为胞内酶,而且至少有3种酶参与了MC-LR的降解,它们是菌体本身的组织酶而非诱导酶。当反应体系pH值为8.0,胞内粗酶液浓度为404.9 mg/L,MC-LR的初始浓度为10 mg/L时降解率最高,16 h可达98.7%。【结论】分离出的MC-LR降解菌为蜡状芽胞杆菌,该菌株对MC-LR有较高的降解能力,并且酶促反应受到反应体系的pH值、胞内粗酶液浓度以及藻毒素初始浓度等因素的影响。     

一株产冠菌素新菌种的分离与鉴定

【目的】从不同样本中分离筛选性能稳定的产冠菌素菌株。【方法】根据冠菌素引起植物叶片产生弥散性黄萎病、块茎膨大的特性,采集各种植物病叶、病枝及感病植物的土壤,采用穿刺法与系列稀释法分离筛选菌株;液相色谱测定菌株产生的冠菌素;在电子和光学显微镜下观察菌体形态;根据生理生化试验、(G+C)mol%含量、16S rDNA序列分析等对菌株进行鉴定;对分离提纯的发酵产物进行紫外、质谱和红外分析。【结果】菌株BBC933为革兰氏阴性菌,端生鞭毛,短杆状,无芽孢。在41℃下不生长,细胞内有聚β-羟基丁酸盐颗粒积累,没有精氨酸双水解酶和氧化酶,不能水解淀粉、明胶,不进行硝酸还原及反硝化作用,过氧化氢酶反应呈阳性。菌株的(G+C)mol%含量为67.2%,根据该菌株16S rDNA序列的同源性分析,构建系统发育树。【结论】菌株BCC933鉴定为洋葱伯克霍尔德氏菌(Burkholder cepacia),具有产冠菌素性能。国内外未曾见报道洋葱伯克霍尔德氏菌产冠菌素。     

一株引起马来甜龙竹组培污染内生菌的分离与鉴定

【目的】对一株引起马来甜龙竹组培污染内生菌的分离与鉴定。【方法】采用改良的NA培养基分离纯化菌株,并通过菌体的形态结构观察、生理生化试验及其16SrDNA序列同源性分析对其进行鉴定。【结果】菌株SWFU01的形态特征及生理生化试验结果与解淀粉芽孢杆菌[Bacillus amyloliquefaciens(Fukumoto)Priest et al.]的描述基本相同;16S rDNA序列分析表明,该菌株与解淀粉芽孢杆菌JS在同一系统发育分支,其同源性为99.28%。【结论】综合形态学特征、生理生化特征以及16S rDNA序列分析的研究结果,菌株SWFU01被鉴定为解淀粉芽孢杆菌。     

内蒙古碱湖中紫色非硫光合细菌的分离和鉴定

从内蒙古碱湖水样中分离得到一株紫色非硫光合细菌,命名为JH1-6.对该菌株进行了形态学观察、生理生化鉴定、活细胞吸收光谱以及16S rDNA序列分析.16S rDNA序列分析结果表明该菌株与沼泽红假单胞菌的16S rDNA序列同源性高达99%,结合形态特征和生理生化特性以及活细胞吸收光谱特征等,确定菌株JH1- 6在分类地位上属于沼泽红假单胞菌(Rhodopseudomonas palustris).     

3-Dehydroquinate synthase in germinating Phaseolus mungo seedlings.

Dehydroquinate synthase, an enzyme catalyzing the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, was detected in cell-free extracts of etiolated Phaseolus mungo seedlings. The reaction product, 3-dehydroquinate, formed from [1-14C]DAHP was identified by paper-radiochromatography. The enzyme required NAD+ and Co2+ for activity.     

Conversion of indole-3-acetaldoxime to indole-3-acetonitrile by plasma membranes from Chinese cabbage

The in vitro conversion of [¹⁴C]-indole-3-acetaldoxime (IAOX) to [¹⁴C]-indole-3-acetonitrile (IAN) by plasma membranes enriched by aqueous two-phase partitioning of Chinese cabbage ( Brassica campestris L. ssp. pekinensis cv. Granat) has been studied. The reaction product was identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). A reducing agent, e.g. ascorbic acid, was needed as cofactor for the formation of IAN from IAOX. Reduction equivalents and metal ions were not involved in the conversion of IAOX to IAN. The pH optimum for the reaction was at 6.0 and the apparent K_m for IAOX was 6.3 μ M . The enzyme was not inhibited by thiol reagents. The pI of the enzyme was determined to be 7.1 by isoelectric focusing (IEF). Gel permeation chromatography showed one major activity peak of 40 kDa. The reaction is considered as part of a channeling process leading from tryptophan to IAN with IAOX as an intermediate. This process is probably regulated by the indole derivatives IAOX and IAN.     

A plasma membrane-bound enzyme oxidizes l-tryptophan to indole-3-acetaldoxime

The in vitro conversion of [¹⁴C]-tryptophan to [¹⁴C]-indole-3-acetaldoxime (IAOX) by microsomal membranes of Chinese cabbage (Brassica campestris ssp. pekinensis cv. Granat) has been studied. The reaction product was identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Furthermore. IAOX was identified as an endogenous compound of Chinese cabbage by mass spectroscopy. The tryptophan-oxidizing enzyme (TrpOxE) was characterized. MnCl₂ was required as cofactor, H₂O₂, and 2,4-dichlorophenol (DCP) stimulated the reaction. The enzyme showed a pH optimum at pH 8–9 and a K_m for l -tryptophan of 20 μ M . The membranes containing TrpOxE activity were identified as plasma membranes by means of aqueous polymer two-phase partitioning. The TrpOxE from Chinese cabbage was purified 3-fold from plasma membranes by solubilization followed by (NH₄)₂SO₄-fractionation, affinity-chromatography with concanavalin A, and native gel electrophoresis. Enzyme activity was reduced by a tunicamycin pretreatment. Several other plant species, e.g. maize (Zea mays L. Inrakorn), sunflower (Helianthus annuus L. cv. Hohes Sonnengold), tobacco (Nicotiana tabacum L. cv. White Burley), and pea (Pisum sativum L. cv. Krombeck) showed a similar conversion of [¹⁴C]-tryptophan to [¹⁴C]-IAOX by phase-partitioned plasma membranes.     

Biosynthesis of sphingomyelin from erythro-ceramides and phosphatidylcholine by a microsomal cholinephosphotransferase

Mouse liver microsomes were shown to be active in the synthesis of sphingomyelin from ceramide and phosphatidylcholine in a reaction independent of CDPcholine. The conversion was not inhibited by calcium chelating reagents, and no evidence for the involvement of phospholipase C activity in the transformation could be adduced. Activity was also demonstrated in monkey liver and heart microsomes. Mouse brain microsomes produced a sphingomyelin analogue, tentatively identified as ceramide phosphorylethanolamine, but not sphingomyelin. Both [14C]ceramide and [G-14]phosphatidylethanolamine were precursors of the brain product, while phosphatidylcholine was inactive. Progress in the partial characterization of the liver enzyme is also described.     

Ring-opening bulk polymerization of epsilon-caprolactone and trimethylene carbonate catalyzed by lipase Novozym 435.

The affects of lipase concentration on ring-opening bulk polymerizations of epsilon-caprolactone and trimethylene carbonate were studied by using Novozym 435 (immobilized form of lipase B from Candida antarctica) as biocatalyst. The polymerization of epsilon-caprolactone was carried out in bulk at 70 degrees C. Three lipase concentrations of 9.77, 1.80 and 0.50 mg/mmol epsilon-CL were used in the experiment. The results showed that increasing the lipase concentration used in the polymerization system resulted in an increased rate of monomer consumption. For an enzyme concentration of 9.8 mg lipase per mmol monomer, an 80% monomer conversion was achieved in a 4-h time period, while for the lower enzyme concentration of 1.8 mg lipase per mmol monomer, 48 h were needed to reach monomer conversion. Linear relationships between Mn and monomer conversions were observed in all three enzyme concentrations, suggesting that the product molecular weight may be controlled by the stoichiometry of the reactants for these systems. At the same monomer conversion level, however, Mn decreased with increasing enzyme concentration. After correcting for the amount of monomer consumed in initiation, the plot of ln[([M]o - [M]i)/([Mt] - [M]i)] versus reaction time was found to be linear, suggesting that the monomer consumption followed a first-order rate law and no chain termination occurred. For the TMC systems, the polymerization was carried out in bulk at 55 degrees C. Similar to the epsilon-CL systems, increasing the Novozym 435 concentration from 8.3 to 23.6 mg/mmol TMC increased the rate of monomer conversion. Unlike the epsilon-CL systems, however, nonlinear relationships were obtained between Mn and monomer conversion, indicating that possible chain transfer and/or slow initiation had taken place in these systems. Consistent with the above result, nonlinear behavior was observed for the plot of ln[[M]o/[M]t] versus reaction time.     

Subsite structure and catalytic mechanism of a new glycosyltrehalose-producing enzyme isolated from the hyperthermophilic archaeum, Sulfolobus solfataricus KM1

A glycosyltrehalose-producing enzyme from Sulfolobus solfataricus KM1 catalyzes a conversion of maltooligosaccharides to glycosyltrehaloses and also hydrolyzes maltooligosaccharides to liberate glucose, as a side reaction. From the sum of the conversion and hydrolysis reaction rates, the rate parameters involved in the "splitting" of the alpha-1,4 glucosidic linkage were calculated. From the data obtained, the subsite structure for maltooligosaccharides was identified. From the analysis of the hydrolysate of maltotriose in [18O labeled H2O, the hypothesis of the C1-O bond splitting and the formation of a glycosyl (maltosyl)-enzyme intermediate was strongly supported. From the analysis of the reaction product in the presence of [3H] labeled glucose, the occurrence of intermolecular transglycosylation was confirmed. These data strongly support the suggestion that the catalytic mechanism of this enzyme is a transglycosylation.     

Canine thyroid fucokinase.

A radiometric assay was developed for fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) based on the conversion of L-[14C]fucose to L-[14C]fucose 1-phosphate which is trapped and counted on ion exchange paper. This assay was used to detect the presence of a fucokinase in canine thyroid tissue which was subsequently purified 2754-fold over the crude tissue extracts. The product of the fucokinase was identified as the beta-anomer. The pH versus activity curve for the enzyme appears biphasic with optima at pH 6.5 and pH 8.25. The enzyme was shown to be highly specific for L-fucose with a Km of 2.6 - 10(-5) M at pH 8.25. It was shown to be absolutely specific for ATP as a phosphate donor with a Km of 6.3 - 10(-4) M at pH 8.25. The enzyme requires a divalent cation. Mg2+ is slightly more effective than Mn2+ in meeting this need. The molecular weight of the enzyme has been determined to be 494 000 +/- 12 400.     

Conversion of L-cystine and L-cysteine to taurin by the enzyme systems of liver cells

The kinetics of conversion of sulfur-containing amino acids L-cystine and L-cysteine to taurin by the enzyme system of cattle liver cells was studied, and a mathematical model was developed. It was shown that L-cystine and L-cysteine conversion obeyed the Michaelis-Menten equations of serial-sequential conversions with regard to inhibition by the final product and inactivation. The yield of taurin under the optimized conditions of L-cystine and L-cysteine conversion (temperature, 40 degrees C; pH 1.5 and 3.0, respectively; and addition of enzyme preparations in five equal portions at 2-h intervals) was in the range 80-85% of the substrate weight.     

Aminoglycoside-3'-phosphotransferase I from aminoglycoside-polyresistant strain E. coli 182]

An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.     

Conversion of proparathyroid hormone to parathyroid hormone by a particulate enzyme of the parathyroid gland.

The conversion of proparathyroid hormone (proparathormone) to parathyroid hormone (parathormone) by subcellular fractions of the bovine parathyroid has been investigated. The identification of the conversion product as parathormone was established by its elution postion during ion exchange chromatography and gel filtration, and by partial amino acid sequence analysis of its NH2-terminal region. Total homogenates and derived subcellular fractions (600 X g pellet, 5,000 X g pellet, 20,000 X g pellet, 190,000 X g pellet, and 190,000 X g supernatant) all catalyzed the conversion of exogenous [3H]- or [14C]prohormone. Over 60% of the converting activity was in the particulate fractions; the 190,000 X g particulate fraction contained the highest specific converting activity. The converting activity appeared to be an integral component of the membranes since it could only be partially removed by extraction with Triton X-100. The production of parathormone by the particulate converting enzyme increased with time and the concentration of enzyme protein. The optimum pH range was between 7 and 9, and the enzyme was inactive below pH 6. Conversion by the particulate enzyme was inhibited by benzamidine or chloroquine, but not by pancreatic trypsin inhibitor, indicating its dissimilarity to trypsin. When a mixture of [14C]proparathormone and [3H]parathormone was used as substrate, the particulate enzyme did not metabolize the hormone despite over 70% conversion of the prohormone to hormone and other peptides. There was a close correlation between the subcellular distribution of converting activity and that of newly formed parathormone found in the membrane fraction. These data suggest that the particulate converting activity is that concerned with the formation of parathormone in vivo.