葡萄糖苷酶在毕赤酵母中的重组表达及一步纯化

从绿色木霉中克隆了葡萄糖苷酶bg基因,构建入表达载体pPIC9k-His6中,然后在AOX1启动子的控制下,在毕赤酵母GS115菌株中表达. 在5 L发酵罐中发酵120 h,重组P. pastoris Mut+菌株湿重达360.6 g/L,葡萄糖苷酶浓度和酶活分别为2.1 mg/mL和73.5 U/mL. 经亲和层析一步纯化后,得到了电泳纯的葡萄糖苷酶. HPLC分析显示其纯度为95.6%,比酶活为71.9 U/mg. 纯化过程酶得率为73.6%,纯化倍数为42.6. 纯酶的等电点为5.0,最适温度为50℃,最适pH为6.5. 金属离子Ag+, Ca2+, Cu2+, Fe2+及SDS对葡萄糖苷酶活性有抑制作用,而Mg2+, Mn2+, K+能增强葡萄糖苷酶活性,其中1 mol/L Mg2+能使酶活提高20%.     

Purification and Characterization of Three Alkaline Endopolygalacturonases from a Newly Isolated Bacillus gibsonii

A newly isolated Bacillus gibsonii, designated as S-2 (CGMCC1215), was cultivated for production of alkaline pectinases utilizing sugar beet pulp as growth substrate. Purification of three alkaline endopolygalacturonases (endoPGs) from the crude pectinases extract was carried out by ultra-filtration, ammonium sulphate fractionation and ion-exchange chromatography, and their enzyme activities characterized. The three purified alkaline endoPGs, designated as S-I, S-II, and S-III, had a molecular weight about 38 kDa as determined by SDS-PAGE. The Km value and optimal temperature for optimal enzyme activities of S-I, S-II and S-III were 1.2 mg/mL and 60℃, 0.9 mg/mL and 55℃, 1.1 mg/mL and 60℃, respectively. Their best performances were given at an optimal pH 10.5, and sodium polygalacturonate was found to be the best substrate. The isoelectric points of S-I, S-II and S-III were 5.4, 7.4, and 8.2, respectively. Surfactants of Tween-80 and Tween-20 and metal ions such as Mg2+ and Ca2+ stimulated the activity of S-I, S-II and S-III, whereas S-III was inhibited by Ca2+, and Mn2+ and Zn2+ ions inhibited the activity of the three enzymes.     

A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg(-1) protein, 20.0 U mg(-1) protein and 11.0 U mg(-1) protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl(2). K(m) and V(max) values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co(2+), Zn(2+), Cu(2+), Hg(2+), Ba(2+), Cd(2+), and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry.     

Thermoanaerobacterium saccharolyticum JW/SL-YS485来源的GH11家族木聚糖酶的克隆表达及其应用

对Thermoanaerobacterium saccharolyticum JW/SL-YS485来源的GH11家族木聚糖酶基因xyn11进行了克隆表达研究。xyn11基因全长636bp,编码211氨基酸。通过构建重组质粒,使其在大肠杆菌中实现了活性表达,酶活达到13.8U/mL。重组酶通过热处理和Ni亲和层析达到电泳纯,SDS-PAGE显示其分子量为20000,与理论分子量吻合。重组酶的最适反应温度为65℃,最适反应pH为4.5,在pH 3.5~6.0范围内保持较高的稳定性,60℃保温1h,酶活还残存60%,Cu²⁺对该酶有激活作用。重组酶底物特异性强,且不能降解木二糖和木三糖。重组酶以榉木木聚糖为底物,其V_max和K_m分别为9809U/mg和5.9mg/mL。榉木木聚糖质量浓度为25g/L,重组木聚糖酶用量为20U/g,在50℃、pH 4.5条件下水解12h低聚木糖的得率为27.8%,水解产物主要由木二糖和木三糖组成,且不产生任何木糖。结果表明,该木聚糖酶催化特性优异,可用于低聚木糖的制备。     

阿魏酸酯酶和木聚糖酶协同降解麦糟

橘青霉以麦糟为唯一碳源培养时,阿魏酸酯酶的最佳发酵时间为60 h,其酶活力可达40.8 mU/mL。在pH值为5.0、45℃、料液比1∶30(g∶mL)条件下,取47.5 U/mL的木聚糖酶粗酶液15 mL,加入1.0 g麦糟的乙醇不溶物,反应12 h后,加入40.8 U/mL的阿魏酸酯酶粗酶液15 mL再反应12 h,阿魏酸和低聚木糖释放率分别为54.1%和161 mg/g(麦糟的乙醇不溶物)。实验结果还表明,阿魏酸酯酶与木聚糖酶存在协同作用,能极大提高麦糟中阿魏酸及低聚木糖的释放率,有利于麦糟的降解。     

Purification and characterization of novel thermo-alkaline lipase and its application

In order to excavate thermo-alkaline lipases from bacterial living in extreme conditions, we try to express new gene from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows in alkaline hot springs Lake Bogoria in Kenya and explore its application in biodiesel production. The lipase gene (tll1) of 1434 bp were ligated at the Nco I / EcoR I sites of the expression vectors pET28a to yield the construct of pET28a-TLL1. The strain harboring pET28a-TLL1 was cultivated for expression at 25℃, the specific activity of 1.99 U/mg protein were detected in disrupted cells. The recombinant lipase TlLipA was purified by a simple two-step procedure involving heat treatment and Ni-chelating affinity chromatography. The subunit of purified TlLipA showed a molecular mass of 53×10³ on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified TlLipA exhibited optimal activity at 65℃ and pH 8.0 and it was stable from 55℃ to 65℃. The enzyme remained above 80% of its original activity at pH ranging from 7.0 to 11.0 and at room temperature for 1 h. The activity of TlLipA was little unaffected by Co²⁺, K⁺, Na⁺, and Ni²⁺, and a little activated by Mg²⁺ and Mn²⁺, ^{but were significantly inhibited by Zn2+, Fe3+, and SDS, and Tween 80 under the assay conditions. The purified recombinant TlLipA had a specific activity of 22.11 U/mg protein using p-nitrophenyl palmitate (p-NPP) as substrate. Determined by Sigma-Plot of reaction rate on p-NPP, the K_m was 0.23 mmol/L, the V_max was 33.50 mmol/(L·min), and the k_cat was 22.83 s-1. The enzyme was also active towards p-NPP, p-nitrophenyl laurate (p-NPL), p-nitrophenyl myristate (p-NPM) and p-nitrophenyl caproate (p-NPC), moreover TlLipA exhibited a strong preference for p-nitrophenol decanoate (p-NPD) and p-nitrophenyl octoate (p-NPO). Using recombinant lipase as a catalyst to prepare biodiesel in a solvent-free system, with a water content of 20%, an enzyme dosage of 200 U/g oil, and an alcohol-to-oil ratio of 4∶1, catalyzed soybean oil reaction at 55℃ for 48 h, the yield can reach 91.75%.}     

新型嗜热耐碱脂肪酶的纯化表征及应用

将来源于解脂嗜热互营杆菌（Thermosyntropha lipolytica）的脂肪酶（TlLipA）基因tll1导入大肠杆菌BL21（DE3）中表达,通过热处理和镍柱亲和层析获得纯酶,并对其酶学性质进行研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示TlLipA分子量为53×10³,其最适反应温度为65℃,最适反应pH为8.0。在55~65℃范围内酶活较高且比较稳定;在pH^{7.0~11.0于室温保存1 h后,残留相对酶活仍达80%以上。1 mmol/L 金属离子Zn2+、Fe3+和试剂SDS,0.05%（质量分数）Tween 80,对酶活力具有强烈的抑制作用,残留相对酶活皆低于15%;1 mmol/L Mg2+、Mn2+对酶活力表现出轻微的激活作用。由底物专一性实验可得,该酶对辛酸对硝基苯酯（C8）和癸酸对硝基苯酯（C10）偏好明显。以棕榈酸对硝基苯酯（p-NPP）为底物,该酶动力学参数K_m值为0.23 mmol/L,V_max为33.50 mmol/(L·min),k_cat为22.83 _S-1。以重组脂肪酶为催化剂在无溶剂体系中制备生物柴油,含水率20%,酶加量200 U/g油,醇油比为4∶1的条件下,在55℃催化大豆油反应48 h,收率可达91.75%。}     

Utilization of feruloyl esterase and xylanase for the degradation of brewers＇ spent grain

橘青霉以麦糟为唯一碳源培养时,阿魏酸酯酶的最佳发酵时间为60 h,其酶活力可达40.8 mU/mL。在pH值为5.0、45 ℃、料液比1∶30（g∶mL）条件下,取47.5 U/mL的木聚糖酶粗酶液15 mL,加入1.0 g 麦糟的乙醇不溶物,反应12 h后,加入40.8 U/mL的阿魏酸酯酶粗酶液15 mL再反应12 h,阿魏酸和低聚木糖释放率分别为54.1%和161 mg/g （麦糟的乙醇不溶物）。实验结果还表明,阿魏酸酯酶与木聚糖酶存在协同作用,能极大提高麦糟中阿魏酸及低聚木糖的释放率,有利于麦糟的降解。     

气相色谱测定消心痛中有机溶剂的残留量

建立了C．C测定消心痛原料药中氯仿、乙酸、乙酸乙酯残留量的方法。采用色谱柱为PEG-20M不锈钢柱(4m×2mm),柱温采用程序升高,起始柱温50℃维持3 min,然后以10℃/min的速率升至180℃维持5 min,再以10℃/min的速率升至280℃维持10 min。FID检测器,检测器温度320℃,进样口温度320℃,氮气为载气,流速50mL/min,氢气流速30 mL/min。空气流速400 mL/min。可使三种有机溶剂完全分离,氯仿在0．0005～0．003 mg/mL的浓度范围内呈良好的线性关系(r=0．9994),回收率为100．3％(RSD＜1．0％)；乙酸乙酯在0．005～0．03 mg/mL的浓度范围内呈良好的线性关系(r=0．9999),回收率为100．5％,(RSD＜1．0％)；乙酸在0．005～0．03 mg/mL的浓度范围内呈良好的线性关系(r=0．9997),回收率为98．7％,(RSD＜1．0％)。     

液相色谱-串联质谱测定印染废水中3种碱性染料

建立了液相色谱-串联质谱法(LC-MS/MS)测定印染废水中碱性橙2、碱性橙21、碱性橙22三种碱性染料的检测方法。采用乙酸乙酯-环己烷混合溶液(体积比1:1)萃取碱性染料,经凝胶渗透色谱净化浓缩,液相色谱-串联质谱仪测定,结果表明:3种碱性染料在5.0~100.0 ng/m L范围内,线性关系良好,相关系数(R~2)大于0.999 6;检出限在2.5~5.0mg/L之间;回收率在88.6%~98.4%之间,相对标准偏差(RSD)在1.3%~2.7%之间。该方法自动化程度高、样品前处理简单、灵敏度高、检出限低、结果准确可靠、回收率和重现性良好,可用于印染废水中三种碱性染料残留量的同时测定。     

环氧氯丙烷改性花生壳吸附水中Cu~(2+)的研究

利用环氧氯丙烷对花生壳改性制备吸附剂,并用其吸附水溶液中Cu2+。实验结果显示,花生壳的改性条件为:花生壳5.0 g,浓度为1.5 mol/L NaOH溶液100 mL,环氧氯丙烷5 mL,反应温度30℃,反应40 min;用上述条件改性花生壳0.3 g,吸附初始浓度50 mg/LCu2+溶液,控制溶液的pH为5.0,吸附时间3.0h,对Cu2+吸附率可达96.0%,高于未改性花生壳的70.4%,使吸附率提高36.4%。     

巴斯德梭菌甘油脱水酶基因的克隆表达、纯化及酶学性质的研究

用聚合酶链反应(PCR)技术,从巴斯德梭菌(C lostridium pasteurianum)扩增出甘油脱水酶基因(dhaBCE),在大肠杆菌中高效表达,SDS-PAGE(聚丙烯酰胺凝胶)电泳结果显示,分别有相对分子质量为66、21、16 kD三条特异性蛋白表达条带。用金属镍亲和层析及S-300H凝胶层析将重组蛋白进行分离纯化,所得纯酶的比活为4.26 U/mg。重组酶的最适反应温度42~44℃,最适作用pH=9.10~9.50,它对三个底物结构类似物的米氏常数(Km)值分别是:1,2-丙二醇0.38 mmol/L,甘油0.29 mmol/L,1,2-乙二醇2.0 mmol/L;对辅酶B12的Km值为0.22μmol/L。     

负载Ag~+ D72树脂色谱柱分离纯化花生四烯酸工艺的建立

目的建立负载Ag+D72树脂色谱柱分离纯化花生四烯酸(Arachidonic acid,AA或ARA)的工艺。方法采用负载Ag+D72树脂色谱柱对微生物油脂中的ARA进行分离纯化,确定最佳分离纯化参数,利用气相色谱法检测ARA的含量。结果负载Ag+D72树脂的最大饱和吸附量约为9 mg/g干树脂,在吸附温度0℃、不饱和脂肪酸甲酯上样质量浓度5 mg/ml,5%丙酮正己烷溶液(体积比)洗脱、解吸温度30℃、洗脱流速2 ml/min的条件下,ARA的纯度达89.4%,收率达79.3%。结论负载Ag+D72树脂色谱柱可有效分离纯化微生物油脂中的ARA,为进一步生产高纯度的ARA,进而规模化生产ARA产品提供了参考。     

海星多糖的提取分离与抗氧化活性研究

目的:从南海海星中提取多糖并研究其抗氧化活性。方法:采用碱提-酶解法提取海星多糖,并采用离子交换层析与凝胶过滤层析法进行纯化;采用醋酸纤维素薄膜电泳及凝胶过滤色谱法鉴定纯度;采用Fenton反应体系研究清除羟基自由基活性,采用邻苯三酚自氧化法研究清除超氧阴离子活性;采用H2O2损伤家兔红细胞模型研究其对生物膜的抗氧化保护作用。结果:海星多糖及阳性对照甘露醇、苯甲酸清除羟基自由基的半数有效浓度EC50分别为5.51 mg/mL、16.33 mg/mL、4.29 mg/mL;海星多糖及阳性对照维生素C清除超氧阴离子自由基的半数抑制浓度IC50分别为32.36μg/mL、18.32μg/mL;海星多糖对H2O2致红细胞损伤有良好的保护作用。结论:海星多糖具有较强的抗氧化活性。     

甘蔗渣纤维素磷酸酯的合成与应用研究

甘蔗渣纤维素为原料 ,用尿素作催化剂 ,研究了甘蔗渣纤维素磷酸酯合成的最佳工艺条件。将 5 .0g甘蔗渣纤维素与w(NaOH) =2 0 %的 3 0mL水溶液反应 1h ,水洗至中性 ,制得碱纤维 ,5 .0g碱纤维再用w (H3PO4) =2 0 %的水溶液 4 0mL进行预膨润 12h ,将滤饼用 5mLw(H3PO4) =85 %的水溶液、0 .3g催化剂尿素、甲苯作溶剂 ,5 0℃下反应 1h。产品对Ag+、Pb2 +、Fe3+、Zn2 +离子的平衡交换吸附量比活性炭平均增加 7.15mg/g ,对X -GRRL阳离子蓝染料的交换吸附性能比活性炭提高了 15 .7%。     

烟酰胺和3-氰基吡啶的HPLC检测

采用高效液相色谱法测定烟酰胺和3-氰基吡啶的含量.色谱条件为:STR-ODSⅡ柱(4.6 mm×150 mm),流动相为甲醇∶水=1 ∶ 3,流速1.000 mL·min-1,检测波长254 nm,柱温30℃.该方法的烟酰胺和3-氰基吡啶检测范围均为1.0～40.0 mg·L-1,烟酰胺的检测限为0.25 mg·L-1...     

仙人掌超氧化物歧化酶的纯化及其部分性质研究

10 0 0g仙人掌茎经匀浆、硫酸铵分级沉淀、SephadexG - 75凝胶层析和DEAE - 5 2离子交换层析 3个步骤 ,将仙人掌SOD纯化到SDS -PAGE均一纯 ,得白色粉状SOD 0 8985g ,比活 910U/mg,活力回收 4 3 1% ,纯化倍数 76。该酶相对分子质量 37 1kD ,H2 O2 和KCN的抑制实验结果表明为Cu、Zn -SOD。该酶的最大紫外吸收在 2 75nm处。 5 0℃保温 15 0min ,SOD的活性不变 ,在pH =5 0～ 9 0时SOD有较好的稳定性     

糙皮侧耳菌漆酶的分离纯化及部分性质研究

对糙皮侧耳菌5.42产生的漆酶粗酶液通过超滤浓缩、DEAE-Cellulose和SephadexA-50纯化,得到纯化18.1倍漆酶,得率36%,用PAGE检测为单一条带,用SDS-PAGE证明漆酶的分子量为61.4 kD,而用经过高效液相色谱分离的酶液进行质谱检测分子量为60.5 kD。该酶最适温度为40℃,最适pH为5.0。金属离子对酶活有很大的影响,其中K+、Mn2+、Cu2+有明显促进作用,Hg2+、Ag+、Ba2+等对酶活有明显的抑制作用。该酶与果胶酶按比例组合在一起,对荨麻生物脱胶取得良好的脱胶效果——木质素去除率为85%。     

源于博伊丁假丝酵母的甲酸脱氢酶的分离纯化与反应机制

使用两步发酵法培养博伊丁假丝酵母(Candida boidinii),菌株细胞经破碎与分离所得粗酶液经DEAE SepHaroses Fast Flow层析快速纯化获得NAD+依赖型的甲酸脱氢酶,酶的比活从0.83 U/mg提高到2.67 U/mg,纯化倍数和回收率分别为3.22倍和29.7%。研究了从反应物消耗到产物生成之间的酶反应历程,确定了甲酸脱氢酶的酶促反应为有序BiBi反应机制,其中NAD+是第一底物,HCOONa 是第二底物,NADH是首先释放的第一产物,HCO3ˉ是随后释放的第二产物;二次作图法求解出Vmax、KiS1、KmS2、KmS1等动力学参数,确定甲酸脱氢酶有序BiBi反应速度方程为 。     

红豆杉单组分多糖的超滤预处理及连续分离工艺

以红豆杉枝叶部位总多糖为原料,采用超滤方法对原料进行预处理,并对工艺进行优化,最佳优化条件为:截留相对分子质量为5kDa,温度为25℃,压力为20psi(1psi=6894.76kPa),pH值为6.8。 在此条件下,将浓度为10g/L的1L红豆杉多糖溶液超滤至125mL,再添加去离子水至1L,反复超滤4次,得到的红豆杉多糖的截留率和脱色率分别为87.2%和75.6%。进一步以红豆杉单组分多糖间歇色谱分离工艺参数为基础,采用自行设计制作的连续色谱设备,建立了连续离子交换色谱分离红豆杉酸性单组分多糖PST-1的分离工艺,处理量达到每批次100.08L,PST-1纯度在99.0%以上,收率达到50.0%。