黑曲霉糖化酶分离纯化与酶学性质研究

纯化了黑曲霉Aspergillus niger FJL0801糖化酶,并对其酶学性质进行研究.粗酶液经纯化后,较粗酶液纯化了38.42倍,酶活回收率达到17.45%.酶最适作用温度为60℃;最适反应pH值为4.5;在60℃下,保温2 h后,相对酶活42%±2.8%.在pH4.5,60℃下,作用时间在60 min以内,其酶活保存89%±2.56%;其酶学性质符合淀粉糖化工业化过程中对酶的要求,该酶比较适合应用于淀粉糖化工业.     

黄粉虫几丁质酶的纯化及性质

以黄粉虫为材料,采用盐析、亲和柱层析、Sephadex G-100分子筛及DE-52离子交换柱层析等制得纯化倍数约33倍,回收率为38%,比活力为294.7U/mg的几丁质酶制剂.在此基础上,以胶体几丁质为底物,考察黄粉虫几丁质酶的性质.结果表明：该酶催化水解几丁质的Km值为71.4mg/L;酶的最适pH值是6.0;最适温度为45℃左右;酶在pH 5.0～7.0,45℃以下酶活力稳定性较好.Na＋和K＋对酶活无影响;Ca2＋、Cu2＋对酶活起先扬后抑作用;Mn2＋、Mg2＋、Fe3＋、Al3＋对该酶活力均具有不同程度的抑制作用.     

含油废水中产脂肪酶微生物的筛选和酶学性质研究

采用罗丹明B平板初筛和摇瓶发酵复筛法从含油废水中筛选出产脂肪酶活性较高的菌株,并对活性最高的菌株F-11进行酶活性条件分析,发现F-11属革兰氏阴性杆菌。对F-11菌株所产脂肪酶进行酶催化特性分析发现,该脂肪酶属中温碱性脂肪酶,反应转速为150 r/min时,其最佳初始pH为7.5,最佳培养温度为30℃,菌株酶活性最高为10.96 U/mL,稳定性良好。     

乳酸脱氢酶的分离纯化及其性质的研究

乳酸脱氢酶是在厌氧条件下能够催化丙酮酸接受NADH上的氢,生成乳酸;当动物体体内缺乏葡萄糖时,还可以氧化乳酸生成丙酮酸并经葡萄糖异生途径转变为葡萄糖的一组同工酶．蛋白质的分离纯化是研究蛋白质的结构与功能的一种有效手段．现已有大量的关于LDH分离纯化及其性质研究的文献报道,本文将其综述如下．     

壳聚糖固定化果胶酶的制备及其酶学性质研究

以1%壳聚糖与5%戊二醛交联8h制得交联壳聚糖载体,1g载体固定10mg的果胶酶,载体先与酶液缓慢振荡混合30min后,在固定化体系(pH3.4,4℃)中固定反应12h,该条件下制得的固定化酶强度大韧性好,酶活力回收率高达56.31%;固定化酶的最适温度50℃,最适pH3.4,Kmapp值为5.42mg·mL-1,连续使用7次后,酶活力还保留70.45%以上,具有较好的操作稳定性。     

荚膜红细菌(Rhodobacter capsulatus)hemA基因的高效可溶表达及其酶学性质对比研究(英文)

研究目的:提供一种酶学性质优良的,可在大肠杆菌体内高活性可溶表达的,具有工业应用前景的5-氨基乙酰丙酸合成酶。创新要点:利用E.coli Rosetta(DE3)表达编码荚膜红细菌5-氨基乙酰丙酸合成酶(RC-ALAS)的hemA基因,实现其在大肠杆菌体内高活性可溶表达。以放射形土壤杆菌和类球红细菌hemA基因表达的5-氨基乙酰丙酸合成酶(AR-ALAS和RS-ALAS)为参照,对分离纯化后的RC-ALAS进行酶学性质对比研究。研究方法:工程菌的构建如图1所示,酶的分离纯化采用镍柱亲和层析和凝胶过滤层析,酶学性质的测定采用对比研究法。重要结论:(1)RC-ALAS酶活性高达198.2 U/mg,比AR-ALAS(151.1 U/mg)和RS-ALAS(116.9 U/mg)分别提高31.2%和69.5%;(2)RC-ALAS对底物琥珀酰辅酶A的专一性常数((kcat/Km)S-CoA)为1.4989,高于AR-ALAS(0.7456)和RS-ALAS(1.1699),具有较高的催化效率;(3)对含有荚膜红细菌hemA基因的工程菌进行补料分批发酵,发酵结束时,ALA的产量高达8.8 g/L(67 mmol/L)。     

双头菌WH-1环氧乙烷二酸水解酶的克隆和酶学性质的研究（英文）

目的:克隆双头菌WH-1环氧乙烷二酸水解酶(ORCH)的基因并研究其酶学性质。创新点:首次获得双头菌ORCH的基因,且该酶催化效率高,热稳定性好。方法:柱层析纯化ORCH后,进行酶学性质研究;通过蛋白末端测序和PCR获得其基因序列;通过二级结构预测和多序列比对进行ORCH序列分析。结论:来源于双头菌WH-1的ORCH是迄今报道的催化效率和热稳定性最好的ORCH,为L(+)-2,3-二羟基丁二酸的生产提供了新的催化剂。     

干酪乳杆菌(Lactobacillus casei)3MP-5-3胞外甘露聚糖酶的分离纯化（英文）

以分离自东北酸菜发酵液中的干酪乳杆菌(Lactobacillus casei)3MP-5-3为起始菌株,在以魔芋粉为唯一碳源的MRS培养基中发酵产酶,以发酵液的上清液为粗酶液来纯化L.casei 3MP-5-3所产的胞外甘露聚糖酶。研究表明：粗酶液与丙酮的最适体积比为1∶1.4。采用阴离子交换层析体系来纯化目标蛋白时,洗脱体系的最适pH为6.5,洗脱能力最强的阴离子为CH₃COO^-。     

“两学一归纳”教学模式的实践与思考——以《反比例函数的图像和性质（第一课时）》为例

“两学一归纳”的课堂教学模式的研究没有终点,永远行走在路上,唯有依靠学生、促进学生发展的“学”．才是最有智慧的学法;唯有生长于课堂、源于教师真实感悟的“教”,才是最有智慧的教法。     

Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

INTRODUCTION Because of its low molecular weight and ability to fluoresce independently (George, 1997), the new molecular tag, green fluorescent protein (GFP), has become more and more popular after Prasher et al.(1992) cloned its cDNA in 1992. There are many reports describing the co-expression of GFP and a specific antibody or cytokine gene, with the fusion protein expressing the fluorescent activity and bio-logical activity of the complement protein (Haraguchi et al., 1999; Mclean…     

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice

Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value.This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds,and a simple process for recovery and purification of rHSA for economical manufacture.An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference(RNAi) cassette suppressing the CYP81A6 gene.This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) cassette which confers glyphosate tolerance.ANX Sepharose Fast Flow(ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance(Butyl HP) hydrophobic interaction chromatography was used to purify rHSA.A transgenic rice line,HSA-84,was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds.This line also demonstrated high sensitivity to bentazon,and thus could be killed selectively by a spray of bentazon.A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%.Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA.This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29"

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel P-100.The protein was absorbed on DEAE-cellulose and Bio-Gel P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pl value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited in-hibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia scle-rotiorum. The 50% inhibitory concentrations (IC50) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B291 also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germi-nated spores.     

Caffeic acid product from the highly copper-tolerant plant Elsholtzia splendens post-phytoremediation: its extraction, purification, and identification

In the current study, caffeic acid was an important metabolite in the highly copper-tolerant plant Elsholtzia splendens. Preparation and purification of caffeic acid were performed on the dried biomass of the plants by means of sonication/ethanol extraction, followed by purification using a macroporous resin (D101 type) column and silica gel chromatography. The faint-yellow caffeic acid product was yielded with a purity of 98.46%, and it was chemically identified from spectra of Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (¹H NMR)/carbon nuclear magnetic resonance (¹³C NMR), and electrospray ionization mass spectrometry (ESI-MS). Caffeic acid is a possible product from the post-harvest processing of Elsholtzia splendens biomass.     

2-苯基-1,3-丙二硫醇的合成

苯乙酸乙酯与金属钠反应所生成的碳负离子与碳酸二乙酯反应生成苯基丙二酸二乙酯,经四氢化铝锂还原、酸化得到2-苯基-1,3-丙二醇.通过四溴化碳/三苯基膦、硫代乙酸钾的羟基官能团转化反应,合成2-苯基-1,3-二溴丙烷和2-苯基-1,3-丙二硫醇乙酸酯,最后用肼还原硫醇乙酸酯,合成了目标产物2-苯基-1,3-丙二硫醇.所有中间产物和目标产物经核磁共振氢谱或红处光谱表征.     

Purification and characterization of keratinase from a new Bacillus subtilis strain

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50℃ was 8.5 and the optimum temperature at pH 8.5 was 55℃. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT),mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO)stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC₅₀) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.     

Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B

INTRODUCTION Chronic hepatitis B virus (HBV) infection is aserious clinical problem because of its wide distribu-tion and possible adverse consequences, such as he-patic decompensation, cirrhosis and/or primary livercancer (PLC). The natural course of chronic HBVinfection is characterized by a series of hepatitic flaresor exacerbations and remissions (Ganem and Prince,2004). The severity, extent, duration and frequency ofhepatic histopathological changes in hepatitic flaresare d…     

Expression, purification and characterization of a phyA(m)-phyCs fusion phytase

The phyAm gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyAm-phyCs gene was successfully overexpressed in P. pastoris as an active and ex-tracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5～6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyAm.