Extraction of Pancreatic Lipase Inhibitor from Rice Germ

以米胚芽为原料,提取具有抑制胰脂肪酶活力的胚芽蛋白.经工艺优化得到提取条件为:原料颗粒60目,料液比1:6,pH 10.18,温度55℃,提取时间4h,水提后离心速度4 456 r·min-1,等电点沉降pH5.1,等电点沉降后离心速度3 692r·min-1.在此条件下提取得到的胰脂肪酶抑制剂的蛋白质纯度为60.7％.15.18 g/L的胚芽蛋白对30.1 U/mg的猪胰脂肪酶的平均抑制率达71.17％.     

海洋野生鱼酶解提取鱼油的工艺分析

海洋野生鱼与养殖鱼比较, 其鱼油中含更多的二十碳五烯酸（EPA）、二十二碳六烯酸（DHA）、脂溶性维生素等活性成分。为提高海洋野生鱼的利用价值, 以野生小带鱼为原料进行酶法提油工艺研究。分析了不同的温度, 时间, pH值等影响因素下的提取、萃取以及离心效果, 以响应面法确定了最佳的酶解工艺条件: 液固比为6、pH7.3、酶量1000 u/g原料、搅拌速度200 r/min、45oC酶解90 min; 最优萃取条件: 萃取剂100 mL(每20 g鱼糜原料)、pH4.0、40oC萃取25 min; 离心条件: 离心速度3000 r/min (1865 g)、离心时间10 min。上述工艺条件下提油率为79.90%。改进了传统的鱼油提取工艺, 在活性成分保护上有较大改善。     

海洋野生鱼酶解提取鱼油的工艺分析

海洋野生鱼与养殖鱼比较, 其鱼油中含更多的二十碳五烯酸（EPA）、二十二碳六烯酸（DHA）、脂溶性维生素等活性成分。为提高海洋野生鱼的利用价值, 以野生小带鱼为原料进行酶法提油工艺研究。分析了不同的温度, 时间, pH值等影响因素下的提取、萃取以及离心效果, 以响应面法确定了最佳的酶解工艺条件: 液固比为6、pH7.3、酶量1000 u/g原料、搅拌速度200 r/min、45oC酶解90 min; 最优萃取条件: 萃取剂100 mL(每20 g鱼糜原料)、pH4.0、40oC萃取25 min; 离心条件: 离心速度3000 r/min (1865 g)、离心时间10 min。上述工艺条件下提油率为79.90%。改进了传统的鱼油提取工艺, 在活性成分保护上有较大改善。     

相思豆毒蛋白的提取及毒理作用研究

目的:探索相思豆毒蛋白的最佳提取条件、蛋白含量、等电点及对动物的毒理作用. 方法:经pH 7.2磷酸缓冲液提取、浓度为30%、50%硫酸铵分级沉淀及甘油透析处理,从去壳相 思豆种子中提取得到相思豆毒蛋白,福林酚法测定蛋白含量,等电聚焦法测定相思豆毒蛋白等电点,小白鼠和家兔灌胃实验研究其毒理作用.结果:从去壳相思豆中 分离提取得到相思豆毒蛋白;相思豆毒蛋白的等电点为6.10和6.24.动物实验表明该毒蛋白对小白鼠、家兔具 有毒性.结论:磷酸缓冲液法是相思豆毒蛋白适宜的提取途径,相思豆毒蛋白对动物具有毒性.     

白地霉脂肪酶的双水相萃取和反胶团提取

对影响双水相萃取和反胶团提取脂肪酶的各种因素进行了探讨,并通过正交实验进一步优化提取条件,PEG浓度15%,(NH4)2SO4浓度22.5%,pH8.0的条件下进行双水相萃取,脂肪酶纯化倍数达到7.5倍;CTAB浓度150mmol/L,相体积比4/2,水相pH8.0,温度40℃的条件下进行反胶团提取,脂肪酶的比活力达到最大,但其比活力稍有下降,约为原来的0.9倍。     

生姜蛋白酶提取及反胶束纯化工艺初步研究

本文研究了生姜中生姜蛋白酶的分布及贮藏中的活力变化 ,研究了从新鲜生姜中提取生姜粗蛋白酶及用AOT 异辛烷和CTAB庚烷 /辛醇反胶束萃取该酶的工艺和方法。实验结果指出 :在贮藏茎中生姜蛋白酶的活力为 2 .7μg/mL·min-1,在膨大茎中该酶活力为 0 .6 8μg/mL·min-1,而在幼嫩茎中活力最低 ,仅为0 .4 8μg/mL·min-1。新鲜生姜在 0℃下贮藏 2 4h即完全丧失活力 ,在室温下贮藏 3d后其活力损失达38 2 5 %。用 10倍 0 .2mol/L、pH =6 .0的磷酸缓冲液 (4℃ )三次提取生姜蛋白酶 ,其提取率分别为 6 4 .75 %、14 .2 8%和 5 .2 %。用 6 5 %饱和度的 (NH4) 2 SO4沉淀提取液中的生姜蛋白酶 ,再以 pH 6 .2、0 .1mol/L的柠檬酸缓冲液溶解 ,其比活力达到 4 .2 1(μgPro/ μgPro·min-1)。生姜蛋白酶的 pI =5 .4～ 5 .5 ,在pH 5 .4以上 ,用AOT 异辛烷反胶束不能萃取出生姜蛋白酶 ,但却可以萃取出 71.86 %的杂蛋白。用CTAB庚烷 /辛醇反胶束二次萃取AOT 异辛烷萃余液 ,其蛋白质萃取率为 6 0 .2 5 % ,萃取液中生姜蛋白酶理论比活力达到 4 9.77(μgPro/ μgPro·min-1)。     

L-脯氨酸羟基化酶的提纯及活性研究

目的,以猪肺为原料,提取能把游离的L-脯氨酸羟化脯氨酸的羟基化酶。方法：把新鲜猪肺在提取液中低温高速匀浆,然后用4000r/min离心分离20min,取上清液进一步用8000r/min高速离心后,分别用盐析和有机溶剂的方法提取羟基化酶;结果;不同的方法都得到棕色的固体。经电泳测定该固体纯度高,溶于水中后能把游离的L-脯氨酸羟化为L-羟脯氨酸。测定其分子量约为66.000;等电点约为4。结论：所采用的提取工艺流程具有简单,效率高和实用的特点,且猪肺便宜,易得,适合较大规模的制备。     

响应面法优化苦荞麸皮粉蛋白质提取工艺

目的：分析苦荞麸皮粉中蛋白含量及其蛋白组成。方法：采用碱法提取-等电点沉淀分离蛋白。在单因素试验基础上,选取料液比（w/v）、pH、时间等3个影响因素,采用响应面法 （Box-Behnken 中心组合）优化苦荞麸皮粉蛋白提取工艺。结果：料液比、pH、时间对麸皮蛋白提率有显著影响,影响顺序为料液比> pH>时间。响应面优化得到最佳提取条件为pH 10.5、料液比1∶35、时间3 h 40 min,此条件下苦荞麸皮蛋白提取率为（97.31±4.64）%。结论：本研究为有效开发利用苦荞麸皮粉的蛋白提供科学依据。     

黑木耳中抑制胰脂肪酶活性物质的提取工艺及体外抑制效果

本文以黑木耳醇提物和水提物为研究对象,以对胰脂肪酶活性的抑制率为指标,分别对其提取工艺进行了单因素和正交试验,选取优化后抑制率高的提取物进行抑制类型和3T3-L1前脂肪细胞方面的研究。结果表明醇提物的最佳提取工艺为提取温度70℃、提取时间1h、乙醇浓度90%、料液比1:20,抑制胰脂肪酶的IC₅₀=681.56μg/mL。水提物的最佳提取工艺为提取温度70℃、提取时间2h、料液比1:40,抑制胰脂肪酶的IC₅₀=850.59μg/mL,醇提物的抑制效果优于水提物。醇提物对胰脂肪酶的抑制类型为非竞争性抑制,抑制常数为4.69mg/mL;醇提物浓度低于1mg/mL时,对3T3-L1前脂肪细胞的活性无影响;浓度高于400μg/mL时即可显著抑制前脂肪细胞的分化。     

斜带石斑鱼2种胰脂肪酶基因全长cDNA序列的克隆与分析

利用RT-PCR和RACE方法克隆得到斜带石斑鱼（Epinephelus coioides）肝胰脏中胆盐活化的胰脂肪酶(bile salt-activated lipase,BSAL)和依赖于辅酶的胰脂肪酶(colipase-dependent pancreatic lipase,PL)基因的全长cDNA序列.BSAL基因全长cDNA序列1 796 bp,编码558个氨基酸,该蛋白序列含有BSAL的全部特征结构区,与其他脊椎动物BSAL的氨基酸序列同源性为49.9%～57.3%.PL基因的全长cDNA序列1 503bp,编码465个氨基酸,该蛋白序列含有PL全部的特征结构区,与其它脊椎动物PL的氨基酸同源性为49.1%～73.9%.系统树分析表明,斜带石斑鱼BSAL和PL与其它物种BSAL、PL和胰脂肪酶相关蛋白(PL-RP)聚于进化树的两个不同分支,属于2种不同的胰脂肪酶.结果证实,在同一鱼类体内也存在BSAL和PL两种胰脂肪酶基因.     

麻疯树叶中过氧化物酶JCP-1的分离纯化和几种特性的检测

麻疯树叶片蛋白粗提液经硫酸铵分级沉淀,强阴离子琼脂糖、强阳离子琼脂糖和交联葡聚糖层析,得到一个比活为4499U·mg-1（蛋白）过氧化物酶,命名为JCP-1。其分子量为49kDa,等电点为pH3.3,最适pH为5.0-6.0。以H2O2为底物的Km为2.14mmol·L-1。JCP-1具有宽泛的最适保存pH（7.0-11.0）和较高的耐热性（80℃高温处理15min,活性保持在90%以上）。30%PEG6000处理模拟干旱胁迫及50℃高温胁迫麻疯树苗,其叶片中JCP-1活性分别提高121%和155%。     

Enhanced production of Penicillium expansum PED-03 lipase through control of culture conditions and application of the crude enzyme in kinetic resolution of racemic Allethrolone

Alkaline lipase production was performed in submerged fermentation by Penicillium expansum PED-03. It was found that the suitable carbon source and nitrogen source for lipase production were 0.5% starch and 4.0% soybean meal, respectively. The maximal lipase activity (850 U/mL) of production was achieved at initial pH 5.5-6.0, 26 degrees C, 72 h. Tween-80 was an effective enhancer for lipase production. Agitation speed of the fermentor played an important role, and the suitable agitation speed for lipase production was 500 r/min. The lipase was stable within the range of pH 7.0-10.0 and 20-40 degrees C, and the optimum conditions for the enzymatic reaction were 35 degrees C and pH 9.5. The enzymatic resolution of racemic allethrolone (4-hydroxy-3-methyl-2-(2-propenyl)-2- cyclopenten-1-one) was carried out by the lipase from P. expansum PED-03, and the conversion reached 48% with excellent enantioselectivity (E > 100), which showed a good application potential in the production of optically pure allethrolone.     

铜藻岩藻黄素提取及纯化工艺研究

为了对岩藻黄素的提取、纯化进行系统研究,进而为高纯度岩藻黄素的工业化生产提供研究基础,筛选了适用于提取铜藻（Sargassum horneri）鲜藻中岩藻黄素的有机溶剂,并通过单因素实验和正交实验确定了最佳的提取溶剂浓度、提取温度、提取时间、料液比等工艺参数。随后采用硅胶柱层析法进行纯化,并通过单因素实验确定了最佳的硅胶柱床高度、上样量和洗脱流速。最后采用制备液相法对经层析纯化的岩藻黄素进一步纯化。结果表明,有机溶剂萃取的最佳工艺条件为：甲醇浓度90%,提取温度50 ℃,提取时间1 h,料液比1∶10,此条件下岩藻黄素提取率达到(0.258 9±0.003 6) mg·g-1鲜重（FW）［(1.078 8±0.015 0) mg·g-1干重（DW）］。硅胶柱层析的最佳工艺条件为：硅胶柱床高度10 cm,上样量6 g,洗脱流速10 mL·min-1,此条件下岩藻黄素得率为0.176 5 mg·g-1FW（0.735 3 mg·g-1 DW）,纯度为87.01%±0.88%。经制备液相进一步纯化后,岩藻黄素得率为0.127 1 mg·g-1 FW（0.529 4 mg·g-1 DW）,纯度为99.27%±0.22%。研究所用工艺简单,岩藻黄素得率高,为高纯度岩藻黄素的制备提供了实验基础。     

Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Abstract

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of conditions showed that the maximal loading of lipase on PA-M reached 230.2 mg g^{? 1} at pH 9.0 and 35°C. An increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed. Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on PA-M was thermodynamically favorable. Covalent crosslinking of the lipase adsorbed extended the pH range and increased the optimal temperature of the lipase activity. The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase) retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase, covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.     

菜豆幼苗EPSP合成酶的分离纯化和它的部分性质

利用硫酸铵分级沉淀,SephedexG-50凝胶柱层析,FPLCMono-Q和磷酸纤维素离子层析法从菜豆幼苗中分离提纯了EPSP合成酶。该酶被纯化2961.6倍,比活性达到6219.4nmolmg-1蛋白min-1。该酶分子量经SDS-PAGE检测为51kD,等电点为pH5.7,酶促反应最适pH7.5,最适温度45℃。6.2μmol/L的除草剂草甘膦能抑制EPSP合成酶活性的50%。     

粗茎鳞毛蕨孢子萌发研究

以经过3年低温储藏的粗茎鳞毛蕨孢子为实验材料,从孢子离心、孢子消毒、培养基种类、光质等4方面对孢子萌发进行研究,结果表明:在离心转数≤14 000 r.min-1、离心时间≤30 min条件下,离心处理对孢子萌发基本无影响;对孢子进行1%NaClO水溶液浸泡处理20～30 min为最佳消毒条件;改良Knop’s培养基为最佳孢子萌发培养基;黑暗条件下孢子不能萌发,但是黑暗处理能够明显提高孢子萌发整齐性;红光比白光能促进孢子提早萌发1 d左右,但对提高萌发率效果不显著。     

聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液(pH4.0),1%PAA选择性沉淀碱性蛋白(等电点pI为8.65~9.30)的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA(1%)用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH>9.0),当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经SephadexG-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

Purification and characterisation of adenosine-3',5'-phosphate-independent protein kinase from wheat germ

cAMP-independent protein kinase was isolated from the wheat germ and purified to electrophoretic homogeneity. The molecular weight of enzyme was approximately 20,000, Km for ATP was (1 +/- 0.2) x 10(-5) M. V was 215 nmol phosphate mg enzyme-1 min-1, and the isoelectric point was at pH 9.2. The enzyme promotes phosphorylation of casein and crude wheat germ ribosomes.     

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.     

Purification and properties of chlorophyllase from greened rye seedlings

1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.