氨基酸制备色谱——丙氨酸、缬氨酸和亮氨酸制备柱柱效率研究

现代液相色谱已从分析色谱发展到制备色谱,为了研究氨基酸制备色谱柱柱效率,我们从液相色谱理论的十几个基本关系式中归纳出五个基本方程式,用以评价丙氨酸、缬氨酸和亮氨酸制备柱柱效率。初步试验结果表明,对强酸性阳离子交换树脂柱而言: 1.凝胶型离子交换树脂柱柱效率高于普通大孔型离子交换树脂柱柱效率,而与本室合成的Dpsc大孔型离子交换树脂柱柱效率相当。 2.凝胶型离子交换树脂中,低交联度离子交换树脂有利于提高缬氨酸和亮氨酸制备柱柱效率,可较好地分离缬氨酸和亮氨酸;较高交联度离子交换树脂有利于提高丙氨酸和缬氨酸制备柱柱效率,可较好地分离丙氨酸和缬氨酸。将不同的离子交换树柱组合起来,可以提高丙氨酸,缬氨酸和亮氨酸的分离度,提高氨基酸的回收率。这项研究成果已应用到从猪血粉、猪毛等天然蛋白质酸水解物中系统分离多种氨基酸,以及分别从缬氨酸、亮氨酸(异亮氨酸)发醇液中分离纯化缬氨酸、亮氨酸(异亮氨酸)。     

从发酵液中高效提取L-缬氨酸的工艺研究

目的:利用有机膜过滤和离子交换法分离提取发酵液中的L-缬氨酸。方法:通过有机膜过滤,除去发酵液中的菌体及蛋白,滤液浓缩结晶获得L-缬氨酸产品,通过离子交换法从结晶母液中回收部分L-缬氨酸。结果:确定了有机微滤膜和超滤膜去除发酵液中菌体蛋白和色素的操作条件;确定了采用离子交换法提取L-缬氨酸的操作条件:选择732强酸性阳离子树脂,料液pH值为3.0左右,用0.4 mol/L的氨水以1.0 mL/min的速度洗脱,L-缬氨酸的收率为89.2%。结论:通过有机膜过滤和离子交换法分离提取发酵液中的L-缬氨酸,可以提高提取收率和产品质量。     

用离子交换树脂分离缬氨酸的研究

<正> 一、前言L-缬氨酸为人体八种必需氨基酸之一,是配制氨基酸输液的重要原料。目前国内采用发酵法的工业化生产报道不多见。我厂采用北京棒状杆菌AS1586发酵法生产缬氨酸的研制工作,虽于1983年底通过鉴定,但由于发酵法生产缬氨酸除因产酸不太高(2%左右)外,还受到多种副生酸的干扰分离,给离子交换树脂提取带来了很大的困难。曾采用串柱方式,但单斑分离周期     

用吸附柱和离交柱层析法从猪血粉水解液中连续分离七种氨基酸

<正> 本文介绍了采用国产活性炭及阳离子交换树脂两种柱体巧妙地组合,并应用不同类型的洗脱剂,变换其浓度和洗脱速度,相互配合,能成功地从猪血粉水解液中一次连续分离和制取L-苯丙氨酸、L-酪氨酸、L-亮氨酸、L-缬氨酸、L-组氨酸、L-赖氨酸和L-精氨酸。多次提取试验表明,工艺操作简单、稳定可靠,苯丙氨酸与酪氨酸、亮氨酸与缬氨酸分离良好,     

用阴阳离子交换树脂分离提纯D-蛋氨酸的比较

应用阴阳离子交换树脂分离、提纯D-蛋氨酸,通过不同的树脂对比实验,初步确定了水解液通过阳离子NH4+型柱吸附,再用0.15 mol.L-1的氨水解析的工艺流程,解析液对水解液的D-蛋氨酸收率为87%。     

单柱法分离L-缬氨酸的工艺研究

本文用离子交换树脂从发酵液中分离中性(支链)氨基酸——L-缬氨酸,进行了一些探讨。将双柱串联分离(Ⅰ—号工艺)改为单柱一次分离(Ⅱ—号工艺) 本工艺的小试与放大试验(65L柱)的数据基本吻合,均达到了:离交周期<30小时。单斑收率约90%,具有降低成本、提高收率,减少投资等优点。还介绍了,以线性梯度洗脱为依据的Ⅲ—号工艺,具有单斑收率也较高,更快速的优点。     

离子交换法分离D-天冬氨酸和L-丙氨酸

对离子交换法分离D-天冬氨酸和L-丙氨酸的工艺条件进行了研究。综合考虑树脂对D-天冬氨酸和L-丙氨酸的吸附容量及相对选择系数,选择了一种适合该体系分离的树脂—XH-1,并对吸附条件及洗脱条件进行了研究,确定了最佳工艺条件,为今后工业应用提供一定的依据。     

L-缬氨酸高产菌株的选育研究

以产L-缬氨酸的谷氨酸棒状杆菌(Corynebacterium glutamicum)为原始菌株,利用注入低能氮离子束进行一系列诱变,获得一株稳定的高产L-缬氨酸突变菌株。摇瓶培养96h后发酵能力可达38.0g·L-1,较出发菌株提高18.01%。通过对摇瓶中葡萄糖、玉米浆浓度及培养条件进行优化,发酵能力达到40.6g·L-1,50L发酵罐的发酵能力可达70g·L-1左右。     

不同营养条件下竹叶眼子菜NH4^＋-N吸收动力学的初步研究

以竹叶眼子菜(Potamogeton malaianus)无菌系种苗为试验材料,研究了不同水体营养浓度水平(低营养:TN 0.213 mg·L-1,TP 0.0093 mg·L-1;中营养:TN 0.71 mg·L-1,TP 0.031 mg·L-1;高营养:TN 7.1 mg·L-1,TP 0.31 mg·L-1)对其生长与NH+4-N的吸收动力学参数的影响.结果表明,不同浓度水体营养对竹叶眼子菜生长的影响较小,而NH+4-N的吸收动力学参数有显著差异.竹叶眼子菜在高、中和低营养培养条件下的NH+4-N最大吸收速率Vmax分别为 41.1、 29.1、 21.1 μmol·g-1·h-1,米氏常数Km分别为 0.356、 0.306、 0.122 mmol·L-1.竹叶眼子菜营养吸收动力学与其生长环境关系紧密,在低浓度生长环境中时,竹叶眼子菜可以通过降低Km值来提高对营养离子的亲和力以满足营养需求;在高浓度生长环境中,该植物通过增大吸收潜力来适应高营养.     

脲酶Km值简易测定方法的改进

对脲酶Km值简易测定方法中存在的问题和(NH4)2SO4标准溶液浓度、尿素浓度、脲酶终止剂、缓冲液系统以及脲酶用量等因素对Km值的影响,进行了多组对比测定.将该方法测定条件优化为:(NH4)2SO4标准溶液浓度0.001 mol·L-1,尿素浓度1/250、1/200、1/150、1/100、1/50 mol·L-1,终止剂10%硫酸锌,缓冲液1/15 mol·L-1Tris-H2SO4(pH 7.0),脲酶用量0.50mL.     

离子交换层析纯化透明质酸

考察6种离子交换树脂的静态吸附解析效果,选出201＊7阴离子交换树脂填柱,确定洗脱流速为0.6mL/min,40mL0.3mol/LNaCl和50mL0.5mol/L NaCl双浓度洗脱,实现透明质酸和杂蛋白的分离。制得透明质酸产品蛋白含量为0.057%,葡萄糖醛酸含量为43%,平均相对分子质量大于1.1×10^6,收率为54%,符合医用级透明质酸行业标准的要求。     

Fermentation and recovery of glutamic acid from palm waste hydrolysate by Ion-exchange resin column

Glutamic acid produced from palm waste hydrolysate by fermentation with Brevibacterium lactofermentum ATCC 13869 is produced with a remarkably high yield compared with that produced from pure glucose as a carbon source. The produce yield is 70 g/L with glucose, wherease, when palm waste hydrolysate is the fermentation medium in the same bioreactor under same conditions, it is 88 g/L. The higher yield may be attributed to the fact that this organism has the ability to convert sugars other than only glucose present in the hydrolysate. Bioreactor conditions most conducive for maximum production are pH 7.5, temperature of 30 degrees rmentation period of 48 h, inoculum size 6%, substrate concentration of 10 g per 100 mL, yeast extract 0.5 g per 100 mL as a suitable N source, and biotin at a concentration of 10 pg/L. Palm waste hydrolysate used in this study was prepared by enzymic saccharification of treated palm press fiber under conditions that yielded a maximum of 30 g/L total reducing sugars. Glutamic acid from fermentation broth was recovered by using a chromatographic column (5cm x 60 cm) packed with a strong ion-exchange resin. The filtered broth containing glutamic acid and other inorganic ions was fed to the fully charged column. The broth was continuously recycled at a flow rate of 50 mL/min (retention time of 55 min) until glutamic acid was fully adsorbed on the column leaving other ions in the effluent. Recovery was done by eluting with urea and sodium hydroxide for total displacement of glutamic acid from the resin. The eluent containing 88 g/L of glutamic acid was concentrated by evaporation to obtain solid crystals of the product. (c) 1995 John Wiley & Sons, Inc.     

Distribution of saccharides and salts on amphoteric ion-exchange resin

An amphoteric ion-exchange resin hardly shrank in 550 and 300 g/L glucose and sodium chloride solutions, respectively; however, the bed packed with a cation-exchange resin shrank considerably. From the distribution coefficients of some saccharides, the swelling pressure of the amphoteric ion-exchange resin was estimated to be 2.0 MPa at 25 °C. The distribution coefficients of glucose, galactose, fructose, and mannose were independent of their concentration and were about 0.621. On the other hand, the apparent distribution coefficients of NaF, NaCl, NaBr, NaI, LiCl, KCl, and CsCl largely depended on concentration. A model for the distribution of salts on the amphoteric resin was proposed, assuming an interaction between the anion of the salt and the positively charged fixed ions with binding constant B. The B values of the chloride salts were nearly the same (1.69–2.94 L/mol), while the values of the sodium salts were largely different depending on the anion.     

Elimination of phenylalanine from amino acid mixtures obtained from yeast hydrolysates and autolyzates]

In order to obtain amino acid mixtures devoid of phenylalanine, adsorption of aromatic amino acids was studied during their chromatography on ion-exchange resin IA-Ip. When 40 g of the amino acid mixture were passed through 400 ml of the swollen ion-exchange resin, phenylalanine and tyrosine were eliminated and 67% of dry substance were yielded. Ion-exchange resin adsorbed phenylalanine and tyrosine could be regenerated. The use IA-Ip is advantageous as compared with that of ion-exchange resin Amberlite IR-45 and activated charcoal.     

重组人源过氧化氢酶表达优化和纯化研究

研究了重组表达人源过氧化氢酶酵母菌株G13的发酵工艺和纯化方法.对接种量、生长期培养基、生长时间、诱导时间等因素进行了优化,并确立了10 L发酵罐的发酵条件.结果表明,菌体最高密度达到了0.15 g/mL,比原重组菌株S4提高了1倍,人源过氧化氢酶表达量也由原来的平均600 U/mL提高到了1500 U/mL.同时建立...     

我国大豆种子中球蛋白2S组分的分离纯化及部分性质的研究

根据大豆种子球蛋白和清蛋白溶解性不同的原理,分离出我国红丰3号大豆种子球蛋白2S粗制剂,再用SephadexG-100柱层析对其进行纯化。纯化后的球蛋白表现SDS-聚丙烯酰胺凝胶电泳非均一性。对这样纯化过的2s球蛋白进行DEAE-纤维素离子交换柱层析分离,用0.1—0.5mol/L pH7.6磷酸盐缓冲液进行线性梯度洗脱,得到四个洗脱峰,每个峰都获得了PAGE单一条带。四个组分分别命名为SⅡP_1,SⅡP_2,SⅡP_3和SⅡP_4。然后对这四种蛋白质的某些性质进行了研究,结果表明四者的分子量按以上顺序分别为22800,21500,19200和17800。所含残基数分别为191,179,163和147个。SⅡP_1,SⅡP_2和SⅡP_3三者的沉降系数(S_(20),w)分别为2.1S,1.9S和1.8S。N-末端分析表明这四种蛋白质的N-末端均为天门冬氨酸.还发现SⅡP_2具有能抑制α-胰凝乳蛋白酶的活性。本实验所提纯的这个抑制剂的一个ATEE(N-乙酰-L-酪氨酸乙酯)单位为0.4μg抑制剂蛋白(仅指对α-chymotrypsin发生作用)。α-chymotrypsin与此抑制剂相互作用时的摩尔数比初步判断为E/I=2/1。     

Extractive lactic acid fermentation using ion-exchange resin

Lactic acid fermentation is an end-product-inhibited reaction. The restriction imposed by lactic acid on its fermentation can be avoided by extractive fermentation techniques. Studies were performed by attaching an ion-exchange resin packed column with a 2-L fermentor for separation of lactic acid. The fermentation, in a conventional batch mode, resulted in a lactic acid yield of 0.828 g . g(-1) and a lactic acid productivity of 0.313 g . L(-1) . h(-1). However, these could be further enhanced to 0.929 g . g(-1) and 1.665 g . L(-1) . h(-1) by extractive fermentation techniques. The effect of temperature on extractive fermentation was remarkable and has been included in this work.     

Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography

A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio A₂₇₈/A₅₉₇ of 1.10±0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - Tris Tris(hydroxymethyl)aminomethane - FSCE free solution capillary electrophoresis     

Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.     

A comparison of the amino acid sequences of Crithidia fasciculata and Trypanosoma rhodesiense cytochromes c

Apocytochrome c was isolated from procyclic trypomastigotes of Trypanosoma rhodesiense EATRO 1895 and purified on Amberlite IRC-50 ion-exchange resin. Tryptic peptides were generated from the purified apoprotein and a partial amino acid sequence was determined. A comparison of the amino acid sequence of Crithidia fasciculata with the partial amino acid sequence of T. rhodesiense reveals significant homology.