Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

从发酵液中萃取L-乳酸的工艺

以L-乳酸发酵液为对象,以正丁醇为萃取剂,在pH为2、温度为25℃、n（乳酸）：n（正丁醇）=1：1、乳酸在发酵液中的质量分数为30％时,经3次萃取后,最终的萃取率可达到75．7％。萃取完成后,不需要将乳酸进行反萃,可将所得到的正丁醇-乳酸的混合体系作为底物进行酯化反应,生成乳酸丁酯,从而避开提取纯乳酸的高操作要求。     

In situ product recovery of n-butanol using polymeric resins

Polymeric resins with high n-butanol adsorption affinities were identified from a candidate pool of commercially available materials representing a wide array of physical and chemical properties. Resin hydrophobicity, which was dictated by the chemical structure of its constituent monomer units, most greatly influenced the resin-aqueous equilibrium partitioning of n-butanol whereas ionic functionalization appeared to have no effect. In general, those materials derived from poly(styrene-co-divinylbenzene) possessed the greatest n-butanol affinity, while the adsorption potential of these resins was limited by their specific surface area. Resins were tested for their ability to serve as effective in situ product recovery (ISPR) devices in the n-butanol fermentation by Clostridium acetobutylicum ATCC 824. In small-scale batch fermentations, the addition of 0.05 kg/L Dowex Optipore SD-2 facilitated achievement of effective n-butanol titers as high as 2.22% (w/v), well above the inhibitory threshold of C. acetobutylicum ATCC 824, and nearly twice that of traditional, single-phase fermentations. Retrieval of n-butanol from resins via thermal treatment was demonstrated with high efficiency and predicted to be economically favorable. Due to its modular nature, the proposed ISPR design exhibits strong potential for compatibility with future n-butanol fermentation efforts.     

Purification of industrial alphaamylase by reversed micellar extraction

The purification of industrial alpha-amylase by liquid-liquid extraction with Aliquat 336 reversed micellar solution as the extractant was studied. Seven kinds of Aliquat 336 reversed micellar solution, formed by using seven kinds of straight chain alkyl alcohols as cosolvent, have been utilized to extract industrial a-amylase. It was found that these seven kinds of reversed micellar solution can all achieve a high protein transfer efficiency in the forward extraction process. After a full forward and backward extraction cycle, however, only the reversed micelles with n-butanol as the cosolvent was found to be able to maintain the activity of alpha-amylase in the stripping solution. By using the reversed micelles of Aliquat 336/isooctane/1% (v/v) n-butanol to perform a full extraction cycle, it was found that 85% of the total activity of alpha-amylase in the industrial a-amylase could be recovered at the end of an extraction cycle and the specific activity of alpha-amylase could be concentrated about 1.5-fold; meanwhile, most of the neutral protease in the industrial a-amylase could be removed. The separation factor of alpha-amylase to neutral protease at the end of an extraction cycle can reach about 10. (c) 1995 John Wiley & Sons, Inc.     

Radioassay of bile acid coenzyme A:glycine/taurine: N-acyltransferase using an n-butanol solvent extraction procedure

A rapid, specific, and sensitive radioassay for measuring bile acid CoA:glycine/taurine: N-acyltransferase (EC 2.3.1) has been developed. In this assay, 3H-labeled amino acids (glycine or taurine) are conjugated with unlabeled bile acid CoA derivatives to form 3H-labeled bile acid amidates. Following incubation, the 3H-labeled bile acid amidate is separated from the unreacted amino acid by an n-butanol extraction method. The extraction procedure was developed by evaluating the effects of buffer concentration and pH on the recovery of radiolabeled bile acid amidate standards in the presence of human hepatic cytosol. Highest recovery (greater than 90%) of bile acid amidate standards occurred under acidic conditions (pH 2) in the presence of 1% (w/v) SDS. When the radioassay and accompanying n-butanol extraction procedure were utilized to study the amidation of glycine or taurine with cholic acid in human hepatic cytosol, a single peak of radioactivity corresponding with either authentic glycocholate or taurocholate was detected in the n-butanol phase by high-performance liquid chromatography. This assay for bile acid CoA:glycine/taurine: N-acyltransferase activity was linear with incubation time and protein concentration. This assay should be useful in the biochemical studies of this enzyme, as well as in the examination of bile acid amidation in clinical liver specimens.     

大蒜化学成分的气-质联用分析

本文采用环己烷、乙酸乙酯和正丁醇对大蒜（Allium sativum L.）新鲜鳞茎的95%乙醇提取物进行萃取,并与水蒸气蒸馏法提取的挥发油成分进行比较,用GC-MS对其成分进行定性和定量分析.在环己烷萃取物中共检出112个成分,鉴定了38个化合物,占环己烷萃取物总量的80.08%;在乙酸乙酯萃取物中检出86个成分,鉴定了26个化合物,占乙酸乙酯萃取物总量的56.70%;在正丁醇萃取物中未检出挥发性成分.在水蒸气蒸馏法提取的挥发油中共检出109个成分,鉴定了29个化合物,占挥发油总量的83.58%.大蒜95%乙醇提取物的环己烷和乙酸乙酯萃取物及大蒜挥发油中皆以含硫化合物为主.在环己烷和乙酸乙酯萃取物中,阿霍烯的含量分别为生药的0.00395%和0.00145%,大蒜中阿霍烯含量达0.00540%.     

Novel prefractionation method can be used in proteomic analysis

For the first time, a novel prefractionation method used in proteomic analysis was developed, which is performed by a novel aqueous two-phase system (NATPS) composed of n-butanol, (NH(4))(2)SO(4), and water. It can separate proteomic proteins into multigroups by one-step extraction. The phase-separation conditions of n-butanol solutions were studied in the presence of commonly used inorganic salts. The NATPS was subsequently developed. Using human serum albumin, zein, and gamma-globulin as model proteins, the separation effectiveness of the NATPS for protein was studied under affection factors, i.e., pH, n-butanol volume, protein, or salt concentration. The model and actual protein samples were separated by the NATPS and then directly used for gel electrophoresis without separating the target proteins from phase-forming reagents. It revealed that the NATPS could separate proteomic proteins into multigroups by one-step extraction. The NATPS has the advantages of rapidity, simplicity, low cost, biocompability, and high efficiency. It need not separate target proteins from the phase-forming reagents. The NATPS has great significance in separation and extraction of proteomic proteins, as well as in methodology.     

人胎盘酸性β-1,4葡萄糖苷酶的纯化及部分性质

 本文以正常人胎盘为材料,通过匀浆、硫酸铵分级分离、20％正丁醇抽提、Sephadex G-200凝胶过滤及Concanavalin A-Sepharose 4B亲和层析等分离纯化步骤,制备获得了纯化3705倍的酸性β-1,4葡萄糖苷酶,其比活性和获得率分别为277261.33n mol·(mg Prot·h)~(-1)和14.3％。 经agarosn-IEF检验,该纯化的酸性β-1,4葡萄糖苷酶已达蛋白电泳单点纯,PI为5.2。经3～28％梯度PAGE,求知该酶单体分子量为74kD。该纯制酶极不稳定,4℃贮存6天后活性即降低50％以上,与其特异性抗体结合后,活性全部被抑制。     

Alkaline phosphatase in Hirudo medicinalis L.: partial purification and study of certain molecular and catalytic properties

The authors carried out extraction and purification of an alkaline phosphatase (AP) from Hirudo medicinalis and studied certain characteristics of the enzyme. After homogenizing specimens of H. medicinalis and centrifuging the homogenate at 70,000 g, the enzyme, likely membrane-bound, was obtained in a soluble form by treating the sediment with n-butanol. It was then purified by acetone fractionation and Sephadex G-200 chromatography. A fairly good purification degree was achieved; the enzyme appears to be in a single form and displays a molecular weight of about 268,000. The optimum pH (9.5) is lower than the ones generally observed as regards APs from both Invertebrata and Mammalia. The studied AP displays a strong substrate inhibition, similar to that concerning Metazoa at a higher evolutionary level (Mollusca, Echinodermata). On the contrary, the enzyme-substrate affinity, as shown by Km value (2.447 mM), is lower than as regards APs from more advanced organisms (Echinodermata, Mammalia).     

Extraction and partial purification of mouse uterine alkaline phosphatase.

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0.2% (v/v) Triton X-100 and extracted wtih 20% (v/v) n-butanol. The procedure, which resulted in 182-fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex G200 gel filtration. Solubilization with Triton X-100 was an important step in the procedure since extraction with n-butanol alone only partially solubilized the enzyme and gave low extraction yields, much of the enzyme activity remaining in association with negatively charged residues. However, butanol extraction of Triton X-100-treated homogenates gave high yields of enzyme and eliminated p-nitrophenyl phosphatases which displayed activity in the pH range 3.0--7.5, together with a large proportion of inactive protein. The activity of the purified enzyme preparations was electrophoretically homogeneous on cellulose acetate membranes, suggesting that the alkaline phosphatase in the mouse uterus exists in a single isozymic form. Polyacrylamide-gel electrophoresis revealed that the purified preparations contained at least one protein as an impurity. Attempts to further purify the alkaline phosphatase by isoelectric focusing were unsuccessful since the enzyme was found to have an isoelectric point of about 5.0 and at this pH it was rapidly inactivated.     

Solvent screening methodology for in situ ABE extractive fermentation

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.     

Extractions of pyrroloquinoline quinone from crude biological samples

The best conditions for extractions of free pyrroloquinoline quinone (PQQ) from crude biological samples were investigated with various organic solvents and Sep-Pak C18 cartridges. PQQ was measured with use of its native fluorescence in aqueous solution. PQQ was well extracted into n-butanol under acid conditions, and addition of NaCl did not improve the solvent extraction. PQQ, which had been extracted into n-butanol, could be re-extracted into an aqueous phase by addition of either n-heptane or pyridine, or combination of them. PQQ, which had been adsorbed to Sep-Pak C18 cartridges, could be eluted with a mixture of pyridine and water with very excellent recovery. The recovery of 1 micrograms PQQ, which had been added to 1 g human liver, brain and 1 ml plasma and had undergone the n-butanol and the Sep-Pak extractions, was 50, 75 and 105%, respectively. From the blank fluorescence, endogenous levels of free PQQ in human liver, brain and plasma were found not greater than 0.41, 0.08 and 0.13 micrograms/g or ml, respectively, if present.     

Solvent extraction method used in separating the protected product of synthetic oligodeoxyribonucleotides

A solvent extraction method for separating synthetic protected oligodeoxyribonucleotides was used in our laboratory based on the lipophilic property of the protecting group of 5'-OH of the oligomers. The extraction of synthetic products protected with MMTr is complete by ether or ether-chloroform (6:1 V/V) for mononucleosides, by chloroform for dinucleoside monophosphates, by dichloromethane:n-butanol (4:1 V/V) for trimer or tetramer, and is nearly complete by dichloromethane:n-butanol (2:1 V/V) for hexamer. The 5'-end phosphorylated nucleotides, oligonucleotides and their symmetrical pyrophosphates remain in water phase. The following synthetic products of protected oligodeoxyribonucleotides have been isolated with this method, all above 85% in purity: (Formula: see text).     

蒙药材瑞香狼毒的化学成分研究

采用溶剂提取及柱色谱等方法,首次对瑞香狼毒Stellera chamaejasme L.的正丁醇萃取部位进行系统研究,分离得到6个苯丙素类化合物,并运用UV、1H NMR、13C NMR等现代波谱技术依次鉴定为伞形花内酯7-O-β-D-吡喃木糖(1→6)-β-D-吡喃葡萄糖苷(1),芥子醇1,3’-双-O-β-D-吡喃葡萄糖苷(2),紫丁香苷(3),(+)-落叶松树脂醇4,4’-O-β-D-吡喃葡萄糖苷(4),(+)-松树脂醇4,4’-O-双-β-D-吡喃葡萄糖苷(5)和(+)-丁香树脂醇-双-O-β-D-吡喃葡萄糖苷(6)。其中,化合物4、6为首次从该药材中分离得到。     

Ile-Ser-bradykinin (T-kinin) and Met-Ile-Ser-bradykinin (Met-T-kinin) are released from T-kininogen by an acid proteinase of granulomatous tissues in rats

An acid proteinase of granulomatous tissues in rats with carrageenin-induced inflammation released kinin from T-kininogen. The kinin isolated by n-butanol extraction was separated by reverse-phase high-performance liquid chromatography into T-kinin and a T-kinin derivative. From determination of its amino acid composition and its immunoreactivity toward anti-bradykinin antiserum, the T-kinin derivative was identified as Met-Ile-Ser-bradykinin (Met-T-kinin).     

Single-phase butanol extraction: A new tool for proteolipid isolation

An alternative method to the chloroform/methanol extraction of proteolipids is presented. The proteolipid fraction from sarcoplasmic reticulum membranes is isolated by a single-phase n-butanol extraction and subsequently precipitated by diethyl ether. The two step procedure described is successfully applied in the purification of the dicyclohexylcarbodi-imide binding protein from submitochondrial particles.     

红豆树种子化学成分及其抗氧化和抑菌活性研究

为研究红豆树种子的化学组成及生物活性。本文采用气相色谱-质谱联用技术(GC-MS)对其甲醇提取物的化学成分进行了鉴定,并首次采用不同极性溶剂对红豆树种子的生物活性物质进行萃取;同时,利用DPPH法、ABTS法和抑菌圈法评价红豆树种子生物活性物质的体外抗氧化及抑菌活性。结果表明,从红豆树种子萃取物中共检测出化合物12个,占萃取物总量的89.03%;种子萃取物的主要成分为5-羟甲基糠醛(52.98%)、D-阿洛糖(7.24%)、2,3-二甲氧基-10,11-二氢二苯并(b,f)恶庚英-10-醇(6.51%)、甲基丁香酚(4.53%)、2,3-二氢-3,5-二羟基-6-甲基-4H-吡喃-4-酮(4.45%)、黄樟素(3.75%)、α-松油醇(3.31%)、2,6-二甲氧基苯酚(2.01%),此8种成分占总量的84.78%。红豆树种子正丁醇、乙酸乙酯和石油醚等萃取物对DPPH和ABTS自由基均具有显著的抗氧化活性,且抗氧化活性与萃取物浓度呈线性相关。当红豆树种子正丁醇、乙酸乙酯和石油醚等萃取物浓度为10.0 mg/mL时,正丁醇萃取物对大肠杆菌、绿脓杆菌和鼠伤寒沙门氏菌的抑菌效果最佳;乙酸乙酯萃取物对枯草芽孢杆菌的抑菌效果最佳;石油醚萃取物对金黄色葡萄球菌和苏云金芽孢杆菌的抑菌效果最佳。研究结果为红豆树种子资源的开发和综合利用提供了数据支持。     

Rapid solubility determination of the triterpenes oleanolic acid and ursolic acid by UV-spectroscopy in different solvents

Salvia triloba (Greek sage) has been used for the treatment of various diseases and contains two bioactive triterpene acids of major interest: oleanolic acid (OA) and ursolic acid (UA). The determination of the solubility of OA and UA in different solvents is a prerequisite to select the optimal solvent. The main goal of this work was to develop a quick method of predicting the solubility of OA/UA in different solvents to get a first indication of which solvents could be considered suitable for extraction from any plant material containing at least one of these triterpenes. A novel and simple ultra-violet spectroscopy method was developed for this purpose.The best solubilities were determined in THF, dioxane and n-butanol as well as in blends of dioxane and n-butanol.     

Isolation of 2-hydroxycarboxylic acids with a boronate affinity gel

Boric acid has been known to make a complex with 2-hydroxycarboxylic acids. Based on this principle, we have developed a new method for isolation of vanillylmandelic acid, p-hydroxymandelic acid, vanillyllactic acid, and p-hydroxyphenyllactic acid in urine. The technique involves two steps: extraction of lipophilic compounds from urine with n-butanol and selective isolation of 2-hydroxy acids in the n-butanol phase on a phenylboronate gel column. 2-Hydroxy acids were eluted from the column with 1.5 ml of 2% acetyl chloride in methanol with the recoveries of ca. 70%. According to analysis of urinary extract by gas chromatography--mass spectrometry, the mean excretion rates of the above four compounds in 12 healthy subjects were 3.68, 1.93, 0.091, and 1.08 mg/day, respectively.     

Enzymatic oxidation of isethionate to sulfoacetaldehyde in bacterial extract.

Isethionate degradation in a bacterial extract was shown by the isolation of enzymes and by identification of an intermediate to take place in two steps; dehydrogenation to sulfoacetaldehyde and desulfonation leading to the formation of sulfite and acetate. The enzyme responsible for isethionate oxidation in the presence of FAD was particulate in nature and a solubilized preparation obtained by extraction with buffer of low ionic strength had oxidizing activities against only isethionate and n-butanol among compounds tested. The enzyme was inhibited by thiol and carbonyl reagents.