酶解法提取纯化虎杖提取物中自藜芦醇的工艺研究

本文对虎杖提取物中虎杖苷的酶解条件及苷元白藜芦醇的提取纯化工艺进行研究,以样品中自藜芦醇的含量为指标,对纤维素酶、β-葡萄糖苷酶、复合酶进行筛选,结果表明以复合酶的水解效率最高:采用正交实验对影响复合酶酶解的因素:加酶量、温度、酶解时间进行考察;并对酶解后提取物中自藜芦醇的提取纯化工艺进行研究.得出如下较理想的酶解条件和提取纯化工艺:虎杖提取物,加水(pH 5)10倍,加20%的复合酶,于50℃保温24 h;酶解后的提取物经水、乙醇-水、碱溶液分步溶解沉淀,得白藜芦醇粗品,含量可达65%,工艺稳定可行.     

A comparison of three methods of glycogen measurement in tissues

Three methods have been used for analysis of glycogen in tissue homogenates: hydrolysis of the tissue in acid and followed by enzymic analysis of the resulting glucose; enzymic hydrolysis with amylo-α-1,4-α-1,6-glucosidase, again followed by enzymic measurement of glucose; and degradation of the glycogen with phosphorylase and debrancher complex coupled to measurement of the resulting glucose-1-P. The two enzymic procedures yielded equivalent results with all tissues examined (brain, liver, muscle and polymorphonuclear leucocytes). Acid hydrolysis of the tissues resulted in higher values for brain tissue only, presumably due to the hydrolysis of the gangliosides and cerebrosides present in brain.     

CHEMICAL AND ENZYMIC DEACETYLATION OF METHYL 2,3,4-TRI-O-ACETYL-β-D-XYLOPYRANOSIDE

The deacetyiation of methyl 2,3,4-tri-O-acetyl-β-D-ylopyranoside was studied under different conditions of alkaline and enzymic hydrolysis. During enzymic deacetylation using porcine liver esterase a considerably higher amount of partially acetylated derivatives was observed in contrast to chemical hydrolysis.     

Hydrolysis of terpenyl glycosides in grape juice and other fruit juices: a review

The importance of monoterpenes on varietal flavour of must and other fruit juices has been reviewed. These compounds were mainly found linked to sugar moieties in grape juice and wines, showing no olfactory characteristics. In this way, analytical techniques developed to study these compounds, in both free or glycosidically forms, are discussed. Mechanisms to liberate terpenes were studied, making a comparative study between acidic and enzymic hydrolysis of terpene glycosides; as enzymic hydrolysis seems to be the most natural way to liberate terpenes, the ability to use glycosidases from grapes, yeasts, bacterial or exogenous, i.e. fungal commercial preparations, were reviewed. Re-arrangements of terpenes after acidic hydrolysis of glycoconjugated are discussed as well as potential adverse effects of enzyme preparations.     

Various lipases for producing products enriched with polyenic acids in fish fat hydrolysis]

The enzymic hydrolysis of fish with lipases from various sources was studied. The lipase from the fungus Rhizopus microsporus preferentially removes saturated fatty acids, while lipase from the pyloric caeca of salmon unsaturated fatty acids upon hydrolysis of fish fats. The enzymes can be used to obtain fatty products enriched with eicosanopentaenoic acid, mono- and diacylglycerols by enzymic hydrolysis of the ivasi fat.     

Complete enzymic digestion of acidic proteins.

Acidic proteins are usually resistant to complete enzymic hydrolysis. The increasing number of "unusual" amino acids, which are unstable to acid hydrolysis, makes it necessary to have a method of enzymic hydrolysis applicable to all proteins. The complete hydrolysis of four acidic proteins by subtilisin plus leucine amino-peptidase plus prolidase followed by carboxypeptidase C, is described. Recoveries of amino acids were in excellent agreement with the expected content from the known sequences.     

Metabolism of exogenous polyisoprenoids by animal cells cultured in vitro

The content and type of dolichols in chicken embryo fibroblasts was estimated and the presence of enzymic activities of various dolichyl phosphate sugar transferases was documented. Chicken embryo fibroblasts effectively take up various polyprenoids from the culture medium and catalyse both formation of polyprenyl fatty acid esters and their hydrolysis. With fully unsatured polyprenols the hydrogenation of the terminal alpha-isoprene residue was observed. In baby hamster kidney cells the formation of mannolipids was enhanced by exogenous polyprenols.     

Use of enzymic preparations during diosgenine isolation from Dioscorea caucasica Lipsky]

The use of enzymic preparations of the cellulolytic and macerating effect was studied as applied to the isolation of diosgenine from rhizomes of Dioscorea caucasica Lypsky. The enzymic treatment of the steroid containing raw material prior to acid hydrolysis increased the yield of diosgenine by 30-48%. It is suggested that additional extraction of diosgenine takes place due to: 1) enzymic hydrolysis of structural polysaccharide components of the plant tissue and intercellular binding materials and 2) disintegration of glycoside bonds of saponins.     

Boundaries of the butyrylcholinesterase anion site from data of a conformational analysis of substrates

By means of molecular mechanics, theoretical conformational analysis has been made of 19 substrates of butyrylcholinesterase - acetylcholine derivatives with different structure of the ammonium group. It was concluded that the anionic point is located in the cavity of the enzymic molecule. Dimensions and shape of this cavity were established which provide satisfactory correlation between its filling by substrate conformers and the rate of their enzymic hydrolysis. Some suggestions were made with respect to the mechanism of the effect of non-productive sorbtion of the substrates on the rate of their enzymic hydrolysis.     

Kinetics of enzymic hydrolysis of malto-oligosaccharides: A comparison with acid hydrolysis

The hydrolysis of malto-oligosaccharides G₃-G₆ catalysed by porcine pancreatic alpha-amylase was investigated kinetically at 25°. Kinetic parameters corresponding to different positions of enzymic attack were determined and product inhibition was evaluated. The enzymic hydrolysis was compared in terms of reaction rate and pattern of action with hydrolysis in 0.1m H₂SO₄ at 70°. Mathematical models for the mechanism of hydrolysis were developed and a good rationalisation of the experimental results was achieved.     

The study of hydrolysis of new lipid-like substrates and trilaurin in monolayers catalyzed with the lipase from Pseudomonas fluorescens

A simple method for determining the enzymic hydrolysis parameters of lipid-like substrates and trilaurin assembled in monolayers at the water-air interface was suggested. At a surface pressure of 10 mN/m, the initial rates of lipolysis were found to be proportional to the decrease in area of the substrate monolayer caused by the enzymic hydrolysis in a single-compartment Langmuir balance. The kinetic parameters for the hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acetylated in position 2 with an amino acid (phenylalanine, leucine, or valine) catalyzed with lipase from Pseudomonas fluorescens were determined. Unlike models of enzymic hydrolysis that neglect the thickness of the substrate monolayer, our method allows the determination of kinetic parameters in standard dimensions. The values of kcat for the synthetic pseudoglycerides were found to be significantly higher than that for trilaurin, while the values of Km(app) were close. This may be due to the presence of positively charged primary amino groups in the molecules of pseudoglycerides.     

2-O-(beta-D-glucuronyl)-7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose: a constituent of the Bordetella pertussis endotoxin.

The presence of bound D-glucuronic acid in the endotoxin of Bordetella pertussis was demonstrated. The branched chain trisaccharide named in the title was isolated after hydrolysis of the endotoxin with 3 M HCl for 2 h at 100 degrees C. Its structure was established by chemical and enzymic degradation.     

The hydrolysis of new lipid-like substrates and trilaurin in monolayers catalyzed with the lipase fromPseudomonas fluorescens

A simple method for determining the enzymic hydrolysis parameters of lipid-like substrates and trilaurin assembled in monolayers at the water-air interface was suggested. At a surface pressure of 10 mN/m, the initial rates of lipolysis were found to be proportional to the decrease in area of the substrate monolayer caused by the enzymic hydrolysis in a single-compartment Langmuir balance. The kinetic parameters for the hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acetylated in position 2 with an amino acid (phenylalanine, leucine, or valine) catalyzed with lipase fromPseudomonas fluorescens were determined. Unlike models of enzymic hydrolysis that neglect the thickness of the substrate monolayer, our method allows the determination of kinetic parameters in standard dimensions. The values ofk _cat for the synthetic pseudoglycerides were found to be significantly higher than that for trilaurin, while the values ofK _m(app) were close. This may be due to the presence of positively charged primary amino groups in the molecules of pseudoglycerides.     

Lability of equol to acidic hydrolysis procedures.

If equol is subjected to acidic hydrolysis by refluxing with 1.5 n HCl, a large proportion of the equol is altered to other substances. Subjection to enzymic hydrolysis by aryl sulfatase does not bring about any such alterations.     

Production of hemicellulosic sugars and glucose from residual corrugated cardboard

Corrugated cardboard samples were subjected to two-step saccharification. A first prehydrolysis stage was carried out to solubilise the hemicellulosic fraction as hemicellulosic sugars, and the solid phase from prehydrolysis was used as a substrate for the enzymic hydrolysis of cellulose. The prehydrolysis step was carried out for 0–180 min in media containing 1–3 wt.% of H₂SO₄ and the fraction of solid recovered after treatments and the compositions of solid and liquid phases from treatments were measured. The susceptibility of prehydrolysed solids towards the enzymic hydrolysis was assessed in further experiments. Under selected prehydrolysis conditions (3% H₂SO₄, 180 min), 78.2% of initial hemicelluloses was saccharified, leading to liquors containing up to 10 g hemicellulosic sugars/l and 9.2 g glucose/l. The corresponding solid phase, enriched in cellulose, showed good susceptibility towards enzymatic hydrolysis, leading to solutions containing up to 17.9 g glucose/l (conversion yield=63.6%) and a glucose/total sugar ratio of 0.93 g/g. Mathematical models assessing the effects of the operational conditions on both the prehydrolysis stage and the susceptibility of substrates towards enzymic hydrolysis have been developed.     

Enzyme-linked immunosorbent assay for the activator protein specific for the enzymic hydrolysis of GM1 in urine

We have established a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of the activator protein which stimulates the enzymic hydrolysis of G_M1 (G_M1-activator) in human urine. The level of G_M1-activator in 19 normal, adult urine samples was estimated to be 370.7±33.2 ng/ml. The amounts of G_M1-activator excreted in 24 h were estimated to be between 0.28 and 1.1 mg. The coefficient of variation for this method is 4.3% for the intra-assay and 14.4% for the inter-assay. Urine samples, without purification, can be used directly for the ELISA.     

An enzymic microassay for starch

Abstract Conditions are described for measuring the starch content of plant tissues or extracts as glucose over the range from 10^?7 mol to 10^?14 mol. The method is based on the hydrolysis of gelatinized starch by amyloglucosidase; the glucose released is measured by reduction of NADP⁺ by coupled enzymic reactions. The NADPH is determined directly either spectrophotometrically or fluorimetrically, or after enzymic amplification. Amyloglucosidases were tested for contaminating enzymes which might degrade glucans other than starch, and a commercial preparation from Rhizopus niveus was found to be suitable for use without pretreatments. Glucose present in tissues and extracts may be measured and subtracted from starch values using appropriate blanks, or first destroyed by dilute alkali and heat. Addition of α-amylase to amyloglucosidase during starch hydrolysis was not found to increase percentage hydrolysis from the normal range of 86–99% from starches of different sources. The procedures described are rapid and several orders of magnitude more sensitive than current methods, and can be used to measure the starch content of single cells.     

Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides.

Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.     

The enzymic hydrolysis of amygdalin

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond beta-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the beta-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components.     

A method for the assay of conjugated 3,4-dihydroxyphenylglycol,a major noradrenaline metabolite in the rat brain

We present the first published procedure for the measurement of endogenous conjugated 3,4-dihydroxyphenylglycol (DOPEG) in the rat brain. Conjugated DOPEG is estimated from brain extracts after enzymic hydrolysis, isolation of hydrolysed DOPEG on alumina, methylation of DOPEG to 3-methoxy-4-hydroxyphenylglycol (MOPEG) and gas chromatographic quantification of MOPEG. The level of conjugated DOPEG in the CNS of rats (65.7 +/- 0.7 ng/g whole brain tissue corrected for recovery) almost equals the level of conjugated MOPEG. The sensitivity of the method is about 6 ng/g brain tissue. After inhibition of monoamine oxidase with clorgyline (30 mg/kg) conjugated DOPEG and MOPEG both disappeared from the brain with a half-life of about 1 h. Turnover calculations indicate that conjugated DOPEG and MOPEG are the two major noradrenaline end-metabolites in the rat brain. The method of estimating conjugated DOPEG also allows the measurement of noradrenaline, dopamine and total MOPEG in an extract from one half of a rat brain.