Separation of mono- and diglycerides by gas--liquid chromatography

The parameters affecting the separation and quantification of trimethylsilyl ethers of mono- and diglycerides have been investigated by gas-liquid chromatography with QF-1 and SE-30 as stationary phases and a flame ionization detector. Results have been compared with those obtained earlier for triglycerides. The isothermal characteristics of a range of trimethylsilyl ethers of mono- and diglycerides on both stationary phases showed that log retention volume was directly proportional to carbon number and inversely proportional to absolute temperature. However, glyceride derivatives with lower carbon numbers deviated from these relationships. By using various rates of programmed temperature rise, we have determined the elution temperatures (Kelvin scale) of the mono- and diglyceride trimethylsilyl ethers relative to that of glycerol trilaurate. The "carbon equivalent of a trimethylsilyl group" is defined and shown to be useful in comparing the chromatographic properties of different glyceride classes. Weight and molar correction factors have been obtained and used to analyze diglycerides derived from egg and bovine brain lecithins.     

Viscosimetric determination of cellulase activity: critical analyses

The mode of expression of cellulase activity obtained by viscosimetricmeasurement is analysed. After testing different types of substrates,it appears that the best one is hydroxyethylcellulose used ata high degree of polymerisation and a high concentration. Comparisonof results obtained with cellulases from Trichoderma virideand extracted from Pisum sativum favours the validity of thedetermination proposed. Possible physiological significanceof the measurements of cellulase activity is also discussed. (Received February 10, 1976; )     

Black substrate for spectrophotometric determination of cellulase activity in coloured solutions.

A simple procedure for spectrophotometric determination of cellulase activity in coloured solutions is described. CM-cellulose, cross-linked with epichlorhydrin in the presence of black drawing ink, is used as an insoluble chromolytic substrate. The absorbance of reaction mixture filtrates are read in the near infra-red region (at 800-900 nm) where the absorbances of contaminating coloured substances are substantially lowered; by contrast, black drawing ink released from the substrate during the action of cellulases absorbs strongly at these wavelengths.     

纤维素分解菌的选育及酶活测定

纤维素是地球上最丰富的有机物质,这些丰富的宝贵资源大部分被浪费了,而且由于部分地区焚烧秸杆造成了严重的环境污染。为了充分利用纤维素,纤维素分解菌的筛选研究逐步展开。通过新华滤纸为唯一碳源的杜氏培养基和刚果红纤维素培养基,从堆肥、污泥、马粪和土壤中分离得到7株纤维素分解菌。以5号菌株为出发菌株,经过紫外线诱变,用刚果红纤维素平板透明圈选育法得到8号菌株。为了评价筛选工作,对8株纤维素分解菌进行酶活测定。结果表明,8号菌株具有最高的CMC酶活和FPA酶活。     

The determination of aminoacyl adenylate by thin-layer chromatography.

A rapid and convenient method is presented by which one may determine the extent of formation of Enz·(AA ∼ AMP) in the presence of isotopic amino acid, isotopic nucleotides, tRNA, enzyme, etc. Separation of the components of the reaction mixture is achieved by chromatography on tlc cellulose. Aminoacyl adenylate separates from other solutes and is characterized by its chemical and enzymatic reactions. The method may be used to determine the equilibrium constant for the synthesis of Enz·(AA ∼ AMP) which is very sensitive to salt concentration.     

The determination of methionine in proteins by gas-liquid chromatography.

Intact methionine residues in proteins were rapidly and precisely determined by measuring methyl thiocyanate released during the reaction with CNBr and separated by g.l.c. Conditions for the reaction and for chromatography on columns of Porapak P-S are described. The recovery of methyl thiocyanate from several methionine derivatives and analogues were examined. Carbamoylmethionine was adopted as a stable primary standard and ethyl thiocyanate as internal standard. The measured methionine content of several isolated proteins was close to the theoretical value indicated by previous work and the results for these and a range of food proteins agreed well with results obtained by ion-exchange chromatography after performic acid oxidation. Since CNBr does not react with methionine sulphoxide and a preliminary hydrolysis is not required, the method discriminates between methionine and any methionine sulphoxide that may be present. It could be useful in studies on the nutritional availability of methionine in processed foods.     

A continuous spectrophotometric assay for the determination of cellulase solubilizing activity

A cellulase assay was developed for the continuous measurement of colored cellulose oligosaccharides (total carbohydrates) released during enzymatic hydrolysis of dyed crystal-line cellulose. Several cellulosic substrates were uniformly dyed by Remalzol brilliant blue R salt without altering their physical properties. Dyed Avicel (6.5%, w/w) was selected as the most representative substrate for the assay procedure. The assay was performed continuously in a simple, thermally controlled apparatus designed for filtration of the reaction mixture via a 5-μm-pore-size nylon filter to retain the crystalline dyed cellulose while spectrophotometrically monitoring the absorbance at 595 nm of the reaction filtrate. Crude supernatant cellulase of Trichoderma viride QM9414 was used to test the assay procedure. The activity of cellulase on dyed Avicel as measured by ΔA_595nm correlated directly with the total carbohydrates formed. The initial reaction rate of cellulase solubilizing activity was readily determined with high sensitivity. The continuous assay has utility for the study of cellulase kinetics and for the comparison of activities from different microorganisms.     

The determination of myoglobin by gel chromatography

The method recently described for the determination of haemoglobin in ovine muscles (1) has now been modified for the measurement of myoglobin. Evidence is presented here for the validity of using it for myoglobin determination in muscles of the rat, pig, sheep, and ox.     

Inhibition of cellulase activity by sun_ower polyphenols

The cellulase-inhibitor binding constant (b) for chlorogenic, ferulic, caffeic and sinapic acids, when these compounds were present in the range of concentrations found during the aqueous extraction of sun_ower oil, was calculated. Chlorogenic acid presented a weak inhibiting power (b = 0.14 L/mmol), whereas sinapic acid conducted as the strongest inhibitor (b = 14.60 L/mmol). The inhibition effects were not additive, since in the presence of mixtures the enzyme retained 75-85% of its activity.     

An automated spectrophotometric flow-injection procedure for the determination of cellulase activity in bioreactor preparations.

A spectrophotometric procedure for the determination of cellulase activity in precipitated bioreactor preparations and culture filtrates is described. It is based on the determination of reducing sugar produced by the action of the enzyme on carboxymethylcellulose. The reducing sugar is derivatized with p-aminobenzoyl-hydrazide and permits a limit of detection of 0.1 U ml-1 cellulase in the presence of background sugar, with a sampling rate of 5 h-1. The method can readily be applied to the determination of any carbohydrase acting on soluble substrates and producing reducing sugars, e.g. amylase, dextranase, xylanase, glucanase and polygalacturonase.     

The cellulase activity of an extreme thermophile

Summary The carboxymethylcellulase activity concentrated from the extremely thermophilic anaerobe H173 was found to have a pH optimum of 6.5–7.0. The enzyme activity was stabilised by the addition of dithiothreitol and CaCl₂·2H₂O and was very stable at 80° C, retaining 77% of the initial activity after 120 min incubtation. At min and after 120 min only 3% of the initial activity remained. With the enzyme dissolved in buffer, glucose and cellobiose were formed from the hydrolosis of Avicel. In culture medium the Avicel-solubilising activity was insensitive to the presence of up tp 50 mm glucose and showed linear glucose accumulation over a period of days at 70° C. HPLC analysis established that glucose was the major end-product of hydrolysis in the culture broths.Offprint requests to: H. W. Morgan     

Rapid isolation and high-throughput determination of cellulase and laminarinase activity in soils

A new method for extracting soil enzymes is described and a microplate method for assaying soil β-1,4-glucanases (cellulases) and β-1,3-glucanases (laminarinases). Soil samples were mechanically disrupted to produce crude enzyme extracts, and diluted preps incubated in microplates containing either carboxymethyl cellulose (CMC) to determine cellulase activity or laminarin substrate to determine laminarinase activity. The resulting glucose was measured using the fluorometric Amplex Red^® glucose assay. The method was reproducible, could be completed in 1 day and measured twice as much enzyme activity than the standard passive soil enzyme extraction procedure. The method described herein facilitates the development of high-throughput soil multiplex enzymatic assays from several soil samples at one time, and is well suited to the study of functional microbial ecology.     

Gas-liquid chromatography for the determination of acetylcholinesterase activity

Method has been modified and used for quantitative gas chromatographic determination of microtraces in a large volume of test mixture under study. The method can be applied for the differentiated determination of genuine and false blood cholinesterase by final products of specific substrate hydrolysis.