The purification of myosin long subfragment 2 by DEAE-Sepharose CL-6B ion-exchange chromatography

A new method for the purification of myosin long subfragment 2 is presented. This method is based on the fractionation, by DEAE-Sepharose CL-6B ion-exchange chromatography, of either chymotryptic hydrolysates of heavy meromyosin or tryptic hydrolysates of myosin total rod. Although emphasis is given to the purification of long subfragment 2, the method could be easily adapted to the purification of short subfragment 2.     

Determination of radioactivity of proline and hydroxyproline in tissue hydrolysates after the elimination of interference by primary amino acids by deamination

A simple method for the determination of radioactivity of proline and hydroxyproline, particularly of small amounts, in hydrolysates of tissues is described. Specificity is assured by eliminating primary amino acids from the hydrolysates by deamination and then extraction before separation of proline from hydroxyproline by paper chromatography. Six to eight tissue samples may be compared simultaneously. The efficiency and reproducibility are good, as indicated by the use of labeled l-proline, labeled dl-hydroxyproline, a hydrolysate of a protein in which the amino acids (and proline) were labeled, and hydrolysates of tissues cultured in media containing radioactive l-proline. The method is particularly useful when ion-exchange column chromatography of amino acids is not in routine use.     

Rapid Quantitation of desmosine content in tissue hydrolysates by high-performance liquid chromatography

The lysine-derived crosslinks in elastin, desmosine, and isodesmosine, are quantitated in tissue hydrolysates by monitoring high-performance liquid chromatography eluents at 275 nm. The results from this method compare favorably with results from the amino acid analyzer. However, this more sensitive method (1) eliminates ninhydrin-positive artifacts which elute with the desmosines from some tissue hydrolysates on the amino acid analyzer, and (2) makes possible elastin quantitation in a tissue with the minimum amount of manipulations.     

Functional,antioxidant and antibacterial properties of protein hydrolysates prepared from fish meat fermented by Bacillus subtilis A26

Composition, functional properties and in vitro antioxidant and antibacterial activities of protein hydrolysates prepared with a proteolytic bacterium, Bacillus subtilis A26, through fermentation of fish proteins were investigated. Fermented fish meat protein hydrolysates (FPHs) were prepared from sardinelle (SPH), zebra blenny (ZPH) goby (GPH) and ray (RPH). The protein content of freeze-dried FPHs ranged from 74.3% to 81%. All fermented hydrolysates had an excellent solubility and possessed interfacial properties. The antioxidant activities of FPHs were evaluated by different methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, β-carotene bleaching and DNA nicking assay. All hydrolysates showed dose-dependent antioxidant activities. Further, FPHs exhibited antibacterial activity and SPH was the most effective, particularly against Gram positive bacteria.     

A method to determine protein of bacteria grown in agar

A method is described to determine the amount of protein of bacteria grown in agar. Amino acid contents of hydrolysates of agar cultures are compared with the amino acids in hydrolysates of the same bacteria grown in a liquid medium. In the latter medium also the amount of bacterial protein is determined. From these data the protein content of bacteria grown in agar can be calculated.     

蛋白水解物制备工艺及其在生物技术领域中的应用研究进展

蛋白水解物是蛋白质经酶、酸、碱和发酵等方法水解得到的混合物,由于其含有丰富的氨基酸和多肽,并为细胞生长提供多种营养成分、贴壁因子及生长因子类似物等,从而在生物技术领域中得到了广泛的应用。本文综述了蛋白水解物制备工艺、在生物技术领域中的应用及其在细胞培养中对细胞增殖、代谢的影响,以期为蛋白水解物的开发和应用提供参考。     

Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Background  

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.     

Improved method of analysis of biomass sugars using high-performance liquid chromatography

The precise quantitative analysis of biomass derived sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time-consuming but the alternative HPLC method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This was demonstrated for both standard sugars and corn stover hydrolysates. Our method can also be used to analyze dimmeric sugars (cellobiose and sucrose).     

High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis

Twelve kinds of marine protein materials, including fish, shrimp, seashell, algae and seafood wastes were selected for the hydrolysis using four different proteases. The IC(50) values for angiotensin-converting enzyme (ACE) inhibitory activity of 48 hydrolysates were rapidly determined by capillary electrophoresis (CE). The values ranged from 0.17 to 501.7mg/ml, and were affected by both the marine protein resources and the selected proteases. Hydrolysates of the lowest IC(50) values were from shrimp (Acetes chinensis), shark meat, mackerel bone, Polysiphonia urceolata and Spirulina platensis, indicating these five kinds of marine food proteins contained beneficial materials for the production of ACE inhibitory peptides by proteolysis. The hydrolysates obtained using proteases Protamex and SM98011 had lower IC(50) values, showing these two proteases were superior to others. The CE method achieved the same sensitivity as the high performance liquid chromatography (HPLC) method. However, the CE method was faster and, as a result, more economical. Therefore, CE had potential for rapid screening of marine protein hydrolysates enriched in ACE inhibitory peptides.     

The nature of the organic nitrogen of soils

Summary Examination of the 6N HC1 hydrolysates from 14 different proteins indicated that a considerable proportion of the total protein nitrogen in the hydrolysates, as determined by the micro-Kjeldahl method, was not accounted for by the NH₄-N and the α amino nitrogen found in the hydrolysates. It seems clear that this hydrolysable unidentified nitrogen (HUN) originates mainly from non-amino nitrogen atoms present in arginine, tryptophan, lysine and proline. These nitrogen atoms do not satisfy the conditions necessary for reaction with ninhydrin. The amounts of each amino acid in a particular protein determine the HUN value which will be obtained for 6N HC1 hydrolysates of that protein. There is good agreement between the HUN values for a wide range of proteins when calculated from the amino acid composition of the protein and when determined experimentally. It is concluded that these findings indicate a considerably higher content of amino acid nitrogen in the organic nitrogen of soils and leaf litter than was previously considered to be the case. It is suggested that the findings support the contention that the organic nitrogen of soils contains leaf protein complexes.     

Preparation of hydrolysates from tobacco stalks and ethanolic fermentation by Saccharomyces cerevisiae

Chipped tobacco stalks were subjected to steam pretreatment at 205 °C for either 5 or 10 min before enzymatic hydrolysis. Glucose (15.4–17.1 g/l) and xylose (4.5–5.0 g/l) were the most abundant monosaccharides in the hydrolysates. Mannose, galactose and arabinose were also detected. The hydrolysate produced by pretreatment for 10 min contained higher levels of all sugars than the 5 min-pretreated hydrolysate. The amounts of inhibitory compounds found in the hydrolysates were relatively low and increased with increasing pretreatment time. The hydrolysates were fermented with baker's yeast. Ethanol yield, maximum volumetric productivity and specific productivity were used as criteria of fermentability of the hydrolysates. The fermentation of the hydrolysates was only slightly inhibited compared to reference solutions having a similar composition of fermentable sugars. The ethanol yield in the hydrolysates was 0.38–0.39 g/g of initial fermentable sugars, whereas it was 0.42 g/g in the reference. The biomass yield was twofold lower in the hydrolysates than in the reference. The fermentation inhibition caused by the tobacco stalk hydrolysates was less than that caused by sugarcane bagasse hydrolysates obtained under the same hydrolysis conditions.     

An enzymatic estimation of free hydroxyproline in tissue hydrolysates

An enzymatic method using 4-hydroxyproline 2-epimerase (EC 5.1.1.9), D-amino acid oxidase (EC 1.4.3.3.), and a colorimetric reagent of the mixture of Ti(IV) and 4-(2-pyridylazo)-resorcinol was found to be useful in the spectrophotometric assay for hydroxyproline in collagenous tissue hydrolysates. It was possible to determine free hydroxyproline in 250 microliter of tissue hydrolysates at concentrations ranging from 1 to 12.5 micrograms. The coefficient of variation was less than 3%, and the correlation between the proposed method and the colorimetric method (K.I. Kivirikko, O. Laitinen, and D. J. Prockop (1967) Anal. Biochem. 19, 249-255) was 0.973. Thus, this method is convenient because it requires less time than the above colorimetric method, and is also reproducible and sensitive.     

HPLC determination of xylulose formed by enzymatic xylose isomerization in lignocellulose hydrolysates

Summary In the concentration range appropriate for enzymatic xylose isomerization, xylulose was measured in a lignocellulose hydrolysate using HPLC with two hydrogen loaded ion exchange columns in series. Spent sulphite liquour (SSL) was used as a model for lignocellulose hydrolysates. In buffer the separation took 22 minutes and in SSL the analysis time was 47 minutes due to the presence of ethanol. The enzymatic isomerization of xylose to xylulose was followed directly in SSL, providing a method for the direct determination of xylose isomerase activity in lignocellulose hydrolysates.     

乳清分离蛋白酶解物的抗氧化活性研究

研究了乳清分离蛋白(WPI)酶解物对DPPH.、超氧阴离子自由基和羟基自由基的清除效果,同时用还原法研究了其抗氧化活性。结果表明,WPI酶解物在体外具有较强的抗氧化能力。木瓜蛋白酶酶解物和胰蛋白酶酶解物对DPPH.、超氧阴离子自由基、羟基自由基的清除能力和还原能力强于胰凝乳蛋白酶酶解物和胃蛋白酶酶解物。     

The identification and quantitation of connective tissue hexosamines using a combination of gas liquid chromatographic and colorimetric procedures.

Due to interactions between amino sugars, amino acids, and/or carbohydrate breakdown products from acid hydrolysis, the quantitation of individual amino sugars from connective tissue hydrolysates, requires a number of indirect steps involving separation and purification of the hexosamines prior to gas-liquid chromatography. In this paper, a method is reported which permits the direct quantitation of galactosamine and glucosamine from connective tissue hydrolysates, utilising a combination of both gas-liquid chromatographic and colorimetric procedures. A two-phase extraction system which selectively eliminates pyridine and amino acids from the T.M.S. ethers of glucosamine and galactosamine is also described.     

Effect of the peptide and vitamin composition of casein hydrolysates on the β-galactosidase activity of Kluyveromyces bulgaricus cells

Instead of yeast extracts, casein hydrolysates from several suppliers were added to culture media and analyzed with respect to their ability to stimulate β-galactosidase activity in Kluyveromyces bulgaricus cells. Four enzymatic casein hydrolysates caused a significantly higher stimulation of enzyme activity, while acid casein hydrolysates clearly reduced the enzyme activity. Enzymatic casein hydrolysates, inducing high and low lactase activity, were analyzed with respect to average peptide length (APL), vitamins (niacin, pathothenate) as well as free and bound amino acids. The molecular weights of these casein hydrolysates were estimated by gel filtration. No correlation was found between the degree of enzyme stimulation and the vitamin contents, the APL values and the free amino acid contents of the casein hydrolysates. Casein hydrolysates stimulating the lactase activity were less soluble in water and, in a gel filtration column, they showed three peaks with slightly lower molecular weights than the three peaks seen in hydrolysates which had no effect on activity. APL values of alcoholic precipitates of casein hydrolysates showed an inverse correlation to lactase activity. The molecular weights of alcoholic precipitates of lactase stimulating digests were also lower compared to non-stimulating ones. Alcoholic precipitates with lactase-stimulating activity were more hydrophilic, as was shown by a smaller proportion absorbed in a C18 column and by amino acid analysis. Our results sugest that alcoholic precipitates could probably be important in the lactase stimulation and their composition should be further investigated. The mechanism is nevertheless complex and may be caused by various simultaneous factors.     

Preparation of fish protein hydrolysates

A process for the preparation of fish protein hydrolysates using commercially available proteolytic enzymes is described. Both non-fatty and fatty species of fish have been used as the raw material and the hydrolysates obtained have been assessed in nutritional studies on neonatal animals. Data on the release of nitrogenous substances during the digestion of cod with the enzyme papain are presented, together with amino acid analyses of the various fractions produced. As far as hydrolysates from non-fatty species are concerned there are no technical difficulties in preparing a spray-dried or concentrated product suitable for animal feeding. For hydrolysates from fatty species, however, it is necessary either to remove the oil mechanically or to stabilise it with suitable antioxidants. For the present exercise, hydrolysates were frozen and stored at ?30°C until required for the nutritional studies. Before any industrial development is considered, further studies on the drying and storing of hydrolysates from fatty species are necessary.     

基因组改组及其在木质纤维素水解液乙醇发酵菌种选育中的应用展望

能高效代谢木质纤维素水解液中的可发酵糖、同时可耐受/分解发酵抑制剂的菌种, 是利用木质纤维素为原料生产燃料乙醇技术的关键。基因组改组技术是近些年发展起来的一项新型育种技术, 该技术已运用于食品和医药行业菌种的改良。本文综述了基因组改组技术的原理、方法、特点、及其运用, 并对其在木质纤维素水解液乙醇发酵菌种选育方面的应用进行了展望。     

活性炭对玉米芯半纤维素稀硫酸水解液的脱毒效果

用稀硫酸对玉米芯半纤维素进行水解是一种常用的方法,但是玉米芯半纤维素在水解成木糖等还原糖的同时还产生了糠醛、乙酸和酚类等抑制水解液发酵的毒物。以混合脱毒法为基础,研究活性炭在脱毒过程中的作用。结果表明,有脱毒效果的活性炭种类是GH-13和GH-15,随着活性炭添加量的增大,脱毒效果增强,但木糖损失也随之增多。其中采用5%GH-15时的脱毒效果最佳,该条件下乙酸去除率为24.60%,糠醛去除率达100%,酚类化合物去除效率R280值0.009,而木糖的损失率为23.70%。