Triacylglycerol and phospholipid hydrolysis in human plasma lipoproteins: role of lipoprotein and hepatic lipase.

To explore the interactions of triacylglycerol and phospholipid hydrolysis in lipoprotein conversions and remodeling, we compared the activities of lipoprotein and hepatic lipases on human VLDL, IDL, LDL, and HDL2. Triacylglycerol and phospholipid hydrolysis by each enzyme were measured concomitantly in each lipoprotein class by measuring hydrolysis of [14C]triolein and [3H]dipalmitoylphosphatidylcholine incorporated into each lipoprotein by lipid transfer processes. Hepatic lipase was 2-3 times more efficient than lipoprotein lipase at hydrolyzing phospholipid both in absolute terms and in relation to triacylglycerol hydrolysis in all lipoproteins. The relationship between phospholipid hydrolysis and triacylglycerol hydrolysis was generally linear until half of particle triacylglycerol was hydrolyzed. For either enzyme acting on a single lipoprotein fraction, the degree of phosphohydrolysis closely correlated with triacylglycerol hydrolysis and was largely independent of the kinetics of hydrolysis, suggesting that triacylglycerol removed from a lipoprotein core is an important determinant of phospholipid removal via hydrolysis by the lipase. Phospholipid hydrolysis relative to triacylglycerol hydrolysis was most efficient in VLDL followed in descending order by IDL, HDL, and LDL. Even with hepatic lipase, phospholipid hydrolysis could not deplete VLDL and IDL of sufficient phospholipid molecules to account for the loss of surface phospholipid that accompanies triacylglycerol hydrolysis and decreasing core volume as LDL is formed (or for conversion of HDL2 to HDL3). Thus, shedding of whole phospholipid molecules, presumably in liposomal-like particles, must be a major mechanism for losing excess surface lipid as large lipoprotein particles are converted to smaller particles. Also, this shedding phenomenon, like phospholipid hydrolysis, is closely related to the hydrolysis of lipoprotein triacylglycerol.     

Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods.

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.     

Three-stage enzymatic hydrolysis of steam-exploded corn stover at high substrate concentration

The feasibility of three-stage hydrolysis of steam-exploded corn stover at high-substrate concentration was investigated. When substrate concentration was 30% and enzyme loading was 15-30 FPU/g cellulose, three-stage (9+9+12 h) hydrolysis could reach a hydrolysis yield of 59.9-81.4% in 30 h. Compared with one-stage hydrolysis for 72 h, an increase of 34-37% in hydrolysis yield could be achieved. When steam-exploded corn stover was used as the substrate for enzyme synthesis and hydrolysis was conducted at a substrate concentration of 25% with an enzyme loading of 20 FPU/g cellulose, a hydrolysis yield of 85.1% was obtained, 19% higher than that the commercial cellulase could reach under the same conditions. The removal of end products was suggested to improve the adsorption of cellulase on the substrate and enhance the productivity of enzymatic hydrolysis.     

Enzyme management in the Iotech process

In the Iotech process, steam explosion of wood chips potentials hydrolysis of the cullulose by enzymes. A small portion of the exploded wood is used as substrate for the production of enzymes for hydrolysis of the remaining wood. Explosions conditions degrade some of the hemicellulose, creating factors (probably related to furfural) that impair enzymatic hydrolysis and destablized the cellulases. Fortunately, simple washing with water paremits more rapid hydrolysis while enzyme stability is greatly improved. Enzymes can be recovered from the hydrolysis residue by adjusting the pH to neutrality, and additional enzymes from hydrolysis filtrate can be adsorbed on fresh exploded wood en route to hydrolysis. A simple mass balance calculation shows proportions of various activities in a mix of fresh and recycled enzymes.     

从葫芦巴种子中提取薯蓣皂甙元方法的研究

为探明葫芦巴种子中药用活性成分薯蓣皂甙元提取的最佳方法,设置了不同的酸解液浓度及酸解时间以了解葫芦巴粉酸解的适宜条件;并在酸化工艺前将脱脂的葫芦巴粉进行自然发酵或接种曲霉发酵以提高薯蓣皂甙元的提取率。结果表明葫芦巴粉酸解液最佳浓度为15%的硫酸溶液,最适宜的酸解时间为6 h。在酸化工艺前增加发酵工艺,可以显著提高有效成份薯蓣皂甙元的提取率,接霉菌发酵比直接酸化提高62%,自然发酵比直接酸化提高48%。     

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.     

Optimized glucuronide hydrolysis for the detection of psilocin in human urine samples

In order to develop a sensitive and reliable analytical method for psilocin (PC) in urine samples, the hydrolysis conditions including the acid, alkaline and enzymatic hydrolyses have been investigated by monitoring not only PC but also psilocin glucuronide (PCG) by liquid chromatography tandem mass spectrometry (LC-MS-MS); PCG was initially identified in a "magic mushroom (MM)" user's urine by liquid chromatography mass spectrometry (LC-MS) and LC-MS-MS. The proposed conditions optimized for the hydrolysis are as follows: hydrolysis, enzymatic hydrolysis; enzyme, Escherichia coli beta-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 degrees C for 2h. The complete hydrolysis of PCG in urine was obtained under these conditions, while the enzymatic hydrolyses with three types of beta-glucuronidases originated from bovine liver (Type B-1), Helix pomatia (Type H-1) and Ampullaria provided uncompleted hydrolysis of PCG. Also, neither the acid nor alkaline hydrolysis was found to be applicable. According to the present method, 3.55 microg/ml of psilocin was detected in the "magic mushroom" user's urine after the enzymatic hydrolysis, though psilocin was not detected without hydrolysis.     

Manometric and spectrophotometric procedures for measurement of procaine hydrolysis by mouse liver in vitro

A decrease in absorbance at 313 nm was used to determine procaine hydrolysis by mouse liver. Results suggested that procaine hydrolysis was not linearly dependent upon the amount of tissue added to the incubate. This resulted from the fact that p-aminobenzoic acid, one of procaine's hydrolysis products, also absorbed at 313 nm. A dual wavelength analysis procedure is described which permitted accurate measurement of procaine hydrolysis by mouse liver without physical separation of procaine from its hydrolysis products. Measurements by the dual wavelength and manometric methods of inhibition of procaine hydrolysis in livers from mice treated with triorthotolyl phosphate indicated that both methods yielded quantitatively comparable results. The dual wavelength procedure is recommended for future studies of procaine hydrolysis by mammalian tissues.     

Lactate inhibits hydrolysis of polysaccharide-rich particulate organic waste

This work reveals that, at pH 5-9, a lactate level of up to 30 g l(-1) retarded hydrolysis rates in polysaccharide-rich potato samples. Lactate substantially limited carbohydrate hydrolysis and enhanced the hydrolysis of proteins. Statistical analysis identified the significance of numerous process factors in substrate hydrolysis. At fixed pH, dissociated lactate affected hydrolysis rates more strongly than its molecular counterpart. At a fixed lactate level, an alkaline environment favors carbohydrate hydrolysis; the effect of pH is secondary. Significant effects of lactate on substrate hydrolysis may be evident in fermenting organic substrates with high carbohydrate content.     

Coupling of ATP hydrolysis to phosphate uptake in Rhodospirillum rubrum chromatophores under the influence of Ca2+ and Mg2+

The Pi-ATP exchange and ATP hydrolytic reactions, by the F0F1 complex, were studied in Rhodospirillum rubrum chromatophores in the dark. An optimal pH between 7.0 and 8.5 was determined for the hydrolytic and exchange reactions. Under these conditions, the hydrolysis/exchange ratio was approximately 2. The kinetic analysis of the hydrolytic and exchange reactions using Mg-ATP as substrate showed a change in the hydrolysis/exchange ratio that varied between 2.0 and 2.8 as the substrate concentration was increased. With Ca-ATP, hydrolysis was not saturated up to a substrate concentration of 5.0 mM, and the hydrolysis/exchange ratios changed from 2 to 240 as the substrate concentration was increased from 0.06 to 5.0 mM. Free Mg2+ inhibited hydrolysis and phosphate uptake without altering the hydrolysis/exchange ratio. Nigericin induced an increase in the hydrolysis/exchange ratio from 2.7 to 130, whereas in the presence of valinomycin, this ratio increased from 2.7 to 21. From these results, it can be concluded that Ca-ATP hydrolysis is loosely coupled to phosphate uptake given that Pi-ATP exchange activity is extremely low, even at high rates of ATP hydrolysis.     

Enzymatic hydrolysis of mycelial waste from the production of tetracycline

The process of enzymatic hydrolysis of the mycelial waste from the manufacture of tetracycline with using Streptomyces aureofaciens was studied. For the enzymatic hydrolysis, neutral and alkaline proteinases were used. It was shown that alkaline proteinase (protosubtilin G10X) provided the most efficient hydrolysis. Optimal conditions for the hydrolysis were determined: a temperature of 42 degrees C, hydrolysis time of 4 to 6 hours and enzyme concentrations of 1.25 to 2.20 mg/ml at a mycecial waste concentration of 12.5 mg/ml. The time course of changes in amino acid and amine nitrogen levels during enzymatic hydrolysis was investigated. It was demonstrated that the hydrolysis efficiency depended on the mode of enzyme addition. The highest efficiency was observed with fractional feeding of the enzyme.     

纤维素生物质新型水解技术的研究进展

纤维素生物质水解技术是生物质资源转化的关键技术之一,在传统的酸水解和酶水解技术基础上,近年来出现了一些新型的水解技术,它们一般都具有绿色高效、对环境友好等特点;回顾并综述了纤维素生物质水解技术的最新进展,并对纤维素生物质水解技术的发展研究方向提出了设想.     

The effect of microwave irradiation on enzymatic hydrolysis of rice straw

A series of experiments involving microwave irradiation were carried out to evaluate the effect of microwave irradiation on enzymatic hydrolysis of rice straw. Compared with microwave irradiation free hydrolysis, rice straw pretreated by combining microwave irradiation with alkali could increase the initial hydrolysis rate but the hydrolysis yield remained unchanged. When the enzyme solution was treated by microwave irradiation, the initial hydrolysis rate increased slightly, but the yield was decreased remarkably. Its optimal hydrolysis conditions were temperature (45 degrees C), pH (4.8) and enzyme loading (20 mg g(-1) substrate), which was determined by an orthogonal experiment. When intermittent microwave irradiation was used, initial hydrolysis rate was greatly accelerated but the yield was decreased slightly. Its optimal hydrolysis conditions were temperature (50 degrees C), pH (4.8) and enzyme loading (20 mg g(-1) substrate), which was determined by another orthogonal experiment.     

鲜鹿茸酶降解过程的研究

研究了胰蛋白酶、Alcalase 碱性蛋白酶、木瓜蛋白酶对鲜鹿茸的降解过程,确定了优化降解工艺条件,具有一定的理论意义和实践价值。确定了Alcalase 碱性蛋白酶的降解效率最高,通过单因素实验确定了降解过程中底物浓度、酶解温度、pH值和酶解时间为影响鲜鹿茸降解率的主要因素。正交试验确定最佳的酶解条件为:底物浓度0.08 g/ml、酶解温度65 ℃、pH 9.0、酶解时间6.0 h。在此条件下,鲜鹿茸降解率高达92.6%,氨基酸产品收率达12.1%。     

Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.     

FEULGEN HYDROLYSIS OF NORMAL CELLS AND MOUSE ASCITES TUMOR CELLS

The effect of HCl hydrolysis on the dye content (Feulgen reaction) of normal cells and mouse ascites tumor cells was examined by means of cytophotometric measurements. After 11 min of hydrolysis, 16-day-old tumor cells showed a hypotetraploid DNA line with doubling peaks. The DNA values were in the ratios of 1:2:4:8 during all the tested hydrolysis times (3 to 21 min). The size of the nucleus and the DNA concentration did not influence the hydrolysis and the dye content. However, the time of the hydrolysis considerably influenced the dye content of normal and tumor cells. The course of the curves obtained by plotting dye absorption against hydrolysis time showed an inflection of the curve at 9 min' hydrolysis time in tumor cells, whereas the inflection occurred at 8 min in mitotic cells. These inflections were statistically significant. The DNA stem-line¹ for tumor cells shifted during different hydrolysis times when compared to normal cells. The possibility is discussed of two types of DNA which differed in their acid sensitivity and which yielded atypical hydrolysis curves.     

Enzymatic hydrolysis of polysaccharide-rich particulate organic waste

Hydrolysis of organic particulates in rapid fermentative processes can be inhibited. The volatile fatty acids (VFA) released during fermentation reduce pH. Whether VFA or the drop in pH inhibits hydrolysis is unclear. The effects of pH and acetate on the enzymatic hydrolysis of a potato sample that contains both carbohydrate and protein were studied at fixed pH (5-9) in the presence/absence of 20 g/L of acetate. Experimental results showed that the effects of pH and acetate on the hydrolysis of carbohydrate differed from those on the hydrolysis of protein. Numerous kinetic models fitted the hydrolysis data obtained during the first 40 h of hydrolysis when inhibitory effects were insignificant. The Chen-Hashimoto model was used herein to fit the hydrolysis data obtained during 144 h of reaction. Also, the non-competitive inhibition model of three inhibitors (H(+), OH(-), total/undissociated/dissociated acetate) successfully described the inhibition of the hydrolysis of both carbohydrate and protein.     

Aminoacyl-nucleotide reactions: studies related to the origin of the genetic code and protein synthesis

In the present paper, we report on the effect of pH and carbonate on the hydrolysis rate constants of N-blocked and free aminoacyl adenylate anhydrides. Whereas the hydrolysis of free aminoacyl adenylates seems principally catalyzed by OH-, the hydrolysis of the N-blocked species is also catalyzed by H+, giving this compound a U-shaped hydrolysis vs. pH curve. Furthermore, at pH's less than 8, carbonate has an extreme catalytic effect on the hydrolysis of free aminoacyl-AMP anhydride, but essentially no effect on the hydrolysis of N-blocked aminoacyl-AMP anhydride. Furthermore, the N-blocked aminoacyl-AMP anhydride is a very efficient generator of peptides using free glycine as acceptor. The possible significance of the observations to prebiological peptide synthesis is discussed.     

The hydrolysis of bile acid conjugates

Studies were made of a) the relationship of bile acid structure and analytical recoveries (measured by 3-hydroxysteroid oxidoreductase) following vigorous alkaline hydrolysis of bile acid conjugates and b) the relationship of structure and hydrolysis time of taurine- and glycine bile acid conjugates in a reaction catalyzed by glycocholic acid hydrolase. Alkaline hydrolysis resulted in good recoveries of hydroxy and 7 and 12- oxo-bile acids but poor recoveries of 3-oxo-bile acids. Borohydride reduction of the 3-oxo-acids prevented these losses. Complete enzymatic hydrolysis of glycine conjugated bile acids was about five times more rapid than that of taurine conjugates. Hydrolysis of conjugates containing oxo groups was slow. Borohydride reduction of oxoacids corrected this and did not inhibit enzymatic hydrolysis. It was concluded that both vigorous alkaline and enzymatic hydrolysis are satisfactory in bile acid assays if borohydride reduction is instituted before the hydrolytic step. However, due to the presence of possible enzyme inhibitors and solubility difficulties, strong alkaline hydrolysis is preferable to enzymatic hydrolysis in fecal bile acid determinations at this time.     

Dependence of energy coupling on nucleotide base structure in the reaction catalyzed by 5-oxo-L-prolinase

5-Oxo-L-prolinase catalyzes the virtually complete hydrolysis of 5-oxo-L-proline (L-pyroglutamate) to L-glutamate. The thermodynamic driving force for this endergonic amide hydrolysis is supplied by the coupled stoichiometric hydrolysis of ATP to ADP and Pi. We report here that the efficiency of the coupling between nucleotide and amide hydrolysis is dependent on the nucleotide base. Thus, with both ATP and dATP there is one to one stoichiometry between nucleotide cleavage and 5-oxoproline hydrolysis. With ITP, GTP, or UTP, however, the hydrolysis of NTP exceeds amide hydrolysis by 6 to 50-fold. In the absence of 5- oxoproline, the enzyme catalyzes a slow ATPase reaction, but it catalyzes very rapid ITPase, GTPase and UTPase reactions. These NTPase reactions, which under some conditions are faster than the ATP-mediated overall coupled reaction, are inhibited by 5-oxoproline and by analogs of 5-oxoproline that bind to the enzyme.