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.     

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.     

Enzymatic deinking of various types of waste paper: Efficiency and characteristics

The aim of the present study was to characterize the enzymatic deinking of various types of waste paper. Studies on the optimization of enzymatic deinking have been performed previously using commercially available enzyme preparations containing cellulase and hemicellulase. The enzymatic deinking of different types of waste paper demonstrated a high efficiency of 86.6% on laser-printed paper, but a low deinking efficiency of 12.9% was obtained with newspaper. All enzymatic treatments significantly improved the drainage rate of the deinked waste paper. Enzymatic deinking increased the tensile index of magazine paper but reduced the tensile index of bubble jet-printed paper, photocopy paper and newspaper. Enzymatic hydrolysis caused a 21.1% reduction in the tear index for bubble jet-printed paper, but a 3.1% increase in the tear index was obtained for laser-printed paper relative to respective blank. In addition, enzymatic hydrolysis increased the burst index by 4.7% relative to blank for laser-printed paper. However, photocopy paper showed the highest reduction (8.3%) in the burst index relative to blank. Taken together, these results suggest that enzymatic hydrolysis is both advantageous and detrimental to the mechanical properties of deinked paper. Thus, the proper regulation of enzymatic hydrolysis is crucial to improve the quality of recycled paper.     

Soluble and membrane-bound forms of lipoprotein lipase in mouse lung tissue

Homogenates of mouse lungs were separated by differential centrifugation into two fractions containing lipoprotein lipase, namely, a soluble and a membrane-bound fraction. Lipoprotein lipase was specifically identified by its inhibition by both protamine sulfate (3 mg/ml) and sodium chloride (0.9 mol/l). The enzymatic activity of each fraction was enhanced when serum was preincubated with the enzyme. Both enzyme fractions showed optimum activity at alkaline pH, but the membrane-bound enzyme showed a higher pH optimum. In addition, the apparent Km of the soluble enzyme was lower than that of the membrane-bound enzyme. It is concluded that there are two different forms of lipoprotein lipase in mouse lung tissue that differ in a number of aspects.     

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.     

酶法制备纳米级珍珠粉工艺研究

以珍珠粉粒径为考察指标,采用食品工业用酶处理,研究了蛋白酶种类、酶解时间、加酶量与珍珠粉粒径分布范围的关系。实验表明：10g珍珠粉,加入50mL去离子水溶解,使用饱和Ca（OH）2溶液调pH值至11．0,再加入0．3g碱性蛋白酶,于40℃水浴保温酶解6h。酶解后约有60％珍珠粉粒径分布小于1μm,已满足国家标准（征求意见稿）的规定要求,为纳米级珍珠粉。     

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%。     

Comparative hydrolysis and fermentation of sugarcane and agave bagasse

Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treatment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Celluclast, Novozyme and Viscozyme L. From alkaline–enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar yield was obtained (11–20%) compared to agave bagasse (12–58%). Selected hydrolyzates were fermented with a non-recombinant strain of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.     

Enzymatic hydrolysis of gelatin layers on used lith film using thermostable alkaline protease for recovery of silver and PET film

To develop a new efficient and potential industrial enzymatic process for the recovery of silver and poly(ethylene terephthalate) (PET) from used lith film for printing, which has not been recycled at all, enzymatic hydrolysis of gelatin layers on lith film was investigated using the thermostabilized mutant enzyme of the alkaline protease from alkaliphilic Bacillus sp. B21-2. The rate of gelatin hydrolysis of lith film in a stirred-tank reactor increased with the temperature and enzyme concentration. The time required to complete the hydrolysis of gelatin on lith film was longer than that on X-ray film because of the tightly cross-linked structure of the gelatin layers of lith film. The time required to complete the hydrolysis by using the mutant enzyme was less than that using the wild-type enzyme. The gelatin hydrolysis of lith film was well explained by a model that took into consideration a number of physical processes in addition to the chemical process.     

Purification and properties of acid alpha-glucosidase (gamma-amylase) from the human liver]

Acid alpha-glucosidase from human liver was 720-fold purified by means of a specific sorption on Sephadex G-150 and a specific desorption from Sephadex by the competitive inhibitor, methyl-alpha-D-glucopyranoside. The preparation obtained was homogenous in ultracentrifuge and polyacrilamide gel electrophoresis. The enzyme possessed both maltase and glucoamylase activities and splitted maltose, amylopectin and glycogen with Km values of 7mM, 7.7 mg/ml and 5 mg/ml respectively. Methyl-alpha-D-glucopyranoside competitively inhibited the enzymatic hydrolysis of polysaccharides (Ki=6.95 mM) and did not affect the maltose degradation. The sedimentation coefficient of the purified enzyme preparation was 5.4 S; in 5 M guanidine. HCl the coefficient decreased to 2.2 S, which testified to the fact that the enzyme molecule consisted of subunits.     

Cloning and characterization of a fish microsomal epoxide hydrolase of Danio rerio and application to kinetic resolution of racemic styrene oxide

A potent bacterial strain, Pseudomonas aeruginosa, has been isolated from the soil which produces extracellular lipase that can carry out the excellent stereospecific hydrolysis of trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM)] to give [(−)-MPGM], an intermediate required in the synthesis of cardiovascular drug, diltiazem. As a preliminary experiment for enzymatic resolution, we characterized the fractionated enzyme. The enzyme had a pH and temperature optima of 8.0 and 60 °C, respectively. The enzyme showed high degree of thermostability. Also, the enzyme was found to be stable in alkaline condition and in organic solvents. The activity of the enzyme increased by the addition of magnesium ions. The small-scale hydrolysis of (±)-MPGM (250 mg) with partially purified enzyme (21,000 U) gave (−)-MPGM with good isolated yield (44%) and excellent enantiomeric excess (99.9%) in a very short time (12 h).     

Mild alkaline/oxidative pretreatment of wheat straw

A new mild alkaline/oxidative pretreatment of wheat straw prior to enzymic hydrolysis was carried out. It consists of a first alkaline (1% NaOH for 24 h) step, which mainly solubilises hemicellullose and renders the material more accessible to further chemical attack, and a second alkaline/oxidative step (1% NaOH and 0·3% H₂O₂ for 24 h), which solubilises and oxidises lignin to minor polluting compounds. The entire process was carried out at low temperature (25–40°C) using a low concentration of chemicals, resulting in a relatively low cost and waste liquors containing only trace amounts of dangerous pollutants derived from lignin. Recovery of cellulose after the double pretreatment reached 90% of that contained in the starting material, with a concomitant 81% degradation of lignin. The action of a commercial cellulase on the cellulose obtained produced a syrup with a high concentration of reducing sugars (220 mg/ml), of which a large percentage was glucose.     

Pretreatment and saccharification of rice hulls for the production of fermentable sugars

Hydrolytic conditions of rice hulls by acid and alkaline treatments before enzymatic saccharification were optimized in this study. Based on the results of single-factor experiments and an orthogonal array experiment, reaction time was found to be the most important factor for the acidic hydrolysis of rice hulls. Maximum yield of sugars from 1 g of rice hulls by acidic treatment under optimized conditions was 213.6 mg. The yield of lignin removal from acidic pretreated rice hulls by alkaline treatment increased with increase in reaction temperature and time. The amount of sugars obtained from 1 g of pretreated rice hulls by enzymatic saccharification was 307.7 mg, and the conversion rate of sugars from crude fibers in pretreated rice hulls was about 72%. Instrumental analyses with FTIR and SEM indicated that lignin in rice hulls was partially removed by alkaline treatment, and the structure of rice hulls became deformed and more fibers were exposed to cellulases after acidic treatment.     

Thermal-alkaline solubilization of waste activated sludge as a pre-treatment stage for anaerobic digestion

This work studied the hydrolysis kinetics and the solubilization of waste activated sludge under a medium range temperature (50-90 degrees C) and pH in the alkaline region (8-11), as a pretreatment stage for anaerobic digestion. The hydrolysis rate for the solubilization of volatile suspended solids (VSS) followed a first-order rate. A linear polynomial hydrolysis model was derived from the experimental results leading to a satisfactory correlation between the hydrolysis rate coefficient, pH, and temperature. At pH 11 and a temperature of 90 degrees C the concentration of the VSS was 6.82%, the VSS reduction reached 45% within ten hours and at the same time the soluble COD was 70.000 mg/l and the total efficiency for methane production 0.28 l of CH4 per g of VSS loading.     

Enzymatic pre-hydrolysis applied to the anaerobic treatment of effluents from poultry slaughterhouses

A pool of hydrolases with 21.4 U g⁻¹ lipase activity was produced through solid-state fermentation of the fungus Penicillium restrictum in waste from the Orbignya oleifera (babassu) oil processing industry. Enzymatic hydrolysis and anaerobic biodegradability tests were conducted on poultry slaughterhouse effluents with varying oil and grease contents (150–1200 mg l⁻¹) and solid enzymatic pool concentrations (0.1–1.0% w/v). Enhanced anaerobic treatment efficiency relative to raw effluent was achieved when a 0.1% concentration of enzymatic pool was used in the pre-hydrolysis stage with 1200 mg oil and grease l⁻¹ (chemical oxygen demand (COD) removal efficiency of 85% vs. 53% and biogas production of 175 ml vs. 37 ml after 4 d).     

Evaluation of the factors affecting avicel reactivity using multi-stage enzymatic hydrolysis

Multi-stage and single-stage enzymatic hydrolysis of cellulose (Avicel PH-101) were conducted to investigate individual factors that affect the rate-reducing kinetics of enzymatic hydrolysis. Understanding factors affecting enzymatic hydrolysis of Avicel will help improve hydrolysis of various biomasses. Product inhibition, enzyme deactivation, and the changes of substrate are potential factors that can affect the hydrolysis efficiency of Avicel. Multi-stage enzymatic hydrolysis resulted in 36.9% and 25.4% higher carbohydrate conversion as compared to a single-stage enzymatic hydrolysis with an enzyme loading of 5 and 20 FPU/g in a 96 h reaction. However, a decline in carbohydrate conversion of 1.6% and 2.6% was observed through each stage with 5 and 20 FPU/g, respectively. This indicated that the substrate became more recalcitrant as hydrolysis progressed. The decreased reactivity was not due to crystallinity because no significant change in crystallinity was detected by X-ray diffraction. Product inhibition was significant at low enzyme loading, while it was marginal at high enzyme loading. Therefore, product inhibition can only partially explain this decreased conversion. Another important factor, enzyme deactivation, contributed to 20.3% and 25.4% decrease in the total carbohydrate conversion of 96 h hydrolysis with 5 and 20 FPU/g, respectively. This work shows that an important reason for the decreased Avicel digestibility is the effect of enzyme blockage, which refers to the enzymes that irreversibly adsorb on accessible sites of substrate. About 45.3% and 63.2% of the total decreased conversion at the end of the 8th stage with 5 and 20 FPU/g, respectively, was due to the presence of irreversibly adsorbed enzymes. This blockage of active sites by enzymes has been speculated by other researchers, but this article shows further evidence of this effect.     

Production, Purification, and Characterization of beta-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching beta-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60 degrees C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K(m) of 7.9 mg/ml and an apparent V(max) of 305 mumol . min . mg of protein. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

Acetone–butanol–ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment

In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone–butanol–ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.     

Enzymatic prebleaching of sulphite pulps

The ability of a crude enzyme preparation ofAureobasidium pullulans containing xylanase [61 units (U)/ml] and xylosidase (3 U/ml) activity to remove pentosans from unbleached sulphite pulps was investigated. Greater amounts of pentosans and reducing sugars were released from the pulp when the enzyme dosages and incubation times were increased. A combination of enzyme hydrolysis and alkali extraction resulted in a greater removal of pentosans than using the enzyme preparation alone. By treatment with a xylanase loading of 450 U/g pulp for 24 h followed by alkaline extraction, 35% of the pentosans were removed. The kappa number decreased up to 30% whereas viscosity was only slightly affected by these treatments. Enzymatic hydrolysis released mainly xylose whereas xylobiose was the main product liberated by alkaline extraction. Scanning electron micrographs indicated improved fibrillation and flexibility of the fibre structure by enzyme treatment.     

Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method.

A crude cell extract from a mixed bacterial culture growing on parathion, an organophosphate insecticide, hydrolyzed parathion (21 C) at a rate of 416 nmol/min per mg of protein. This rate of enzymatic hydrolysis, when compared with chemical hydrolysis by 0.1 N sodium hydroxide at 40 C, was 2, 450 times faster. Eight of 12 commonly used organophosphate insecticides were enzymatically hydrolyzed with this enzyme preparation at rates ranging from 12 to 1,360 nmol/min per mg of protein. Seven pesticides were hydrolyzed at rates significantly higher (40 to 1,005 times faster) than chemical hydrolysis. The pH optimum for enzymatic hydrolysis of the eight pesticides ranged from 8.5 to 9.5, with less than 50% of maximal activity expressed at pH 7.0. Maximal enzyme activity occurred at 35 C. The crude extract lost its activity at the rate of only 0.75%/day when stored at 6 C. Eight organic solvents, ranging from methanol to hexane, at low concentrations stimulated enzymatic hydrolysis by 3 to 20%, whereas at higher concentrations (1,000 mg/liter) they inhibited the reaction (9 to 50%). Parathion metabolites p-nitrophenol, hydroquinone, and diethylthiophosphoric acid, at up to 100-mg/liter concentrations, did not significantly influence enzyme activity.