The use of crude lipase in deprotection of C-terminal protecting groups

A crude lipase, Newlase F, was used to remove C-terminal protecting groups from dipeptide esters. Hydrolysis of dipeptide n-heptyl esters with Newlase F was conducted in aqueous media containing acetonitrile. The optimum pH and temperature of lipase in Newlase F were 7.0 and 30 °C, respectively. Low level acetonitrile promoted the hydrolysis of dipeptide n-heptyl esters, while high level acetonitrile inhibited the hydrolysis. However, the protease activity in Newlase F was significantly inhibited by acetonitrile. Lipase in Newlase F worked better in a medium containing water-miscible organic solvents than in water-immiscible ones. N-terminal protecting groups were not affected by the protease in the crude enzyme. It was found that the protease in Newlase F did not hydrolyze amide bond with hydrophilic amino acids on either side under these conditions (pH 7.0, room temperature). Newlase F may consequently be used widely in the synthesis of peptide conjugates. The crude enzyme was immobilized on SBA-15 mesoporous molecular sieve. The lipase activity of immobilized preparation was more active on hydrolysis of C-terminal protecting groups and stable than the free enzyme. The immobilization also reduced the protease activity.     

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.     

Optimization of immobilized enzyme hydrolysis combined with high-performance liquid chromatography/thermospray mass spectrometry for the determination of neuropeptides

Peptidases, including chymotrypsin, thermolysin, trypsin, V8 protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with HPLC/thermospray MS for the analysis of neuropeptides. The optimal operating conditions for each immobilized enzyme bioreactor were determined. Optimal hydrolysis usually occurred at the highest percentage of aqueous solution in the mobile phase at pH 7-8 and 40-50 degrees C. Often post-HPLC column addition of aqueous solutions before the bioreactor could improve activity and thermospray sensitivity without changing the HPLC separation. Enzymatic hydrolysis requirements were compatible under conditions for HPLC separation and thermospray MS detection of the selected neuropeptides. Synthetic alpha-, beta-, and gamma-endorphins were the primary neuropeptides used to evaluate on-line immobilized enzyme bioreactor/MS. HPLC followed by peptidase hydrolysis produced characteristic hydrolysis products for confirming the peptides' identity using thermospray MS detection. Furthermore, the peptide formed from enzymatic hydrolysis resulted in a MS ion current 10-40 times higher than that of the [M + 2H]2+ ion for unhydrolyzed beta-endorphin. The increased sensitivity achieved for detecting the hydrolysis products permits detection and quantitation of synthetic peptides down to 800 fmol.     

Pseudomonas aeruginosa A2 elastase: purification, characterization and biotechnological applications

An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34 kDa. Temperature and pH with highest activity were 60 °C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 40 °C with an enzyme/substrate (E/S) ratio of 5 U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes.     

Enzymatic synthesis of polyaniline film using a copper-containing oxidoreductase: bilirubin oxidase.

Electroactive polyaniline films have been synthesized by using a copper-containing oxidoreductase, bilirubin oxidase (BOD). Enzymatic polymerization took place on the surface of BOD-adsorbed solid matrix which was in contact with a buffer solution containing aniline. Optimum conditions for enzymatic polymerization of aniline were investigated. Elemental analysis and IR spectroscopy indicated that the enzymatically synthesized film was polyaniline. The cyclic voltammetric studies demonstrated that the polyaniline film was electrochemically reversible in the redox properties in acidic aqueous solutions. Since the film retained enzymatic activity of BOD which was employed as a catalyst for polymerization, enzymatic polymerization seems promising in preparation of immobilized enzyme membranes.     

Activities of Candida rugosa lipase and other esterolytic enzymes coated on glass beads and suspended in substrate and water vapor: enzymes in thin liquid films.

Candida rugosa lipase was enzymatically active when coated on glass beads and exposed to mixtures of substrate and water vapor over a range of relative humidities up to 100%. Evidence was obtained for operation of the enzyme in a thin liquid film of concentrated buffer on the surface of the glass beads. Formation of the thin film was associated with hygroscopicity of the buffer used to suspend the enzyme in preparation of the enzyme-coated beads. At some buffer concentrations estimated to be on the bead surface, the enzyme was partially soluble and both soluble and insoluble forms were enzymatically active. The vapor mode of operation over a range of relative humidities had comparatively small effects on kinetic constants for hydrolysis of ethyl acetate, which were also similar to those in phosphate buffer. The extent of reaction occurred in the order hydrolysis greater than alcoholysis greater than ester interchange greater than esterification. Reaction preference between alcoholysis and hydrolysis changed as acyl chain length of substrate increased with C. rugosa lipase, as well as with Rhizopus arrhizus lipase and porcine liver esterase, with details depending on the enzyme. The vapor mode approach has the potential of being used with a wide variety of substrates, as shown by the ability to obtain hydrolysis at 30 degrees C with substrate vapor pressures as low as 0.08 mm Hg and with substrates with boiling points as high as 206 degrees C.     

Purification and Properties of Mucor pusillus Acid Protease

The protease produced by Mucor pusillus was recovered from a wheat bran medium by treatment with ammonium sulfate, ethyl alcohol, gel filtration and ion-exchange chromatography. The yield of the enzyme was 55%. The overall increase in the specific activity of the protease was 34-fold. The purified protease was most active at pH 3.8 and 5.6 against hemoglobin and casein, respectively. Optimal hydrolysis of casein was observed at 55 C. The enzyme was stable from pH 3.0 to 6.0. Enzyme inactivated by metal ions was reactivated by ethylenediaminetetraacetate and o-phenanthroline. Reducing agents and thiol poisons had no effect on the protease, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inhibit the protease, indicating the probable absence of serine in the active center. The Michaelis-Menten constant for casein was 0.357%. Electrophoretic analysis of active protein recovered by ion-exchange chromatography showed that the protease preparation was homogeneous.     

Production and Characteristics of Avicel-Disintegrating Endoglucanase from a Protease-Negative Humicola grisea var. thermoidea Mutant

Mutational experiments were performed to decrease the protease productivity of Humicola grisea var. thermoidea YH-78 using UV light and N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, no. 140, exhibited higher endoglucanase activity than the parent strain in mold bran culture at 50°C for 4 days. The culture extract rapidly disintegrated filter paper but produced a small amount of reducing sugar. About 30% of total endoglucanase activity in the extract was adsorbed onto Avicel. The electrophoretically homogeneous preparation of Avicel-adsorbable endoglucanase (molecular weight, 128,000) showed intensive filter-paper-disintegrating activity but did not release reducing sugar. The preparation also exhibited a highly synergistic effect with the cellulase preparation from Trichoderma reesei in the hydrolysis of microcrystalline cellulose. This endoglucanase was observed via scanning electron microscopy to disintegrate Avicel fibrils layer by layer from the surface, yielding thin sections with exposed chain ends. A mutant, no. 191, producing higher protease activity and an Avicel-unadsorbable, Avicel-nondisintegrating endoglucanase was isolated. The purified enzyme (molecular weight, 63,000) showed no disintegrating activity on filter paper and Avicel and a less synergistic effect with the T. reesei cellulase in hydrolyzing microcrystalline cellulose than did the former enzyme. Endoglucanase was therefore divided into two types, Avicel disintegrating and Avicel nondisintegrating.     

Production and Characteristics of Raw-Starch-Digesting alpha-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher alpha-amylaseactivity than the parent strain under submerged culture at 30 degrees C for 24 h. About 70% of the total alpha-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable alpha-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable alpha-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal alpha-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Immobilization of halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. EMB9 protease on functionalized silica nanoparticles and application in whey protein hydrolysis

The present work targets the fabrication of an active, stable, reusable enzyme preparation using functionalized silica nanoparticles as an effective enzyme support for crude halophilic Bacillus sp. EMB9 protease. The immobilization efficiency under optimized conditions was 60 %. Characterization of the immobilized preparation revealed marked increase in pH and thermal stability. It retained 80 % of its original activity at 70 °C while t _1/2 at 50 °C showed a five-fold enhancement over that for the free protease. Kinetic constants K _m and V _max were indicative of a higher reaction velocity along with decreased affinity for substrate. The preparation could be efficiently reused up to 6 times and successfully hydrolysed whey proteins with high degree of hydrolysis. Immobilization of a crude halophilic protease on a nanobased scaffold makes the process cost effective and simple.     

Immobilization of the lytic enzyme complex lysoricephine]

Entrapping of the lysoenzyme complex of lysoricephine in solutions of hydrophilic polymers and its immobilization on dressing materials were performed. Immobilized preparations that retained 80-100% of the lytic activity and stable during storage were obtained. Properties of the immobilized preparations: dependence on pH and temperature, stability in acidic medium, and effect of gamma-radiation were studied. It was shown that coimmobilization with protease C induced a 1.5-1.7-fold increase in the lytic activity of the immobilized preparation compared to the native enzyme complex of lysoricephine.     

Effect of water on the surface molecular mobility of poly(lactide) thin films: an atomic force microscopy study

Physical properties associated with molecular mobility on the surface of thin films with 300 nm thickness for poly(lactide)s (PLAs) were studied under vacuum conditions as well as under aqueous conditions by using friction force mode atomic force microscopy (AFM). Two types of PLAs were applied for the experimental samples as uncrystallizable PLA (uc-PLA) and crystallizable PLA (c-PLA). The friction force on the surface of thin films was measured as a function of temperature to assess the surface molecular mobility both under vacuum and under aqueous conditions. A lower glass-transition temperature of the uc-PLA surface in water was detected than that under vacuum conditions. In the case of the c-PLA thin film, change in friction force was detected at a lower temperature under aqueous conditions than in vacuo. A morphological change was observed in the c-PLA thin film during heating process from room temperature to 100 degrees C by temperature-controlled AFM. The surface of the c-PLA thin film became rough due to the cold crystallization, and the crystallization of c-PLA molecules in water took place at a lower temperature than in vacuo. These friction force measurements and AFM observations suggest that molecular motion on the surface of the both uc- and c-PLA thin films is enhanced in the presence of water molecules. In addition, in situ AFM observation of the enzymatic degradation process for the c-PLA thin film crystallized at 160 degrees C was carried out in buffer solution containing proteinase K at room temperature. The amorphous region around the hexagonal crystal was eroded within 15 min. It has been suggested that the adsorption of water molecules on the PLA film surface enhances the surface molecular mobility of the glassy amorphous region of PLA and induces the enzymatic hydrolysis by proteinase K.     

Characterization of beta-galactosidase from a special strain of Aspergillus oryzae.

beta-Galactosidase [EC 3.2.1.23] was isolated from a partially purified preparation obtained from cultured cells of a special strain of Aspergillus oryzae, RT 102 (FERM-P1680). The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and was free from alpha-galactosidase, alpha- and beta-mannosidase, alpha- and beta-N-acetylhexosaminidase, and protease activities. The beta-galactosidase was capable of acting on aryl beta-galactosides, lactose, and lactosides. It also hydrolyzed beta-galactosyl linkages in urinary glycoasparagines and asialo alpha1-acid glycoprotein. The enzyme was rather stable in aqueous solution, retaining full activity at 4 degrees for at least several months. At pH 4.5, the optimum pH for the enzyme activity, and 37 degrees, full activity was maintained for several days.     

Enzyme activity in organic solvent as a function of water activity determined by membrane inlet mass spectrometry

Summary Membrane inlet mass spectrometry (MIMS) is introduced as a method for measuring water activity in nonpolar solvents, aqueous solutions and gas phase. The determination of the rate of hydrolysis of diphenyl carbonate by porcine liver esterase as a function of water activity in diisopropyl ether is presented as an example. A linear relationship is found between the enzyme activity and the water activity.     

Glycolytic activity of enzyme preparation from the red king crab (Paralithodes camtschaticus) hepatopancreas

Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas. The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation. HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the dependence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chitinase and protease activities are exhibited by different enzymes.     

A new <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> mutant strain producing serine protease efficient for hydrolysis of soy meal proteins

Induced mutagenesis with γ-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366 D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheniformis-145. Samples of dry concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was established. Alkaline serine protease subtilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolicheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited proteolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5–11) and temperature (30–70°C). Efficient hydrolysis of extruded soybean meal protein at high concentrations (20 to 50%) in the reaction mixture was the main advantage of the Protolicheterm-145 preparation. Compared to the preparation obtained using the initial strain, the new preparation with increased proteolytic activity provided for more complete hydrolysis of the main non-nutritious anti-nutritional soy proteins (glycinin and β-conglycinin) with the yield of soluble protein increased by 19–28%, which decreased the cost of bioconversion of the proteinaceous material and indicated promise of the new preparation in resource-saving technologies for processing soybean meals and cakes.     

Stabilization of proteolytic enzymes in solution

The stability of the neutral and alkaline proteases in a Bacillus subtilis enzyme mixture was studied in aqueous solutions at room temperature. Stabilization of the proteases in solution for periods up to 25 days was achieved by the addition of various protein preparations including casein and soya protein. The degree of stabilization by casein was concentration dependent to about 2% protein. The instability of the neutral protease in solutions of the B. subtilis enzyme mixture was shown to be due primarily to proteolysis by the alkaline protease since the diisopropylfluorophosphate-treated enzyme was quite stable. Formulation of such enzyme solutions at low pH gave greater stability as did solutions containing an alkaline protease inhibitor from potatoes. A Conceptual approach to the formulation of enzyme solutions containing proteolytic enzyme to ensure maximum stability is proposed.     

THE PROBLEM OF DEMINERALISATION IN THIN SECTIONS OF FULLY CALCIFIED BONE

Thin sections of embryonic avian bone decalcify during preparation for electron microscopy, creating a false impression of mineral distribution. The results of the experiments reported herein show that viscous embedding materials do not penetrate compact formed bone, and so, in thin sections, the calcium apatite crystals may be leeched out by water, both in the collecting trough and in aqueous solutions of stains used to enhance tissue electron opacity. To prevent decalcification, a simple technique is described in which the aqueous fluids that come in contact with thin sections are saturated with respect to calcium and phosphate ions, thereby preventing solution of the bone mineral. The theoretical basis of this technique is briefly discussed.     

Enzymatic surface modification of poly(ethylene terephthalate)

This study unambiguously confirms hydrolysis using cutinase of the persistent synthetic polymer poly(ethylene terephthalate), the most important synthetic fiber in the textile industry by direct measurement and identification of the different hydrolysis products. In this aqueous heterogeneous system, dissolved cutinase from Fusarium solani pisi acts on different solid poly(ethylene terephthalate) substrates. The extent of hydrolysis was detected by measuring the amount of (soluble) degradation products in solution using reversed-phase HPLC. Crystallinity greatly affects the capability of the enzyme to hydrolyze the ester bonds, displaying relatively high activity towards an amorphous polyester film and little activity on a highly crystalline substrate. The enzyme is sufficiently stable, hydrolysis rate on the amorphous substrate maintained at sufficient high level over a long period of time of at least five days. From an industrial point of view it is highly recommended to increase the hydrolysis rates.     

Enzyme processing of textiles in reverse micellar solution

Scouring of cotton using pectinase enzyme, bioscouring, in reverse micellar system was studied. The effectiveness of bioscouring was evaluated by measuring weight loss of cotton, analyzing pectin and cotton wax remaining and by wetness testing. Pectinase enzyme showed excellent activity even in organic media, and the effectiveness of scouring was equivalent or better than that achieved by conventional alkaline process or bioscouring in aqueous media. Enzymatic modification of wool using protease enzyme in the same system was also studied. It has found that felting property and tensile strength of wool fabrics treated by protease in reverse micellar system were superior to those in aqueous media. Possibilities of utilization of the same system for the subsequent textile dyeing process were also investigated. It was found that cotton and polyester fabrics were dyed satisfactorily by reverse micellar system compared to conventional aqueous system.