Effect of low and high temperatures on chymotrypsin from Atlantic cod (Gadus morhua L.); comparison with bovine alpha-chymotrypsin

1. Cod chymotrypsin displays higher enzyme activity compared to bovine alpha-chymotrypsin when assayed at low temperatures (3-15 degrees C). 2. Both enzymes are inactivated when incubated at temperatures between 60 and 70 degrees C. 3. When incubated at 99 degrees C the cod enzyme retains about 50% of the initial activity measured at room temperature. 4. Preincubation at boiling temperature renders the cod chymotrypsin active at 70 degrees C whereas the bovine enzyme is rapidly inactivated.     

三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization

A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60–65 ^∘C and retained 100% activity after incubation at 60 ^∘C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ^∘C and 23 min incubation at 80 ^∘C. Catalytic function of lipase was activated by Mg⁺⁺ (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, βME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m and V _max of 0.5 mM and 0.139 μM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.     

Glass-immobilized glycated-trypsin: A novel modified trypsin that is remarkably thermostable

Porcine trypsin was glycated with glucose and covalently immobilized through its carboxyl groups onto aminated glass beads to produce porcine immobilized glycated-trypsin (IGT). On incubation at 60 °C and pH 8, IGT retained its full activity for 8 h and 50% of its activity after 24 h. In comparison, under the same conditions porcine native trypsin lost 80% of its activity in 2 h and was completely inactivated in less than 4 h. The rate of autolysis of porcine glycated-trypsin at 37 °C was 40% that of native trypsin and with IGT there was no significant autolysis, even at elevated temperatures as high as 60 °C. Glycation significantly increased the stability of trypsin and immobilization also significantly increased the stability of trypsin. The remarkable thermostability of IGT is attributed to a synergistic effect when these two modifications are combined. Tryptic fragmentation of denatured proteins with IGT can be performed at 60 °C for shorter digestion times and with smaller amounts of enzyme than normally employed to achieve complete digestion with soluble forms of trypsin. Prior denaturation of proteins for tryptic digestion is not required with IGT as in situ denaturation and digestion can be achieved simultaneously at 60 °C with an enzyme:protein mass ratio as low as 1:1000.     

Chemical modification of tryptophanase from E. coli with polyethylene glycol to reduce its immunoreactivity towards anti-tryptophanase antibodies

Escherichia coli tryptophanase was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2, MW 5,000 x 2). The modified tryptophanase, in which approximately 43% of the total 120 amino groups and 38% of the total 16 sulfhydryl groups in the molecule were coupled, completely lost the immunoreactivity towards anti-tryptophanase serum from rabbit. Approximately 10% of the enzymic activity was retained. The modified enzyme showed the same physicochemical properties as the native enzyme: Km value for L-tryptophan (0.3 mmol/l), optimum pH (8.0) and optimum temperature (50 degrees C). The modified enzyme was more resistant than the native counterpart against proteolytic digestion with trypsin.     

Purification and characterization of benzoyl-L-tyrosine ethyl ester hydrolase from the yolk sac membrane of chicken egg

An enzyme which hydrolyzes benzoyl-L-tyrosine ethyl ester (BTEE) was purified from yolk sac membranes of day-18 chick embryos. The purified BTEE hydrolase has a molecular weight of 110,000, being composed of 70,000 and 40,000 subunits, and preferred synthetic substrates for chymotrypsin to those for trypsin. The optimum pH and temperature of this enzyme were 6.5-7.0 and 40 degrees C, respectively. The Km value for BTEE of the enzyme was 16 mM at pH 6.5 and 30 degrees C. The enzyme was inhibited markedly by some chymotrypsin inhibitors but scarcely inhibited by trypsin inhibitors. Magnesium ion acted as potent activator, depending on the enzyme purity and its concentration, whereas p-chloromercuribenzoate and zinc ion inactivated the activity markedly. The BTEE hydrolase was found to hydrolyze proteins such as casein and hemoglobin. These data indicated that the enzyme is a proteinase similar to chymotrypsin. This proteinase could act on yolk proteins, suggesting that it plays an important role in the metabolism of yolk at the yolk sac membrane layer.     

Carnosine promotes the heat denaturation of glycated protein

Glycation alters protein structure and decreases biological activity. Glycated proteins, which accumulate in affected tissue, are reliable markers of disease. Carnosine, which prevents glycation, may also play a role in the disposal of glycated protein. Carnosinylation tags glycated proteins for cell removal. Since thermostability determines cell turnover of proteins, the present study examined carnosine's effect on thermal denaturation of glycated protein using cytosolic aspartate aminotransferase (cAAT). Glycated cAAT (500 microM glyceraldehyde for 72h at 37 degrees C) increased the T(0.5) (temperature at which 50% denaturation occurs) and the Gibbs free energy barrier (DeltaG) for denaturation. The enthalpy of denaturation (DeltaH) for glycated cAAT was also higher than that for unmodified cAAT, suggesting that glycation changes the water accessible surface. Carnosine enhanced the thermal unfolding of glycated cAAT as evidenced by a decreased T(0.5) and a lowered Gibbs free energy barrier. Additionally, carnosine decreased the enthalpy of denaturation, suggesting that carnosine may promote hydration during heat denaturation of glycated protein.     

Transglutaminase-catalysed glycosidation of trypsin with aminated polysaccharides

Summary Dextran (MW=7.2×10⁴), carboxymethylcellulose (MW=2.5×10⁴, substitution degree=0.7) and Ficoll (MW=6.9×10⁴) were derivatized with 1,4-diaminobutane and covalently attached to bovine pancreatic trypsin through a transglutaminase-catalysed reaction. The conjugates contained an average of 0.7–1.8 mol of polymers per mol of protein, and retained about 61–82% of the original esterolytic activity of trypsin. The optimum pH for trypsin was shifted to alkaline values after enzymatic glycosidation. The thermostability of the polymer–enzyme complexes was increased in about 13.7–50.0 °C over 10 min incubation. The prepared conjugates were also more stable against thermal incubation at different temperatures ranging from 50 °C to 60 °C. In comparison with native trypsin, the enzyme-polymer complexes were about 22- to 48-fold more resistant to autolytic degradation at pH 9.0. Transglutaminase-catalysed glycosidation also protected trypsin against denaturation in surfactant media, with 9- to 68–fold increased half-life times in the presence of 0.3% (w/v) sodium dodecylsulfate.     

Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A

An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.     

Purification of chymotrypsin from bovine pancreas using precipitation with a strong anionic polyelectrolyte

The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01 mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity.     

Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives.

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.     

Estimation of protein digestibility—III. Studies on the digestive enzymes from the pyloric ceca of rainbow trout and salmon

Digestive proteinases were isolated and partially purified from the pyloric ceca of trout and salmon. Their stability and some catalytic properties were compared with those of a three-enzyme system that is used for determination of in vitro protein digestibility. In contrast to the three-enzyme system, pyloric ceca trypsin and total proteinase activity were least stable at pH values below 5.0 and most stable under alkaline conditions up to pH 10.0. Thermal inactivation (50%) occurred in 60 min at 55°C for trypsin activity of trout and salmon ceca proteinases and at 40°C for the three-enzyme system at the pH (8.0) of the in vitro assay. Thermal inactivation (50%) of total proteinase activity occurred in 60 min at about 55, 50 and 35°C for chinook, trout and three-enzyme preparations, respectively. SDS-PAGE zymograms of the ceca enzymes showed the presence of several proteolytic activity bands. Two of the bands corresponded in molecular weight to trypsin and chymotrypsin. Ceca proteinases differ from the three-enzyme system in their response to inhibitors; in particular, the ceca proteinases are much more sensitive to soybean trypsin inhibitor than the procine trypsin used in the three-enzyme system when assayed for trypsin, but less sensitive when assayed for total proteinase. The distinctive properties of ceca enzymes help explain why they are more appropriate than the three-enzyme system, and other enzyme cocktails for in vitro protein digestibility assay of saunonid feed components.     

Characterization of a collagenolytic serine proteinase from the Atlantic cod (gadus morhua)

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH₄)₂SO₄ fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (±0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-l-Ala-l-Ala-l-Pro-l-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50°C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30°C.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.     

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.     

Inhibitory effect of glycation on catalytic activity of alanine aminotransferase

Non-enzymatic glycation is a common post-translational modification of tissue and plasma proteins which can impair their functions in living organisms. In this study, the authors have demonstrated for the first time an inhibitory effect of in vitro glycation on the catalytic activity of alanine aminotransferase (ALT, EC 2.6.1.2), a pyridoxal phosphate enzyme with several lysine residues in the molecule. The porcine heart enzyme was incubated with 50 mmol/l D-fructose, D-glucose, D,L-glyceraldehyde, or D-ribose in 0.1 mol/l phosphate buffer (pH 7.4) at 25°C for up to 20 days. The strongest glycation effect was shown by D,L-glyceraldehyde, which caused complete enzyme inhibition within 6 days. After 20 days of incubation, the ALT activity in samples with D-fructose and D-ribose was less than 7% of the initial enzyme activity. A statistically significant effect of D-glucose on the enzymatic activity of ALT was not found. Incubation of ALT with D-fructose, D,L-glyceraldehyde and D-ribose minimized its catalytic activity both in the glycated and non-glycated fractions of the samples. Markedly higher activity was found in the glycated fraction with glucose. The inhibitory effect of glycation of ALT with D-fructose and D-ribose was found to be more intensive in the presence of L-alanine and weaker in the presence of 2-oxoglutarate. The findings suggest that glycation of the e-amino group of Lys313 as a crucial part of the catalytic site of ALT may contribute to ALT inactivation in the presence of glycating sugars. Nevertheless, glycation of lysine residues outside the active center of ALT seems to be primary.     

A thermally sensitive loop in clostridial glutamate dehydrogenase detected by limited proteolysis

The structural flexibility and thermostability of glutamate dehydrogenase (GDH) from Clostridium symbiosum were examined by limited proteolysis using three proteinases with different specificities, trypsin, chymotrypsin, and endoproteinase Glu-C. Clostridial GDH resisted proteolysis by any of these enzymes at 25 degrees C. Above 30 degrees C, however, GDH became cleavable by chymotrypsin, apparently at a single site. SDS-PAGE indicated the formation of one large fragment with a molecular mass of approximately 44 kDa and one small one of <10 kDa. Proteolysis was accompanied by the loss of enzyme activity, which outran peptide cleavage, suggesting a cooperative conformational change. Proteolysis was prevented by either of the substrates 2-oxoglutarate or l-glutamate but not by the coenzymes NAD(+) or NADH. Circular dichroism spectroscopy indicated that the protective effects of these ligands resulted from fixation of flexible regions of the native structure of the enzyme. Size-exclusion chromatography and SDS-PAGE studies of chymotrypsin-treated GDH showed that the enzyme retained its hexameric structure and all of its proteolytic fragments. However, circular dichroism spectroscopy and analytical ultracentrifugation showed global conformational changes affecting the overall compactness of the protein structure. Chymotrypsin-catalyzed cleavage also diminished the thermostability of GDH and the cooperativity of the transition between its native and denatured states. N-terminal amino acid sequencing and mass spectrometry showed that heat-induced sensitivity to chymotrypsin emerged in the loop formed by residues 390-393 that lies between helices alpha(15) and alpha(16) in the folded structure of the enzyme.     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

Physical and chemical characterization of glutamine synthetase purified from Mycobacterium phlei

Glutamine synthetase (L-glutamate: ammonia ligase [ADP forming]) [EC 6.3.1.2] has been purified from a Gram-positive, acid-fast bacterium, Mycobacterium phlei, by simple procedures with 57% recovery. The enzyme resembled that from Mycobacterium smegmatis in the subunit size (56,000), molecular weight (670,000), amino acid composition, the amino acid sequence of the NH2-terminal, and the secondary structure. The enzyme activity was regulated by adenylylation of each subunit in the dodecameric molecule. M. phlei glutamine synthetase possesses two useful characteristics: high thermostability and resistance to protease digestion. The enzyme was not inactivated on exposure to 60 degrees C for 2 h or 37 degrees C for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37 degrees C for 12 h, pH 7.8. With saturating substrate levels, the Arrhenius plot was nonlinear and concave downward with an intersection point at 45 degrees C, and the activation energies were calculated to be 3.2 and 9.6 cal/mol from the slopes. The specific activity of the highly adenylylated enzyme (E10.7) was remarkably lower than that of the slightly adenylylated enzyme (E2.5); however, both enzymes show similar profiles of the Arrhenius plot. These results indicate that the adenylylation of the enzyme does not affect its activation energies.