Preparation and catalytic properties of trypsin immobilized on cryogels of polyvinyl alcohol

Commercial preparations of trypsin, varying in activity, were immobilized on a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In organic media, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-arginine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.     

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

Protein hydrolysis by immobilized and stabilized trypsin

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.     

Studies on the proteolytic activity of Bacteroides fragilis

The proteolytic activity of the intestinal bacterium Bacteroides fragilis NCDO 2217 was cell-bound during exponential growth, but was progressively released from the cells in stationary phase. Proteins hydrolysed included casein, trypsin, chymotrypsin, azocasein and the proteins in azosoya bean flour. Collagen, azocoll, elastin, gelatin, ovalbumin and bovine serum albumin were either weakly degraded or completely refractory to proteolysis. Arylamidase activity was exhibited against leucine p-nitroanilide (LPNA), leucine beta-naphthylamide, glycyl-proline p-nitroanilide and valyl-alanine p-nitroanilide. The bacterium grew with ammonia, peptone or casein as sole nitrogen source. Azocasein- and LPNA-hydrolysing activities were consistently higher when grown on casein. Cell-bound protease activity increased concomitantly with growth rate in both carbon- and nitrogen-limited continuous culture. Leucine arylamidase activity was also growth-rate-dependent, being 3-fold greater at D = 0.18 h-1 compared to D = 0.03 h-1. Extracellular proteolytic activity was only detected at low growth rates, accounting for about 25% of total protease activity.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Acceleration of enzymatic reaction of trypsin through the formation of water-soluble complexes with poly(ethylene glycol)-block-poly(alpha,beta-aspartic acid)

The amidase activity of bovine pancreas trypsin in water-soluble complexes with poly(ethylene glycol)-block-poly(alpha,beta-aspartic acid) (PEG-PAA) was evaluated by a colorimetric assay using L-lysine p-nitroanilide as a substrate. The enzymatic reaction of trypsin was accelerated through the complexation with PEG-PAA. By determining the kinetic parameters of the enzymatic reaction of trypsin, it was confirmed that the catalytic rate constant of the complexed trypsin was 15 times higher than that of the native trypsin. From the evaluation of pH dependence of initial reaction rate, it was indicated that this acceleration was induced by a stabilization of the imidazolium ion of the His residue in the catalytic site, the Asp-His-Ser triad, of trypsin due to the Asp units of PEG-PAA. The hydrogen bonded Asp-His pairs are critical constituents in several key enzymatic reactions including serine protease and apurinic endonucleases, and it was expected that the acceleration of the catalytic reaction might occur for other enzymes by the formation of water-soluble complexes with PEG-PAA.     

Biocomplex of subtilisin Carlsberg with chitosan as an effective biocatalyst for hydrolysis and synthesis of peptides

New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplex (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocata-lytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6 : 4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min.     

Biocomposite of subtilisin Carlsberg with chitosan as an effective biocatalyst for hydrolysis and synthesis of peptides

New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplosite (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocatalytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6:4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min.     

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.     

Increased activity and stability of poly(ethylene glycol)-modified trypsin.

The reaction of trypsin with activated monomethoxypoly(ethylene glycol) with various molecular masses led to the development of a series of poly(ethylene glycol)-modified trypsins (PEG-trypsins). On determining the catalytic properties of PEG-trypsin using N-benzoyl-L-arginine p-nitroanilide as a substrate, a three- to fourfold increase in the maximal velocity of hydrolysis was found to occur, whatever the size of the PEG moiety used. PEG-trypsin with higher molecular mass moieties showed lower Michaelis constant values. The activation of trypsin was neither reversed by nucleophiles such as hydroxylamine, nor prevented when modification was carried out in the presence of benzamidine or in the presence of the polypeptidic soybean trypsin inhibitor. Chemical modification of about 80% of the free amino groups with PEG chains significantly improved the resistance to heat and detergents. This might result from the formation of a highly hydrogen-bonded structure around the enzyme.     

Properties of trypsin and of acid phosphatase immobilized in sol-gel glass matrices.

Trypsin and acid phosphatase-containing silica sol-gel glasses were obtained by mixing a solution of an enzyme with polyethylene glycol (PEG) 6000 and tetramethoxy orthosilicate at room temperature, followed by gelation and drying. Activity of the immobilized trypsin toward small substrates, such as N-benzoyl-L-arginine-4-nitroanilide at its Km, for the best preparations equaled that of the soluble enzyme. Polylysine (M(r) less than or equal to 13,000) and aprotinin (M(r) = 6,500) inhibited this activity. Larger polylysines as well as soybean trypsin inhibitor (M(r) = 20,100) were ineffective. The sol-gel-entrapped trypsin activity was stable when sol-gel glasses were incubated at ambient temperature (pH 7.5) for several months. In comparison, trypsin, immobilized in sol-gel glass by surface adsorption and incubated under the same conditions overnight, was completely autodigested. The firm interaction between the protein molecules and the silica matrix stabilized the enzymes. Thus, the half-life of sol-gel-entrapped acid phosphatase at 70 degrees C (pH 8.0) was two orders of magnitude larger than that of the enzyme in solution. Transparent, mechanically and chemically stable bioactive sol-gel glasses may be used for the development of robust on-line biochemical photodetection sensors and for the purposes of chemical catalysis.     

Stability and specificity of extracellular protein inhibitor for trypsin from Actinomyces janthinus 118]

Some properties of protein inhibitor for trypsin (TI) from Act. janthinus 118 were studied. It was shown that TI has an antitrypsin activity within a wide pH range with a maximum at about 9,5. At 4 degrees and 20 degrees C TI is stable for 24 hours within the pH range of 6,0--11,0. At 100 degrees C TI is more stable in the slightly acid region of pH than at neutral or alkaline conditions. Trypsin and chymotrypsin inactivate the inhibitor for 8 hours. TI inhibits trypsin, fibrinolysin, subtilisin, pronase and terrilytin, but have no effect on chymotrypsin, thrombin, papain and pepsin. The dissociation constants for the trypsin-inhibitor complex were found to be 1,7.10-8 M, 4,1.10-9 M and 2,4.10-10 M, with casein, p-nitroanilide benzoylarginine and tosylarginine methyl ester used as substrates, respectively. The corresponding dissociation rate constants for the subtilisin-inhibitor complex were equal to 1.10-9 M and 4.10-10 M with casein and carbobenzoxy-L-alanyl-L-alanyl-L-leucin p-nitroanilide used as substrates, respectively.     

An inexpensive device for sample stirring in VSU Zeiss spectrophotometers. Its use for kinetic measurements and active site titrations with immobilized enzymes.

The construction of a simple and effective sample stirring device for commercial spectrophotometers and its use for continuous kinetic measurements and active site titrations with immobilized enzymes is described. Sepharose-bound leucine aminopeptidase and trypsin were selected as model enzymes to test the performance of the magnetic stirring equipment. Kinetic parameters of insolubilized leucine aminopeptidase using L-leucine p-nitroanilide as substrate and the catalytic site concentration of matris-bound trypsin using p-nitrophenyl p'-guanidinobenzoate as active site titrant could be evaluated without significant interference from the turbidity of the stirred Sepharose suspension. The problem of grinding of the support material could be overcome. Both unbound native and carrier-fixed enzyme may be reacted under identical conditions with similar convenience and sensitivity.     

N-succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide as a sensitive substrate for assaying activity of cysteine proteinases

N-Succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide has been found to be a very sensitive chromogenic substrate for the assay of cysteine proteinase papain, ficin and bromelain. N-Succinyl-alanyl-S-benzylcysteine p-nitroanilide and N-succinyl-alanyl-alanyl-S-benzylcysteine p-nitroanilide are also suitable for this purpose. These substrates were hydrolyzed only very slightly or not hydrolyzed at all by trypsin.     

Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity.

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.     

Properties of lactate dehydrogenase from the isopod, Saduria entomon

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.     

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.     

Purification of pancreatic kallikrein by the method of affinity chromatography]

Pancreatic Kallikrein was purified by affinity chromatography on BPTI-Sepharose. Immobilized BPTI was prepared via three stages: a) formation of the BPTI--acetylated trypsin complex; b) coupling of the resultant complex with CNBr-activated Sepharose; c) dissociation of the bound complex at pH 2.0 to yield immobilized BPTI and acetylated trypsin. The final product contained 59 X 10(-3) micronmoles insolubilized BPTI/ml Sepharose. Trypsin occurring in commercial Kallikrein preparations was separated by the batch procedure with ovomucoid-Sepharose. This method allows 100-fold purification of commercial Kallikrein.     

Protease activity in gut of Daphnia magna: evidence for trypsin and chymotrypsin enzymes

Two major protease activities were present in gut homogenates of the cladoceran crustacean Daphnia magna: (i) a trypsin activity that hydrolysed the synthetic substrate N-benzoyl-dl-arginine p-nitroanilide and was strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF) and not inhibited by chymostatin; and (ii) a chymotrypsin activity that hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, did not hydrolyse N-benzoyl-l-tyrosine p-nitroanilide, and was strongly inhibited by chymostatin and not by TLCK and APMSF. Both activities had alkaline pH optima (pH 7-10), but were shown to be due to distinct types of proteases. These two enzyme activities accounted for 75-83% of the proteolytic activity of gut contents. Substrate SDS-polyacrylamide gel electrophoresis revealed nine different proteases ranging from 15 to 73 kDa.