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.     

Enzymatic catalysis in heterogenous mixtures of substrates: The role of the liquid phase and the effects of "Adjuvants"

The physicochemical mechanism of protease-catalyzed peptide synthesis in heterogenous etuectic mixtures of substrates has been examined by a combination of microscopic techniques. Using a number of model reactions of dipeptide amide synthesis, it was determined that liquid phase catalysis was mostly, if not exclusively, responsible for the observed conversion of substrates. Furthermore, the formation of liquid or semiliquid eutectics was an important requirement for the occurrence of those reactions where both substrates were solids in the pure state. The addition of small quantities of hydrophilic solvents (adjuvants) often resulted in significat improvements in the rates and yields of the reactions. This was due to the ability of these adjuvants to promote the formation of eutectics, thereby increasing the proportion, as well as affecting the composition the properties, as well as affecting the composition and properties of the liquid phase. (c) 1994 John Wiley & Sons, Inc.     

Native subtilisin Karlsberg and modified subtilisin 72 as effective catalysts of peptide bond formation in organic media

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z-Ala-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P ₁ and P ₁ ^′ (Glu, Arg, and Asp) containing unprotected side ionogenic groups.     

Subtilisin Carlsberg in complex with sodium dodecyl sulfate is an effective catalyst for the solid phase segment coupling of peptides on polyvinyl alcohol cryogel

Subtilisin Carlsberg (SC) was shown to catalyze the solid phase segment coupling of peptides in complex with sodium dodecyl sulfate (SDS) in an organic medium on Aminosilochrom and polyvinyl alcohol (PVA) cryogel activated with glutaraldehyde or divinylsulfone. Diamines of different lengths with a general formula NH₂-(CH₂) _n -NH₂ (n = 2, 4, and 6) were used as spacers between the PVA cryogel and the peptide. A model reaction of enzymatic attachment of the Dnp-Ala-Ala-Leu-OMe tripeptide to the PVA cryogel was carried out by treatment with the SDS-SC complex in a mixture of anhydrous ethanol and DMSO (7 : 3, v/v) using a tenfold excess of the carboxyl component. The molar enzyme-substrate ratio was 1 : 88. The effect of the method of matrix activation, length of a spacer, and reaction time on the coupling efficiency was studied. Hexamethylenediamine was found to be the most effective spacer for the enzymatic coupling on the PVA cryogel activated with glutaraldehyde (the reaction proceeded with the highest yield of 60%). The reaction efficiency was considerably lower in the case of ethylenediamine and tetramethylenediamine (10 and 15%, respectively). The best results were obtained on the PVA cryogel activated by divinylsulfone with hexamethylenediamine as a spacer. A two-step condensation of tripeptides was carried out on this support. The second step of condensation was shown to proceed better (in 85% yield) in comparison with the first step (37% yield).     

Peptide Synthesis in Organic Media with the Use of Subtilisin 72 Immobilized on a Poly(Vinyl Alcohol) Cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate.     

Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family

A chemoenzymatic synthesis was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are o-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for substrate (I). The lowest detectable papain concentrations were 2.4 × 10^?10 M for (I) and 1.2 × 10^?11 M for (II). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.     

Dimethyl sulfoxide-induced high enantioselectivity of subtilisin Carlsberg for hydrolysis of ethyl 2-(4-substituted phenoxy)propionates

The enantioselectivity for subtilisin-catalyzed hydrolysis of ethyl 2-(4-substituted phenoxy)propionates in an aqueous buffer solution was improved by addition of DMSO (54–56% v/v). On the basis of the conformational change of subtilisin Carlsberg observed for FT-IR and CD spectra, the high enantioselectivity for subtilisin-catalyzed hydrolysis of racemic ethyl 2-(4-ethylphenoxy)propionate could be related to a partial decrease of the tertiary structure of the enzyme protein arising from an increase of the ratio of DMSO in the reaction medium. This mechanistic model for the enantiorecognition can also be supported by the discussion based on the value of the Michaelis constant (K _m) obtained for each enantiomer of the substrate.     

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Synthesis of aspartame precursor with an immobilized thermolysin in tert-amyl alcohol

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetner asparatame, was synthesized from N-(benzyloxycarbolyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) with an immobilized thermolysin in various organic solvents. We found that in tert-amyl alcohol containing a small amount of water the immobilized enzyme showed a high activity comparble to that in ethyl acetate with quite a high stability. The immobilized enzyme was fully stable up to 70 degrees C in tert-amyl alcohol in the absence of the subatrate, and up to 50 degrees C in the presence of the substrate. The high stability in the presence of the substrate was found due to the fact that the release of calcium ions, the stabilizing factor of thermolysin, is suppressed.The substrate concentration dependence of the initial synthetic rate with the immobilized enzyme was quite different from that with the free enzyme in the biphasic system, in contrast to that in ethyl acetate. Finally, Z-AspPheOMe was continuously synthesized in a column reactor using 200 mM PheOMe and 120 mM Z-Asp as the substrate for over 300 h at 45 degrees C and a space velocity of 1 h(-1) without any loss of acivity. (c) 1994 John Wiley & Sons, Inc.     

Native and Modified Subtilisin 72 as a Catalyst for Peptide Synthesis in Media with a Low Water Content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content >80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, Lys, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70–98% in DMF–MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

One-pot synthesis and characterization of cross-linked quaternized chitosan microspheres as protein adsorbent

The one-pot synthesis and characterization of cross-linked quaternized chitosan microspheres (CQCM) as a protein adsorbent are presented. First of all, chitosan particles were prepared by spray drying method, and then they were quaternized and cross-linked in turn with glycidyltrimethylammonium (GTMAC) chloride and glutaraldehyde in isopropanol containing 10% water in one-pot. The effect of the reaction temperature, reaction time and the amounts of added GTMAC and glutaraldehyde on the protein adsorption ability of CQCM was investigated. The adsorption behavior of the CQCM prepared in the optimum synthetic conditions was well described by the Langmuir isotherm with maximum adsorption capacity equal to 1424 mg BSA/g dry weight. The particle size ranged from 7.6 to 48.9 μm. The mechanism of adsorption-desorption of BSA to the CQCM was ion-exchange. Finally, the extraction of soybean peroxidase from crude soybean peroxidase solution using the CQCM was performed.     

Reversal of enzymatic hydrolysis: Rate and extent of ester synthesis as catalyzed by chymotrypsin and subtilisin Carlsberg at low water concentrations

Immobilized chymotrypsin catalyzes esterification of N-acetyltyrosine in a medium containing high concentrations of alcohols. The hydrophilic support and inclusion of glycerol protect the enzyme activity and allow catalysis to proceed in the presence of only 10% (v/v) water. The same equilibrium concentration of ester is obtained whether reaction proceeds from ester or from free acid. Hates of ester synthesis and hydrolysis are similar when measured under the same conditions, but are at least one order of magnitude slower than optimal rates of hydrolysis. Subtilisin Carlsberg in the free, unmodified form catalyzes ester synthesis at even lower water concentrations; optimal rates are obtained at 5–15% H₂O. Hydrolytic enzymes can thus be utilized as catalysts of synthesis reactions in nonaqueous solvents where synthesis is thermodynamically favored over hydrolysis; in some cases this may provide economic and/or energetic advantages over conventional techniques.     

Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.     

Enzymatic synthesis of esters using an immobilized lipase

Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from 25 to 37 degrees C. Ethylpropionate was formed from ethanol and propionic acid at a rate of 0.017 mol/h g immobilized protein. Different esters were formed at comparable rates and equilibrium conversions could generally be approached in less than 10 h in a batch reaction system. The immobilized lipase catalyst was quite stable and retained about one third of the initial activity after repeated experiments during the course of 72 days. A stirred tank continuous flow reactor was used successfully for the continuous production of esters.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

Pulsed column reactor as a novel tool for continuous enzymatic synthesis of peptide in an aqueous/organic biphasic medium

We propose a novel effective method for a continuous peptide synthesis in an aqueous/organic biphasic medium using a pulsed column reactor. N-Formyl-l-aspartyl-l-phenylalanine methyl ester was enzymatically synthesized continuously. With this extractive method using a pulsed column reactor, we can synthesize peptides with a stable performance even if a peptide (or a peptide-amino acid complex) is precipitated due to its high hydrophobicity.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Development of the efficient preparation method for thermoresponsive elastin-like peptides using liquid-phase synthesis combined with fragment condensation strategy

Elastin-like peptides (ELPs) are synthetic peptides that mimic the characteristic hydrophobic amino acid repeat sequences of elastin and exhibit temperature-dependent reversible self-assembly properties. ELPs are expected to be used as temperature-responsive biomolecular materials across diverse industrial and research fields, and there is a requirement for a straightforward method to mass-produce them. Previously, we demonstrated that phenylalanine-containing ELP analogs, namely, (FPGVG)_n, can undergo coacervation with short chains (n = 5). The Fmoc solid-phase peptide synthesis method is one strategy used to synthesize these short ELPs. However, owing to its low reaction efficiency, an efficient method for preparing ELPs is required. In this study, efficient preparation of ELPs was investigated using a liquid-phase synthesis method with a hydrophobic benzyl alcohol support (HBA-tag). Because HBA-tags are highly hydrophobic, they can be easily precipitated by the addition of poor solvents and recovered by filtration. This property allows the method to combine the advantages of the simplicity of solid-phase methods and the high reaction efficiency of liquid-phase methods. By utilizing liquid-phase fragment condensation with HBA-tags, short ELPs were successfully obtained in high yield and purity. Finally, the temperature-dependent response of the ELPs generated through fragment condensation was assessed using turbidity measurements, which revealed a reversible phase transition. Consequently, the ELPs exhibited a reversible phase transition, indicating successful synthesis of ELPs via fragment preparation with tags. These findings provide evidence of the potential for mass production of ELPs using this approach.     

Enzymatic synthesis of an acylated phloroglucinol derivative with phloroglucinol and vinyl octanoate in acetonitrile

Monooctanoyl phloroglucinol with anti-microbial activities was synthesized from phloroglucinol (50 mM) and vinyl octanoate (150 mM) with agitation at 40 °C using 10 mg lipase AK (Pseudomonas fluorescens) in acetonitrile (1 ml, aw=0.07). The yield was approximately 70% as conversion rate of phloroglucinol over 3 days. The product, at 205 g ml^–1, was bacteriocidal against Escherichia coli and Staphylococcus epidermidis.