Thermolysin, immobilization

CBZ-Ser-Leu-OMe synthesis has been carried out starting from CBZ-Ser and Leu-OMe, using free thermolysin in a monophasic system, and thermolysin immobilized-stabilized on agarose gels, in a biphasic system. The influence of different variables such as pH, polarity of the reaction medium, temperature and substrate concentrations on the dipeptide yield and reaction rate has been studied. 

Thermolysin immobilized on agarose gels as described in the experimental section was used as catalyst in a biphasic reaction system (acetate buffer 0,1 M and 10 mM CaCL/ethyl acetate (1 1) (v/v)). This medium could prevent the secondary reactions [1] and [2], since the dipeptide is more soluble in the organic phase than in water, and it allows to achieve high synthetic yields by removing the product from the aqueous phase. 

TABLE 1 Effect of Organic Solvents on Relative Remaining Activity of Thermolysin Immobilized onto XAD-7 and Solubility of Water... 

FIG. 12 Relationship between the relative remaining activity of thermolysins immobilized onto Amberlite XAD-7 [1,2], Amberlite XAD-8 [3], Amberlite XAD-5003 [4], and CPG-7.5 [5], and CPG-300 [6] and water content inside the immobilized enzyme after 5-h incubation at 70°C in ethyl acetate (open circles) and in ferf-amyl alcohol (closed circles). The figures in parentheses indicate the water content in the bulk organic solvent phase. (From Ref. 59.)... 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

K. Kihara, andT. Hashimoto, Synthesis of an aspartame precursor by immobilized thermolysin in an organic solvent,... 

First and foremost, cross-linked enzyme crystals are crystals. Within the crystal lattice the concentration of protein approaches the theoretical limit. This is important to the process development chemist, who would much rather use a small quantity of a very active catalyst in a reactor than fill it with an immobilized enzyme. Typically an immobilized enzyme contains only 1-10% by weight enzyme, with the remaining carrier material simply occupying valuable reactor space. The crystallinity is absolutely required to achieve the stability exhibited by CLCs [8], Cross-linked soluble thermolysin and cross-linked precipitate of thermolysin are no more stable than the soluble enzyme. Crystals of proteins... 

Other important applications in the food industry running at a large scale are the production of L-aspartic add with Escherichia coli entrapped in polyacrilamides [6], the immobilization of thermolysin for the production of aspartame [14], The production of L-alanine by Tanabe Seiyaku [7], the production of frudose concen-centrated syrup [3], the production of L-malic acid by the use of Brevibacterium ammoniagenens immobilized in polyacrilamide by entrapment immobilization methods [11] and L-aminoacids production by immobilized aminoacylase [5],... 

Recently the possibility of total enzymatic synthesis of Aspartame has been suggested (44). It was shown that immobilized penicillinacylase would hydrolyze JJ-phenacetyl L-L-aspartame thus, if thermolysin will accept phenacetyl L-Asp, use of the carbobenzoxyl protecting group and its subsequent removal by hydrogenation would not be required. 

The use of enzymes to stereospecifically form amide bonds has been described in many texts (115) however, the commercial availability cf cross-linked enzyme crystals (CLECs), for example, PeptiCLEC-TR, which is an immobilized form of Thermolysin protease, has been used in the synthesis of D2163 (68), a novel matrix metalloproteinase inhibitor (116). In vitro enzyme screening identified the all-natural SSS-isomer as the active product. The elegant CLEC (117) technology used in this example makes the enzyme stable to typical organic reaction conditions and enables facile removal of the enzyme at the end of the reaction by simple filtration. On this basis, it is... 

Immobilization of Enzymes. Enzymes (carboxypeptidase A, B, and Y, chymotrypsin, thermolysin, trypsin, and V -protease), obtained from Sigma were applied directly for immobilization. About 20 mg of each enzyme was dissolved into 0.1N phosphate buffer pH 7.0, and placed into a 10 x 75 mm test tube with 1 g of succinylamidopropyl glass beads. After degassing, 0.02 umole of l-ethyl-3-(3-dimethylamlnopropyl)-carbodilmide (EDC) (Sigma Chemical Co.) was added to the tube which then was sealed with paraffin and rotated at 4°C overnight for simultaneous activation/immobilization. 

Three fragments of the delicious peptide sequence lysine-glycine, serine-leucine-alanine and aspartic acid-glutamic acid-glutamic acid possess separately umami/salty, bitter and sour taste respectively but mixtures or combinations of them produce a similar taste of that corresponding to the complete octapeptide. The synthesis of CBZ-Lys-Gly-OMe and CBZ-Ser-Leu-OMe were carried out using immobilized trypsin and thermolysin. We have studied the influence of reaction medium (pH, temperature and substrate concentration) on the yield and initial reaction rate of synthesis. 

The synthesis was also carried out in similar stoppered flasks placed in a thermostated bath. The reaction medium (aqueous pH 7 and biphasic acetate buffer 0.1 M, CaCk 10 mM/ethyl acetate (1 1) (v/v)), containing the substrates were adjusted to the desired pH. Reactions were started by adding 1100 units of immobilized thermolysin. Aliquots were taken at different times and analyzed by HPLC as above mentioned. In this case the eluent was phosphate buffer 0.1 M (pH = 2.3) and acetonitrile (62 38) (v/v). 

A lipase has been used to convert solid triolein (glycerol trioleate) to the monooleate by treatment with glycerol at 8°C.75 Other lipases have been used in the hydrolysis and transesterification of oils, as well as in the esterification of fatty acids without solvents.76 Peptides can be produced from eutectic mixtures of amino acid derivatives with the addition of a small amount of solvent.77 Immobilized sub-tilisin and thermolysin were used with 19-24% water or an alcohol to produce polypeptides. Subtilisin on celite (a di-atomaceous earth) was used to convert a mixture of L-phenylalanine ethyl ester and L-leucinamide containing 10% triethyleneglycol dimethyl ether to L-phenylala-nineleucinamide in 83% yield. Addition of 30% 2 1 ethanol/water reduced the time needed from 40 to 4 h. The enzyme could be used three more times. These reaction mixtures contained 0.13-0.75 g peptide per g reaction mixture compared with 0.015-0.035 when the reaction was... 

Production of the artificial low-calorie sweetener aspartame from Z-L-aspartate and D/L-phenylalanine methylester by peptide bond formation with immobilized thermolysin from Bacillus thermoproteolyticus (Tosoh Corp., Ajinomoto, Toyo-Soda, DSM, annual world production approx. 10000 tons). Aspartame is about 200 times as sweet as sucrose, and is used in drinks such as Coca Cola and Pepsi Cola Light. In contrast to the older chemical process, the enzymatic process can - due to the L-selectivity of the enzyme - use the cheaper D/L-phenylalanine methylester instead of the pure L-form. The enzymatic process (Fig. 15) yields a-aspartame exclusively, whereas the chemical route yields a mixture of a-aspartame and bitter-tasting (5-aspartame, thus requiring an additional separation step. 

Murakami Y, Yoshida T, Hayashi S et al. (2000) Continuous enzymatic production of peptide precursor in aqueous/organic biphasic medium. Biotechnol Bioeng 69 57-65 Nakanishi K, Takeuchi A, Matsuno R (1990) Long term continuous synthesis of aspartame precursor in a column reactor with an immobilized thermolysin. Appl Microbiol Biot 32 633-636 Nardin EH, Calvo-CaUe JM, Oliveira GA et al. (1998). Plasmodium falciparum polyoximes highly immunogenic synthetic vaccines constructed by chemoselective ligation of repeat B-ceU epitopes and a universal T-ceU epitope of CS protein. Vaccine 16 590-600 Nilsson B, SoeUner M, Raines R (2005) Chemical synthesis of proteins. Annu Rev Biophys Biomol Struct 34 91-118... 

The first enzyme-catalyzed reaction to be effectively performed in ionic liquids was described in 2000. The reaction of two amino acid derivatives was catalyzed by thermolysin in [bmim][PF6] (containing 5% water) to give aspartame 106 (Scheme 45). Although the rates were comparable to those observed in a mixture of ethyl acetate and water, the enzyme exhibited much higher stability in the ionic liquid and did not require immobilization. ... 

Using the reverse of the hydrolytic reaction, proteases are capable of catalyzing the formation of peptide bonds. Thermolysin is a powerful enzyme in catalyzing the synthesis of various useful oligopeptides, either in a free form or in an immobilized form in organic solvent media [58]. Recently, the systematic study about the stability of immobilized thermolysin has been reported [59]. In this study, the authors described the mechanism and kinetics for the inactivation of immobilized thermolysin with respect to the effects of organic solvents, kinds of support, water content, and so on. The strategy and results of their approach are discussed next. 

The immobilized thermolysin was prepared by adsorption of the enzyme onto the supports, polyacrylic ester resins (AmberUte series) and pore glass particles (CPG series), followed by crosslinking with glutaraldehyde in aqueous solutions. The activity of the immobilized enzyme was assayed by measuring the initial synthetic rate of fV-(benzyloxycarbonyl)-L-phenylalanyl-L-phynylalanine methyl ester in the biphasic reaction system. 

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