Industrial Alkaline Protease Research Articles

SYNTHESIS OF A PRECURSOR TRIPEPTIDE Z-Asp-Val-Tyr-OH OF THYMOPENTIN BY CHEMO-ENZYMATIC METHOD

The precursor tripeptide of thymopentin was synthesized by a combination of chemical and enzymatic methods. First, Val-Tyr-OH dipeptide was synthesized by a novel chemical method in two steps involving preparation of NCA-Val. Second, the linkage of the third amino acid Z-Asp-OMe to Val-Tyr-OH was completed by an enzymatic method under kinetic control. An industrial alkaline protease alcalase was used in water–organic cosolvent systems. The synthesis reaction conditions were optimized by examining the effects of several factors including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-Tyr-OH. The optimum condition is of pH 10.0, 35°C, acetonitrile/Na2CO3-NaHCO3 buffer system (85:15, v/v), and reaction time of 2.5 hr, which achieves tripeptide yield of more than 70%.

Sucrose monolaurate synthesis with Protex 6L immobilized on electrospun TiO2 nanofiber

TiO(2) nanofibers with uniform diameter about 125 nm were prepared based on sol-gel process and electrospinning technology. Protex 6L, an industrial alkaline protease, was covalently immobilized on TiO(2) nanofiber through γ-aminopropyltriethoxysilane modification and glutaraldehyde crosslinking. With 2 (v/v)% glutaraldehyde as crosslinker, the enzyme loading is about 201 mg (g nanofiber membrane)(-1), and the specific activity of the immobilized Protex 6L is 2.45 μmol h(-1) ml(-1) mg(-1) protein for synthesis of sucrose monolaurate from sucrose and vinyl laurate. The optimal condition for sucrose monolaurate production is 5% (v/v) water content in DMSO/2-methyl-2-butanol solvent mixture and 50 °C. Under this condition, 97% conversion was achieved within 36 h by nanofibrous Protex 6L, which is corresponding to a productivity 34 times higher than that of most widely used Novozym 435. After 10 cycles reuse, nanofibrous Protex 6L retained 52.4% of its original activity.

Alcalase®‐catalysed synthesis of the precursor tetrapeptide N‐benzoylarginylglycylaspartylserinamide (Bz‐RGDS‐NH2) of the cell‐adhesion peptide arginylglycylaspartylserine (RGDS)

In the present study, a precursor tetrapeptide Bz-RGDS-NH2 (N-benzoylarginylglycylaspartylserinamide) of cell-adhesion peptide RGDS (arginylglycylaspartylserine) was synthesized by a novel route. First of all, the precursor tripeptide GDS-NH2 (glycylaspartylserinamide) was synthesized by a chemical method only using aspartic acid and serine at gram scale in four steps. The linkage of the fourth amino acid Bz-Arg-OEt (N-benzoyl-L-arginine ethyl ester) to GDS-NH2 was completed by an enzymatic method under kinetic control in water-miscible organic media. An industrial alkaline protease, Alcalase, with a wide substrate specificity, was used as the catalyst. The effects of organic solvents, pH value, reaction temperature, water content and molar ratio of substrates on the yield of Bz-RGDS-NH2 synthesis were examined. The optimum reaction conditions were found to be pH 10.0, 35 degrees C, 8 h, in an acetonitrile/(Na2CO3/NaHCO3) buffer system (93:7, v/v) with a maximal yield of 65.2%. We found that secondary hydrolysis of the peptide product did not take place in these water-miscible organic solvents.

Protease-Catalyzed Synthesis of a Precursor Dipeptide, Z-Asp-Val-NH2 of Thymopentin, in Organic Solvents

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide, Z-Asp-Val-NH2 of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH2 were used as the acyl donor and the nucleophile, respectively. An industrial alkaline protease alcalase was used to catalyze the synthesis of the target dipeptide in water-organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-NH2. The optimum conditions using alcalase as the catalyst are pH 10.0, 35°C, in acetonitrile/Na2CO3-NaHCO3 buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptde product was confirmed by LC- MS.

Synthesis of a Precursor Dipeptide of Thymopentin in Organic Solvents by an Enzymatic Method

The protease‐catalyzed, kinetically controlled synthesis of a precursor dipeptide of thymopentin(TP‐5), Z‐Arg‐Lys‐NH2 in organic solvents was studied. Z‐Arg‐OMe was used as the acyl donor and Lys‐NH2 was used as the nucleophile. An industrial alkaline protease alcalase and trypsin were used to catalyze the synthesis of the target dipeptide in water‐organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z‐Arg‐Lys‐NH2. The optimum conditions using alcalase as the catalyst are pH 10.0, 35°C, in acetonitrile/DMF/Na2CO3‐NaHCO3 buffer system (80∶10∶10, V/V), 6 h, with the dipeptide yield of 71.1%. Compared with alcalase, the optimum conditions for trypsin are pH 8.0, 35°C, in ethanol/Tris‐HCl buffer system (80∶20, V/V), 4 h, with the dipeptide yield of 76.1%.

Synthesis of a precursor dipeptide of RGDS (Arg‐Gly‐Asp‐Ser) catalysed by the industrial protease alcalase

Synthesis of Bz-Arg-Gly-NH(2) (N-benzoylargininylglycinamide) [a precursor dipeptide of RGDS (Arg-Gly-Asp-Ser)] catalysed by protease in water/organic co-solvent systems was studied. Starting substrates were N-benzoyl-L-arginine ethyl ester hydrochloride (acyl donor) and glycinamide (nucleophile). Acetonitrile was selected as the organic solvent. Alcalase, an industrial alkaline protease, was applied to the synthesis of the target dipeptide. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including water content, temperature, pH, molar ratio of the substrates and reaction time, on the yield of Bz-Arg-Gly-NH(2). The optimum conditions were established to be pH 10.0, 45 degrees C, in acetonitrile/0.1 M Na(2)CO(3)/NaHCO(3) buffer system (90:10, v/v) for 1 h with a dipeptide yield of 82.9%.

Alcalase‐Catalyzed, Kinetically Controlled Synthesis of a Precursor Dipeptide of RGDS in Organic Solvents

The protease‐catalyzed, kinetically controlled synthesis of a precursor dipeptide of RGDS, Z‐Asp‐Ser‐NH2 in organic solvents was studied. Alcalase, an industrial alkaline protease, was used to catalyze the synthesis of the target dipeptide in water‐organic cosolvents systems with Z‐Asp‐OMe as the acyl donor and Ser‐NH2 as the nucleophile. Acetonitrile was selected as the organic solvent from acetonitrile, ethanol, methanol, DMF, DMSO, ethyl acetate, 2‐methyl‐2‐propanol, and chloroform tested under the experimental conditions. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including water content, temperature, pH, and reaction time on the Z‐Asp‐Ser‐NH2 yields. The optimum conditions are pH 10.0, 35°C, in acetonitrile/Na2CO3‐NaHCO3 buffer system (85:15, v/v), 6 h, with a dipeptide yield of 75.5%.

Synthesis of tripeptide RGD amide by a combination of chemical and enzymatic methods

The tripeptide Bz-Arg-Gly-Asp( NH 2) OH was synthesized by a combination of chemical and enzymatic methods in this study. Firstly, Gly-Asp-(NH 2) 2 was synthesized by a novel chemical method in three steps including chloroacetylation of l-aspartic acid, esterification of chloroacetyl l-aspartic acid and ammonolysis of chloroacetyl l-aspartic acid diethyl ester. Secondly, the linkage of the third amino acid (Bz-Arg-OEt) to Gly-Asp-(NH 2) 2 was completed by enzymatic method under kinetic control condition. An industrial alkaline protease alcalase was used in water–organic cosolvents systems. The synthesis reaction conditions were optimized by examining the effects of several factors including water content, temperature, pH and reaction time on the yield of the synthesis product Bz-Arg-Gly-Asp( NH 2) OH. The optimum conditions are pH 8.0, 35 °C, in ethanol/Tris–HCl buffer system (85:15, v/v), 8 h with the tripeptide yield of 73.6%.

Proline as nucleophile in kinetically controlled peptide synthesis catalyze by alcalase in 2-methyl-2-propanol.

Procedures have been developed to synthesize proline-containing peptides in good yields and high purities via a kinetically controlled approach using an industrial alkaline protease, alcalase, as a catalyst in anhydrous 2-methyl-2-propanol. The yield of the reaction was dependent on the structure of the acyl-donor and the water content in the solvent. Using tripeptide as an acyl donor, the yield was higher than that using dipeptide, and when amino acid derivatives were used the yield was the lowest. The yield was also dependent on the concentration of water present in the solvent. The higher the water concentration in the solvent, the lower was the yield in the reaction. Both L-proline or D-proline derivatives could be used as nucleophiles in the reaction.

Facile optical resolution of amino acid esters via hydrolysis by an industrial enzyme in organic solvents

AbstractRacemic amino acid esters were optically resolved via hydrolysis in organic solvents by the catalysis of an industrial alkaline protease, “Alcalase”. The products which were composed mainly of L‐amino acids were insoluble and easily separated by filtration. The activity of the enzyme and enantiomeric excess of the products were significantly dependent on the nature of solvent and the water content in the reaction media. Generally, high values of enantiomeric excess were obtained at low water contents. Many natural and unnatural amino acids were resolved by this method.

Kinetic resolution of esters of amino acids in t‐butanol containing 5% water catalyzed by a stable industrial alkaline protease

AbstractWe developed a procedure for the resolution of esters of amino acids in 95% t‐butanol, followed by saponification of the unreacted esters to afford both enantiomers with high yield and optical purity. The hydrolysis, catalyzed by alkaline protease, was conducted in a mixture of t‐butanol (95%) and water (5%) at 25°C, with a pH controlled at pH 8.5 by the addition of NaOH (2 M). The hydrolyzed L‐amino acid, which was insoluble under these conditions, precipitated during the course of hydrolysis. After separation of the precipitate, the pH of the filtrate was adjusted to 11.5 to saponify the unreacted ester. The D‐antipode precipitated at pH 6.2–6.5. Both optically pure antipodes were obtained with high enantiomeric excesses and yields by simple filtration. © 1994 Wiley‐Liss, Inc.

Alcalase catalyzed peptide bond formation between Asp and D-Ala in anhydrous t-butanol

Procedures for preparing precusors of the artificial sweetner, Moz-Asp(Bzl)- D-Ala-R, R=NH2, n-Propyl, isopropyl, in anhydrous t-butanol catalyzed by an industrial alkaline protease “alcalase” using a kinetic-controlled approach have been developed.

Probing the S-1′ subsite selectivity of an industrial alkaline protease in anhydrous t-butanol

Study of the selectivity of s-1′-subsite of alcalase catalyzed reaction in t-butanol shows that the rate of product formation depends on the size of the nucleophiles. D- and L-amino acids as nucleophile form the product with the same reaction rate. Interpretation of this selectivity by molecular modeling using crystal structure of subtilisin Carlsberg complexed with it's substrate gives clear evidence.

Facile Amide Bond Formation From Esters of Amino Acids and Peptides Catalyzed by Alkaline Protease in Anhydroustert-Butyl Alcohol Using Ammonium Chloride/Triethylamine as a Source of Nucleophilic Ammonia

An industrial alkaline protease «Alcalase», stable and active in tert-butyl alcohol, was used to catalyze the synthesis of N-protected amino acids or peptide amides in anhydrous tert-butyl alcohol using ammonium chloride/triethylamine as source of nucleophilic ammonia

Peptide Synthesis in Organic Solvents Catalyzed by an Industrial Alkaline Protease “Alcalase”

AbstractAn industrial alkaline protease “Alcalase” is stable (half life > 5 days in ethanol or in t‐butanol) and active in alcoholic solvents (except methanol), and usable as a catalyst for kinetically controlled peptide bond formation in these solvents. We give an overview of our use of alcalase as catalyst in peptide synthesis; it includes 1) the stability of alcalase in various organic solvents; 2) the resolution of N‐protected amino acids at large concentration of organic solvent; 3) the saponification of Fmoc‐peptide esters in high concentration of organic solvent; 4) kinetically controlled peptide bond formation in alcoholic solvent, and 5) enzymatic synthesis of a Cathepsin inhibitor, peptidyl Q‐acyl hydroxamates.

Kinetic resolution of N-protected amino acid esters in organic solvents catalyzed by a stable industrial alkaline protease

An industrial alkaline protease “Alcalase” has been found to be very stable in organic solvents and usable as a catalyst for resolution of N-protected amino acids, in both aqueous solution and organic solvent with high yield and optical purity. Only the L-amino acid ester has been hydrolysed.

The reaction of trans-cinnamoyl-papain with a series of polyglycinamides of varying chain length

Procedures have been developed to synthesize proline-containing peptides in good yields and high purities via a kinetically controlled approach using an industrial alkaline protease, alcalase, as a catalyst in anhydrous 2-methyl-2-propanol. The yield of the reaction was dependent on the structure of the acyl-donor and the water content in the solvent. Using tripeptide as an acyl donor, the yield was higher than that using dipeptide, and when amino acid derivatives were used the yield was the lowest. The yield was also dependent on the concentration of water present in the solvent. The higher the water concentration in the solvent, the lower was the yield in the reaction. Both L-proline or D-proline derivatives could be used as nucleophiles in the reaction.