Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

Abstract

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield.

Enzymatic strategies and biocatalysts for amide bond formation: tricks of the trade outside of the ribosome

“…Thus, several strategies for improving operational stability of proteases in non-aqueous or biphasic systems have been reported, including the use of a variety of immobilization processes such as covalent attachment on various surfaces to encapsulation in polymers. 61 In addition to structural stability, immobilization offers the added advantage of easy recovery and separation as well as economic viability. Protein engineering has also been routinely used to improve catalytic properties—for example, the introduction of a methyl group to the ε-2 N of the active His in chymotrypsin improves aminolysis over unwanted hydrolysis 62 .…”

Production of an alkaline protease using Bacillus pumilus D3 without inactivation by SDS, its characterization and purification

“…Effect of pH on enzyme production was determined by adjusting pH of basal medium from 5 to 12, using Na 2 HPO 4 -NaH 2 PO 4 buffer solution (pH [5][6][7][8] and NaHCO 3 -NaOH buffer solution (pH [9][10][11][12].…”

“…Alkaline proteases, physiologically and commercially important catalysts, are active in a neutral to alkaline pH range, holding 450% of the total enzyme market 9 . Moreover, alkaline proteases are used in dehairing, peptide synthesis in organic solvents, fortification of fruit juice, manufacturing of protein-rich therapeutic diets 10 , preparation of slow-release dosage forms of therapeutic agents 11 , bioprocessing of used X-ray films for silver recovery 12 .…”

Thermophilic biodegradation of BTEX by two Thermus Species

“…This view is supported by our finding vents. 19,34,35 "Each serum bottle contained I .O mL of base medium and 1.5 X lo7 cells precultured on either the nominal medium or the indicated carbon source and received 65 KL (583,000 cpm) of stock [ring-14C]toluene solution to provide 15 mg of toluene per liter of medium (aqueous concentrations of 0.68 and 1.24 mgiL at 70 and 60°C, respectively). Subsequent incubations were the same as in Table IV. bCalculated based on the total cpm of [ring-"CC]toluene that was added and the amounts that were biodegraded as measured independently by gas chromatography.…”