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

Abstract

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%.     

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-NH(2) of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH(2) 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-NH(2). The optimum conditions using alcalase as the catalyst are pH 10.0, 35 degrees C, in acetonitrile/Na(2)CO(3)-NaHCO(3) buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptide 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 degrees 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 degrees C, in ethanol/Tris-HCl buffer system (80:20, V/V), 4 h, with the dipeptide yield of 76.1%.     

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%.     

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 degrees C, in acetonitrile/Na2CO3-NaHCO3 buffer system (85:15, v/v), 6 h, with a dipeptide yield of 75.5%.     

Trypsin-catalyzed kinetically controlled synthesis of a precursor dipeptide of thymopentin in organic solvents, using a free amino acid as nucleophile

Trypsin-catalyzed, kinetically controlled synthesis of a precursor, dipeptide of thymopentin (TP-5), Bz-Arg-Lys-OH (or-OEt) in organic solvents was studied. Bz-Arg-OEt was used as the acyl donor and Lys-OH and Lys-OEt were used as the nucleophiles. Ethanol was selected as the organic solvent from ethanol, methanol, acetonitrile, and ethyl acetate tested under the experimental conditions. As expected, Lys-OEt is not a suitable nucleophile in trypsin-catalyzed reaction, due to its competition with the protective Arg-OEt as acyl donor for the active site of trypsin, while Lys-OH does not have this problem. The optimal reaction condition for the synthesis of Bz-Arg-Lys-OH was set up as 20% Tris-HCl buffer, pH 8.0, 35 degrees C for 6 h with the yield of 52.5%, or for 18-24 h with the yield of about 60%.     

在AOT/异辛烷反相胶束体系中酶法合成RGD前体二肽

近十年来,在有机相中利用酶法合成短肽技术取得了长足的发展．但对于在有机相中合成含有亲水氨基酸的短肽,仍然是一个难题．利用反相胶束可以解决亲水氨基酸在有机相中的低溶解性问题［１］．Ａｒｇ－Ｇｌｙ－Ａｓｐ（ＲＧＤ）是近年来发现的一种具有粘合细胞作用的三肽...     

Enzymatic acylation of polar dipeptides: Influence of reaction media and molecular environment of functional groups

The enzymatic acylation of polar dipeptides was investigated. First, the Novozym435^®-catalyzed acylation of Lys-Ser, HCl exhibiting three potential acylable sites was carried out in organic media (2-methyl-2-butanol, oleic acid) and in an ionic liquid ([Bmim]⁺[PF₆]^?). In these reactions, the chemo-selectivity of the acylation was exclusively in favour of the N?-lysine acylation and the efficiency (substrate conversion) was demonstrated to be under control of the peptide solubility. The use of [Bmim]⁺[PF₆]^? permitted to significantly improve the dipeptide solubility, and to enhance both substrates conversion and initial rates of acylation reaction. In the three reaction media used, the O-acylated derivative of the dipeptide was never detected suggesting a weak reactivity of the serine hydroxyl group due to its molecular environment and particularly to the presence of a free carboxylic group known for its electroattractor property.Last, the acylation of a natural dipeptide (carnosine), exhibiting a very low solubility in organic solvents, was also performed. Carnosine was successfully N-acylated in 2-methyl-2-butanol, and a yield of 39% was obtained when improving the substrate solubility: a better dispersibility was obtained by application of a high pressure on the reaction medium just before starting the reaction.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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

The tripeptide Bz-Arg-Gly-Asp(NH₂)OH was synthesized by a combination of chemical and enzymatic methods in this study. Firstly, Gly-Asp-(NH₂)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-(NH₂)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(NH₂)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%.     

Facile biocatalytic synthesis of a macrocyclic lactone in sub- and supercritical solvents

Abstract

High yield and selectivity in the intramolecular synthesis of the macrocyclic lactone oxacyclohexadecan-2-one were achieved via a straightforward biocatalytic synthesis utilising non-conventional solvents: supercritical carbon dioxide (scCO₂), ethane, fluoroform and 1,1,1,2-tetrafluoroethane. Batch and continuous syntheses were demonstrated with a higher yield attained in a continuous synthesis using in-situ product extraction by scCO₂. A remarkably high yield was obtained in 1,1,1,2-tetrafluoroethane at batch conditions.     

Thermostability of α-chymotrypsin in water/organic solvent systems

Summary The influence of pH, temperature, substrate concentration and organic solvents (dimethylformamide, dimethylsulfoxide) on the -chymotrypsin stability in a water/organic solvent system was studied. The enzyme activity was measured as the dipeptide, AcPheLeuNH₂ synthesis and the ester substrate hydrolysis. Enzyme stability was enhanced by lower pH and temperature values and higher substrate concentrations. Dimethylsulfoxide allowed an higher enzyme stability than dimethylformamide. -Chymotrypsin displayed an higher stability in the water medium when it was compared to the organic system.     

Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Abstract

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Y_max) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100?mM Tris/HCl buffer pH 7.0, 30?°C, 2.7% (v/v) DMSO, 20?mM MPOA, 0.3 UI/ml of SlPVA), Y_max was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Y_max and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.     

Improved production of (R)-2-octanol via asymmetric reduction of 2-octanone with Oenococcus oeni CECT4730 in a biphasic system

Abstract

Oenococcus oeni CECT4730, which catalyses the asymmetric reduction of 2-octanone to (R)-2-octanol with high enantioselectivity, was further studied to exploit its potential for production of (R)-2-octanol in an aqueous/organic solvent biphasic system. Variables such as the volume ratio of aqueous to organic phase (V_a/V_o), buffer pH, reaction temperature, shaking speed, co-substrates and the ratio of biocatalyst to substrate were examined with respect to the molar conversion, the initial reaction rate and the product enantiomeric excess (e.e.). Under the optimized conditions (V_a/V_o=1:1 (v/v), buffer pH=8.0, reaction temperature=30°C, shaking speed=150 rev/min, ratio of glucose to biomass=5.4:l (w/w), ratio of biocatalyst to substrate=0.51:l (g/mol)), the highest space time yield of (R)-2-octanol, 24 mmol L^?1 per h, and >98% product e.e. were obtained at a substrate concentration close to 1.0 mol L^?1 after 24 h reduction.     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.     

Urease-catalyzed urea synthesis.

The equilibrium of hydrolytic reactions can be shifted toward condensation by carrying out the reaction at low water concentration. The rate and yield of urease-catalyzed urea synthesis from (NH₄)₂CO₃ or NH₄HCO₃ has been examined as a function of water concentration (in mixtures with organic solvents), substrate and H⁺ concentration, and polarity of the nonaqueous component of the solvent. Similar effects of organic solvents are observed on the reaction rate in both directions; the results suggest that at least in some conditions the reaction proceeds through nonenzymically formed carbamate. The equilibrium concentration of urea, in 50% ($\text{v}\text{v}$) water, varies over 10-fold, depending on the nature of the nonaqueous component of the solvent; nonhydroxylic solvents such as acetone given the highest yield. Solubility measurements suggest that the interactions of the solvent mixtures with (NH₄)₂CO₃ (or carbamate), rather than urea, are responsible for the variations in urea yield. Activities of water and the ionic components of the equilibrium are strongly influenced by the nature of the nonaqueous component of the solvent, as well as its concentration.     

Immobilization of goat brain aminopeptidase B in calcium alginate beads

Aminopeptidase B, an arginyl aminopeptidase, was purified from goat brain with a purification factor of ～280 and a yield of 2.7%. It was entrapped in calcium alginate together with bovine serum albumin. The optimal conditions for immobilization for maximum activity yield were 1% CaCl₂ and 2.5% alginate. The immobilized enzyme retained ～62% of its initial activity and could be used for five successive batch reactions with retention of 30% of the initial activity. The pH and temperature optima of the free and immobilized enzyme were pH 7.4, 45°C and pH 7.8, 50°C respectively, while the pH and thermal stability as well as the stability of the enzyme in organic solvents were improved significantly after entrapment. The K_m value for the immobilized enzyme was about twofold higher than that of the soluble enzyme. Because of this increased stability, the immobilized enzyme may be useful in the meat processing industry.     

Purification and characterization of a mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30?KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mass of 133?KDa by SDS-PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The K_m and V_max of purified enzyme when using pNPL hydrolysis were 4.0?mM and 73.53?nmol/ml/min, respectively. The pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity. Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27?°C. The enzyme was stable within a pH range of 6.5 to 7 at 27?°C for 1?h. The enzyme activity was enhanced up to 36% by KCl, BaCl₂, MgCl₂ and CaCl₂ while obviously inhibited (10–20%) by CoCl₂, ZnCl₂, MnCl₂ and CuCl₂. No inhibitory effects were observed with 1.0 and 5.0?mM of 2-mercaptoethanol and EDTA. Similarly, SDS at 1.0?mM does not affect the enzyme activity while high reduction (80%) was observed at 5.0?mM SDS concentration. The enzyme was active against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in organic synthesis where such hydrophilic organic solvents are used as a reaction media.     

Stability and catalytic properties of chemically modified pig trypsin

Pig trypsin was chemically modified with the bifunctional compound ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) to yield EG-trypsin. EG-trypsin showed greater thermal stability (100% active beyond 100 min at 55°C; native only 53% active at 100 min) together with slightly increased tolerance toward some organic solvents. Arg/Lys hydrolysis ratio changed little. Esterase/amidase activity ratio of EG-trypsin in buffer was 11-fold greater than that of native pig trypsin, but 5-fold less in 30% v/v acetonitrile. In buffer, EG-trypsin synthesized the dipeptide benzoyl-Arg-Leu-NH₂ at a 3-fold higher rate than native trypsin, but native trypsin outperformed EG-trypsin in 30% v/v acetonitrile.     

Characterization of a thermostable and solvent-tolerant laccase produced by Streptomyces sp. LAO

Objectives

Decaying wood samples were collected, and actinomycetes were isolated and screened for laccase production. The identity of the efficient laccase-producing isolate was confirmed by using a molecular approach. Fermentation conditions for laccase production were optimized, and laccase biochemical properties were studied.

Results

Based on the 16S rRNA gene sequencing and phylogenetic analysis, the isolate coded as HWP₃ was identified as Streptomyces sp. LAO. The time-course study showed that the isolate optimally produced laccase at 84 h with 40.58?±?2.35 U/mL activity. The optimized physicochemical conditions consisted of pH 5.0, ferulic acid (0.04%; v/v), pine back (0.2 g/L), urea (1.0 g/L), and lactose (1 g/L). Streptomyces sp. LAO laccase was optimally active at pH and temperature of 8.0 and 90 °C, respectively, with remarkable pH and thermal stability. Furthermore, the enzyme had a sufficient tolerance for organic solvents after 16 h of preincubation, with laccase activity?>?70%. Additionally, the laccase maintained considerable residual activity after pretreatment with 100 mM of chemical agents, including sodium dodecyl sulphate (69.93?±?0.89%), ethylenediaminetetraacetic acid (93.1?±?7.85%), NaN₃ (96.28?±?3.34%) and urea (106.03?±?10.72%).

Conclusion

The laccase's pH and thermal stability; and robust catalytic efficiency in the presence of organic solvents suggest its industrial and biotechnological application potentials for the sustainable development of green chemistry.