Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB⁺-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P′₁ position and it has a wide specificity for the P₁ position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH₂). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P′₁ position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P₁ position. A synthesis was demonstrated with αAbu, Nle, Nva and Phg.     

Inhibitors of aminoacyl-tRNA synthetases as novel anti-infectives

Resistance to existing antibiotics has emerged as a major problem in healthcare. Novel antibiotics for which bacteria have not yet acquired resistance need to be developed to combat drug-resistant pathogens. Aminoacyl-tRNA synthetases are leading targets for novel anti-infectives. The validation of aminoacyl-tRNA synthetases as drug targets for anti-infectives has been established in an animal system. Using several conceptually distinct approaches, new inhibitors of synthetases have been developed as drug prototypes.     

A Novel Method for the Synthesis of Kyotorphin,Tyr-Arg,and 3H-Tyr-Arg,Catalyzed by Tyrosyl-tRNA Synthetase from Bacillus stearothermophilus

A novel method of dipeptide synthesis is described that can be carried out in aqueous solution and does not require complicated protecting and deprotecting procedures. An analgesic neuropeptide named kyotorphin, H-Tyr-Arg-OH, was synthesized from unprotected tyrosine and arginine in a new enzymatic reaction catalyzed by immobilized tyrosyl-tRNA synthetase from Bacillus stearothermophilus. The reaction could be a useful tool in the syntheses of radioisotope-labeled oligopeptides to be used in receptor binding assays. ³H-Kyotorphin was prepared by this method at a yield of 72% and could be used in receptor binding assays after a single chromatographic separation.     

嗜热脂肪芽胞杆菌生产1，6—二磷酸果糖的研究

研究了嗜热脂肪芽胞杆菌发酵生产1，6-二磷酸果糖（FDP）的工艺条件和工艺特点，采用活化培养工艺，改变菌体细胞透性和生理状态，获得高产率的FDP发酵液（产量为60mg/ml)。本法生产FDP具有副产物少，转化率高，发酵稳定的优点。     

固定化嗜热脂肪芽孢杆菌生产1,6-二磷酸果糖的研究

采用卡拉胶包埋嗜热脂肪芽孢杆菌，制成块状固定化细胞，对固定化细胞包埋方法及其用于生产1，6－二磷酸果糖（FDP）的工艺条件作了探索，在固定化细胞包埋前，包埋后及用于生产FDP 10批次后采用活化培养工艺，均可提高固定化细胞生产FDP的能力，转化培养液中的FDP产量达到40－65mg/ml，固定化细胞用于生产FDP的批次可达到20次左右。     

Synthesis of tetrapeptide p-nitroanilides catalyzed by pepsin

Swine pepsin at pH 5 efficiently catalyzes a condensation between Z-Ala-Ala-Phe-OH and p-nitroanilides of Leu, Phe, Val, Ala and Arg that leads to formation of corresponding benzyloxycarbonyl-tetrapeptide p-nitroanilides with yields of 70–90%. These reactions are complicated by co-precipitation of pepsin and the reaction products that necessitates the use of a relatively high concentration of pepsin.     

Enzymatically active subunits of Bacillus stearothermophilus enolase bound to Sepharose

The octameric enolase from Bacillus stearothermophilus was immobilized onto Sepharose 4B activated by the cyanogen bromide reaction under conditions for achieving essentially a single-point attachment. The immobilized enzyme was dissociated with guanidine hydrochloride to yield bound monomeric enolase. The Sepharose-bound subunit regained activity upon removal of the denaturant. It was also possible to rehybridize immobilized monomers to native octamers. Of note, the thermal stability of the immobilized enolase subunit does not appreciably differ from that of the parent soluble octameric enzyme. Thus, these results indicate that single subunits of thermophilic enolase are active and that oligomerization is not a prerequisite for the enzymic activity as well as for thermal stability.     

Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase

We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH₂Tyr¹,D-Ala²,Ala¹⁵]-GRF(1-29)-NH₂ ( 4 ), from the precursor, [Ala^15,29]-GRF(4-29)-OH ( 1 ). C-Terminal amidation of 1 to form [Ala¹⁵]-GRF(4-29)-NH₂ ( 2 ) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala²⁹-OH for Arg-NH₂. The target analog 4 was then obtained by acylation of segment 2 with desNH₂Tyr-D-Ala-Asp(OH)-OR ( 3 ) (R = CH₃CH₂ or 4-NO₂C₆H 4 CH₂) catalyzed by the V8 protease. In this paper we report on the use of the recently isolated Glu/Asp-specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3 . GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 °C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp³-Ala⁴ and Asp²⁵-Ile²⁶. However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R. A systematic study of the kinetics of the GSE-catalyzed segment condensations of 2 and a series of tripeptide esters, desNH₂Tyr-D-Ala-Asp(OH)-OR ( 3 ) [R = CH₃CH_2- ( 3a ), CH_3- ( 3b ), ClCH₂CH_2- ( 3c ), C₆H₅CH_2- ( 3d ), 4-No₂C₆H₄CH_2- ( 3e )] revealed that the rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4 , increase with increasing specificity (V_max/K_m) of GSE for the tripeptide ester. The specificity varies in the order 3e>3d>3c>3b>3a and appears to depend on an increase in the maximum turnover rate (V_max) with increasing basicity of R. This work demonstrates the coupling of a small peptide segment containing unnatural amino acids to the N-terminus of an intermediate-length polypeptide, without side-chain protection, by GSE, a potentially less costly and more stable alternative to the V8 protease.     

Peptide synthesis catalyzed by papain at alkaline pH values

The synthesis of peptides in the presence of papain at pH 8–9.5 is described. Starting substances are acylamino acid alkyl esters (the carboxyl component) and amides or tert.-butylesters of amino acids, as well as peptide (the amino component). Under such conditions secondary hydrolysis is not essential, making the synthesis of peptides soluble in aqueous medium. The yield of peptides is 50–94%. The effect of different factors (temperature, solvents, reagent concentrations) on the result of the reaction has been studied. It has been found that an excess of the carboxyl component is expedient to increase the yield of peptides.     

In vivo and in vitro effects of methylmercury on the activities of aminoacyl-tRNA synthetases in rat brain

The activities of six aminoacyl-tRNA synthetase species were determined using enzyme preparations partially purified from the brains of control and methylmercury (MeHg)-treated rats. The activities of Asp-, Leu- and Tyr-tRNA synthetases were significantly reduced in the brains of MeHg-intoxicated rats, whereas those of Lysand Met-tRNA synthetases remained unchanged. In contrast, the activity of His-tRNA synthetase was significantly increased in the symptomatic phase of MeHg intoxication. The activities of these six aminoacyl-tRNA synthetases in the control brains were affected to different extents on the direct addition of MeHg to the assay system in vitro. No positive correlation was observed between the in vivo and in vitro effects of MeHg on the enzyme activities. These results indicate that the aminoacylation of tRNA is one of the actions of MeHg, which leads to inhibition of protein synthesis, and it is suggested that the syntheses of cellular proteins may be modified in different ways by MeHg, depending on their amino acid compositions.This work was supported in part by a grant from the Japanese Environmental Agency.     

Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan.

Microorganisms are very powerful tools for the supply of information about the toxic effects of lipophilic compounds, since an impairment of cell growth usually occurs as a result of perturbations related, in most cases, with the partition of toxicants in membranes. The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify - and β-endosulfan interactions with the membrane possibly related with the insecticide toxicity. Two approaches have been pursued: (a) bacterial growth is followed and the effects of endosulfan isomers determined; (b) biophysical studies with the fluorescent fluidity probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to assess the effects of - and β-endosulfan on the organization of the membrane lipid bilayer. The effects on growth were quantitatively evaluated by determination of growth parameters, namely the lag phase, the specific growth rate and the cell density reached by cultures in the stationary phase. Growth inhibition by and β-endosulfan dependent on the concentration is diminished or removed by the addition of 2.5 m Ca²⁺ to bacterial cultures. Fluorescence DPH polarization consistently showed opposite effects of Ca²⁺ and - and β-endosulfan on the physical state of bacterial polar lipid dispersions.     

Chymotrypsin-catalyzed peptide synthesis

The kinetics of peptide-bond formation catalyzed by δ-chymotrypsin has been studied for a number of peptide products of different length using fixed concentrations of the acyl component (Ac-Phe-OMe. Ac-Ala-Ala-Phe-OMe, or Ac-Ala-Ala-Tyr-OMe) and varying concentration of the amino component (H-Ala-NH₂ or H-Ala-Ala-NH₂). The time course of the reactions was followed by monitoring ester consumption and peptide product formation by analytical HPLC. On the basis of a plausible four-centre mechanistic model, the theoretical time course of these reactions was calculated using rate and equilibrium constants determined by separate kinetic experiments. The excellent agreement observed between the theoretical and the experimental time courses supports the proposed mechanism and provides evidence for the validity of the present kinetic approach. By focusing attention on the rate constants which are critical for efficient synthesis, this mechanistic information constitutes a valuable basis for the use of the enzymatic peptide synthesis in preparative applications.     

Comparative study of the effect of ochratoxin a analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells

Ochratoxin A (OTA), a naturally occurring mycotoxin of Aspergillus and Penicillium species, consists of a 5 ' chlorinated dihydromethyl isocoumarin linked to l,ß-phenylalanine by an α-amide bond. 8 analogues of OTA were prepared in which the phenylalanine was always substituted by another amino acid. The effects of these analogues on yeast tRNA amino acylation reaction and on growth and protein synthesis of hepatoma culture cells were compared with those of OTA. In addition, Ochratoxin B (OTB) and ochratoxin α (OTα) were examined. All the analogues of OTA had inhibitory effects in the 3 test systems, although to a lesser degree than OTA. The degree of inhibition depended on the kind of substituted amino acid, the tyrosine, valine, serine and alanine analogues being most effective, in contrast to the proline analogue. OTB and OTα were ineffective.     

Pepsin as a catalyst of peptide synthesis Enzyme co-precipitation with emerging peptide products

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH₃ formation, an equilibrium yield was attained at 1:3. 10⁵ enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.     

Enzymatic approach to the synthesis of taurine-containing peptides

Subtilisins (subtilopeptidase A, nagarse) and proteinase K were able to catalyze the synthesis of taurine-containing peptides from various N-acylated amino acid or peptide esters and nonprotected taurine. The synthesis was optimized using a model reaction between Boc-Tyr-OMe and taurine. The best results were obtained under strongly alkaline conditions in acetonitrile with low water content as the reaction medium. The choice of the base added to the reaction medium had a substantial effect on the product yield. A preparative synthesis of Tyr-Tau and Ala-Phe-Tau is described.     

Enzymatic synthesis of dipeptide units of the d-d-configuration in aqueous media

The enzymatic synthesis of dipeptide units of the d -d -configuration in aqueous media, catalysed by muramoyl-pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac-l -Lys(Ac)-d -Ala-d -Lac-OH and Ac-d -Ala-OMe were used as acyl-components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the d -enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%     

Side reaction in peptide synthesis

2-Oxazolidone derivatives formed through an intramolecular reaction in the process of alkaline treatment of urethane-type N-protected peptides of which the N-terminal residues were Ser or Thr having unprotected hydroxyl groups. In order to avoid this side reaction, the esters of these peptides could be cleaved by enzymatic hydrolyses instead of saponification.     

Solid-phase synthesis of bombesin by continuous flow procedure using Fmoc-amino acids*

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.     

Peptide N-alkylamides by solid phase synthesis*

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH₂ CH₃, NH₂ CH₂ CH₃, NH₂ CH₂ CF₃) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [D Glu⁶, Pro⁹-NHCH₂ CH₃]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [D Glu⁶]-GnRH. Other analogs including [D Trp⁶, Pro⁹-NHCH₂ CH₃]-GnRH, [D Ala⁶, Pro⁹-NHCH₂ CF₃]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

豆粕的不同酶解液发酵苏云金芽孢杆菌对Zwittermicin A产量的影响

目的 研究培养基中豆粕所含蛋白质的不同存在形式对zwittermicin A产量的影响.方法 豆粕经过3种不同的复合蛋白酶(碱/中蛋白酶、中性蛋白酶、酸/中蛋白酶)处理后形成了蛋白质、寡肽和氨基酸含量均不同的3种酶解液,即DPI,DP2和DP3.以苏云金芽孢杆菌库斯塔克亚种(Bacillus thuringiensis sp.kurstaki D1-23)为菌种,分别将这3种豆粕酶解液作为其氮源进行发酵.结果 从含有DP1的培养基D-1 中获得的苏云金芽孢杆菌生物量最低;含有DP2的培养基D-2中的zwittermicin A含量最高;从含有DP3的培养基D-3中得到的生物量最大.结论 氮源的存在形式不同,对Bacillusthuringiensis sp.turstaki D1-23 菌体繁殖和其次级代谢产物zwittermicin A的合成有不同影响.