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1.
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%.  相似文献   

2.
Efficient development of enzymatic synthesis in two-phase systems is closely related with appropriate selection of the reaction medium (especially the solvent and phase ratio). A selection procedure based on the calculation of the theoretically allowable conversion and product concentration is presented and applied to a peptide synthesis using papain. For the synthesis of the dipeptide Boc-Gly-Phe-OMe, the operating conditions have been determined, and the two-phase system to be used has been successfully selected (with trichloroethylene being the best solvent).  相似文献   

3.
在AOT/异辛烷反相胶束体系中酶法合成RGD前体二肽   总被引:1,自引:0,他引:1  
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...  相似文献   

4.
The enzymatic synthesis of the seven consecutive dipeptide fragments of the cholecysto kinin C-terminal octapeptide (CCK-8) in organic media is reported. The influence of the reaction medium composition, the protease, and the structure of N-α and C-α protecting groups of both carboxyl and amino components was evaluated. α-Chymotrypsin, papain and thermolysin adsorbed on Celite were used as catalysts, under thermodynamic and kinetic control. The carboxamidomethyl, methyl and allyl ester derivatives of acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl and fluoren-9-ylmethoxycarbonyl amino acids, were assayed as carboxy components. Amino acid amide and ester derivatives were employed as nucleophiles with a preference for the latter, since the dipeptide product obtained could be used directly, without any further chemical modification, as acyl-donor in subsequent coupling steps. All dipeptides selected were successfully synthesized, using the optimal combination of protecting groups, reaction media and enzyme different for each coupling reaction. The information gained with this study should be instrumental in designing an optimal strategy for the total enzymatic synthesis of cholecystokinin C-terminal octapeptide (CCK-8).  相似文献   

5.
纤维素固相化木瓜蛋白酶   总被引:1,自引:0,他引:1  
 本文用叠氮法制备了纤维素固相化木瓜蛋白酶(简称CMCP)。与相应酶液水解酪蛋白的反应相比,它表现出较低的酶活性,较高的最适pH值和较高的稳定性。CMCP的比活回收率约为24%,最适pH值向碱性范围移动约为0.5个单位。CMCP经60℃热处理,持续3h活性无明显下降,在4℃下保存127天,活性只下降了40%左右。对这些参数,本文都根据CMCP的结构特点进行了分析。 CMCP柱还表现出明显的对啤酒的防浊能力。过柱的啤酒,氨基酸的含量大大增加。  相似文献   

6.
Abstract

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.  相似文献   

7.
以自制的壳聚糖作为载体,用戊二醛作交联剂,优化了固定化条件,研制成壳聚糖固定化木瓜蛋白酶。其活性回收率达到42—53%,操作半衰期达到一个月以上,对热、乙醇以及尿素的稳定性有很大的提高,Km值为0.67×10~2mg/mL,最适温度65—70℃,最适pH8.0,能使啤酒中的蛋白质浓度从56.5mg/L减少到2.7mg/L,可以消除啤酒的低温混浊现象。  相似文献   

8.
金属螯合载体定向固定化木瓜蛋白酶的研究   总被引:11,自引:1,他引:10  
以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体(IDACu2+)与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu2+1.5×10-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4%,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。  相似文献   

9.
在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0~7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。  相似文献   

10.
海藻糖酶法合成途径及其酶基因的重组表达研究   总被引:1,自引:0,他引:1  
在生物抗逆研究中,海藻糖合酶基因是继甘露醇、脯氨酸、甜菜碱合成酶基因之后又一个与抗逆相关的基因。海藻糖具有独特的生物学功能,能提高生物体对干旱、高温、冷冻和渗透压的抗性,发现以来就受到人们的普遍关注。随着对海藻糖化学性质、生理功能、作用机理及代谢途径等方面研究的深入,其在生物制品、食品、医药、作物育种及精细化工等领域广阔的应用前景日益显现。就海藻糖在生物体中的合成途径,以及海藻糖合成酶的基因工程研究进展进行了综述。  相似文献   

11.
Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.  相似文献   

12.
本文从酶、酶的作用底物和合成体系三个方面综述了酶法合成天冬甜精中的几个问题。  相似文献   

13.
Lipases and proteases from various sources were tested in aromatic polyester synthesis in organic solvents. A commercial protease from Bacillus licheniformis efficiently catalyzed the transesterifica-tion of a diester of terephthalic acid and 1,4-butanediol in anhydrous tetrahydrofuran (THF). This protease was used as a catalyst in the synthesis of aromatic polyesters in THF. Oligomers with average molecular weights from 400 to 1000 daltons were obtained using various diols and aromatic diesters.  相似文献   

14.
尼龙固定化木瓜蛋白酶及其应用研究   总被引:1,自引:0,他引:1  
 尼龙经CaCl_2和H_2O的甲醇溶液处理,稀HCl水解用戊二醛交联以制备固定化木瓜蛋白酶。在溶液酶浓度为1mg/mL pH7.5—8.0、4—15℃条件下固定3h,活力回收42.5%,相对活力46%,偶联效率52%,半衰期72天。溶液酶Km值和固定化酶K_m~(aPP)值(底物酪蛋白W/V,%)分别为0.28%和0.35%。溶液酶和固定化酶分别在pH6.5和pH8.0以下活力稳定;最适pH分别为7.0和8.0;在65℃处理30min活力分别为原有活力的89%和66%。当酪蛋白浓度为1.5%和2.5%以上活力分别受到抑制。固定化酶在6mol/L脲中连续浸洗5次共6h其活力稳定,仍有原活力的44.4%;用以处理啤酒浊度比对照下降了2-11倍;蛋白质含量下降了55%;冷藏(4℃)120天,无冷混浊发生;同时各项理化指标和风味不变。  相似文献   

15.
氨基化二氧化硅颗粒固定木瓜蛋白酶研究   总被引:9,自引:2,他引:9  
采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3~0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。  相似文献   

16.
17.
Enzymatic synthesis of l-ascorbyl linoleate in organic media   总被引:1,自引:0,他引:1  
A novel l-ascorbyl fatty acid ester, l-ascorbyl linoleate was successfully prepared by enzymatic esterification and transesterification in a non-aqueous medium using immobilized lipase as biocatalyst. Changes in enzymatic activity and product yield were studied for the following variable: the nature of the fatty acid, the fatty acid concentration and water content. The yield of synthesis for the C18 unsaturated fatty acids were higher than for the C18 saturated fatty acid. Initial enzyme concentration does not affect the equilibrium of the reaction. And the product yield (33.5%) in the transesterification was higher than that of the esterification (21.8%) at a high-substrate concentration 0.3 M. The medium water content was found to have a distinct influence on the l-ascorbyl linoleate synthesis.These authors contributed equally to the article.  相似文献   

18.
The aim of this work was to study different immobilization strategies on silica supports in order to obtain robust biocatalysts from latex proteases of Asclepias curassavica L., a South American native plant. Immobilized enzyme performance was evaluated under harsh reaction conditions such as the synthesis of the antihypertensive peptide N-α-CBZ-Val-Gly-OH.Proteases from A. curassavica, named asclepain, were immobilized (0.51–5.56 mg of protein/ g of support) in non-functionalized silica (S), in glyoxyl-silica (GS) and in octyl-glyoxyl-silica (OGS), by adsorption, and multi-point covalent attachment on mono and hetero-functional supports, respectively, under previously determined optimal immobilization conditions. Immobilization yields were expressed as activity yield (Ya) and protein yield (Yp).Asclepain-OGS showed the highest Ya (178 ± 1.62 %) meaning an expressed activity 1.8 times higher than the offered activity, while Yp was 75 ± 0.4 %. Ya for asclepain-S and -GS were 64 ± 1.45 % and 16 ± 0.37 %, respectively. Best results were attributed to the ability of OGS support to guide the enzyme before covalent attachment, increasing its reactivity. Asclepain-OGS led to product yield of 95.5 ± 0.14 %, five times higher than soluble asclepain in the synthesis of N-α-CBZ-Val-Gly-OH, after 3 h in 30 % methanol in 0.1 M Tris-HCl buffer pH 8.  相似文献   

19.
We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.  相似文献   

20.
The role of acyl donor structure on the course of peptide bond formation catalyzed by SDS-subtilisin in ethanol was investigated. In the reaction Z---Ala---Ala---Leu---OR+H---Phe---pNA→Z---Ala---Ala---Leu---Phe---pNA, nearly quantitative product yields were observed after 2 h, regardless of whether an activated (R=CH3, p-C6H5Cl) or non-activated (R=H) acyl donor was used. It was found that the enzyme can accept as acyl donors N-protected tri-peptides containing basic or acidic amino acid residues in the P1-position. Tetra-peptides of general formula Z---Ala---Ala---P1---P1′---pNA, where P1=Glu, Asp, Lys, Arg or His and P1′=Phe, Arg or Glu have been obtained in good yield.  相似文献   

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