以L-天冬氨酸为原料制备D-天冬氨酸的新方法

以L-天冬氨酸为原料经过酯化、消旋、拆分和水解制备D-天冬氨酸。使L-2,3-二苯甲酰酒石酸(L-DBTA)与DL-天冬氨酸-β-甲酯在水溶液中于65~70℃反应形成非对映体盐,冷却到室温,D-天冬氨酸.L-DBTA盐析出,过滤后再经水解得到D-天冬氨酸,收率78.2%,旋光纯度达到99%以上。     

以DL-丝氨酸为原料制备D-丝氨酸的新方法

以DL-丝氨酸为原料经过酯化、拆分和水解制备D-丝氨酸。使L-2,3-二苯甲酰酒石酸(L-DBTA)与DL-丝氨酸甲酯在无水乙醇中于60℃反应形成非对映体盐,冷却到0℃,D-丝氨酸甲酯.L-DBTA二盐析出,过滤后再经水解,得到D-丝氨酸,总收率为74.8%,旋光纯度达到98%以上。     

D-氨基酰化酶拆分D,L-苯丙氨酸制备D-苯丙氨酸

进行了以D,L-苯丙氨酸为原料经D-氨基酰化酶制备D-苯丙氨酸的研究。乙酰-D,L-苯丙氨酸浓度为0.5mol.L-1,给酶量为3×104U.L-1时,24 h拆分率可达到97%。采用阳离子交换树脂进行了拆分液中的D-苯丙氨酸的分离,D-苯丙氨酸的收率为95.4%。采用醋酸酐作为催化剂,在145℃的条件下,乙酰-L-苯丙氨酸可以消旋成乙酰-D,L-苯丙氨酸继续拆分。     

酶法拆分制备D-异亮氨酸的工艺研究

以DL-异亮氨酸为原料经过乙酰化、氨基酰化酶拆分和水解制备D-异亮氨酸.使乙酸酐与DL-异亮氨酸在0～10℃下反应生成乙酰-DL-异亮氨酸,在氨基酰化酶的作用下,温度为37℃搅拌反应3d处理得到L-异亮氨酸和乙酰-D-异亮氨酸,乙酰-D-异亮氨酸经过盐酸水解得到D-异亮氨酸,收率为97.4％,光学纯度达到98％以上.另...     

基于微生物同化作用的D-丙氨酸生产工艺研究

以L-丙氨酸为唯一碳氮源,从采集的若干土壤中初筛出能够降解L-丙氨酸的菌株;再以D-丙氨酸为唯一碳氮源,复筛出降解L-丙氨酸而不降解D-丙氨酸的菌株。依据菌种对DL-丙氨酸的不对称降解活性,筛选出具有最高的L-丙氨酸降解活性的菌株,并对菌株同化L-丙氨酸的反应条件进行了研究。结果表明:编号为ALA-D82的菌株具有最高的降解L-丙氨酸的能力,经鉴定为酵母菌属。在30℃,控制pH6.0,通气比1:1(V/V)和转速900 r.min-1的条件下,L-丙氨酸降解的速度最大。在最适条件下,1500 g DL-丙氨酸分两部分添加入7 L的反应液中。反应72 h后溶液中的L-丙氨酸被完全降解,提取得到D-丙氨酸晶体,产率和光学纯度分别达到92.13%和99%。     

离子交换法分离D-天冬氨酸和L-丙氨酸

对离子交换法分离D-天冬氨酸和L-丙氨酸的工艺条件进行了研究。综合考虑树脂对D-天冬氨酸和L-丙氨酸的吸附容量及相对选择系数,选择了一种适合该体系分离的树脂—XH-1,并对吸附条件及洗脱条件进行了研究,确定了最佳工艺条件,为今后工业应用提供一定的依据。     

猪毛中胱氨酸的分离和复合氨基酸的制备

本文研究了以猪毛为原料,经过水解、赶酸、中和、结晶、精制提取出胱氨酸纯品;并从分离胱氨酸后的母液中,经过脱色、离子交换、浓缩、结晶、精制,制备出复合氨基酸.在本工艺条件下,胱氨酸产品的收率为4.8%,纯度在99%以上;复合氨基酸产品的收率为41%,纯度在83%以上.本文为扩大试验打下了基础.     

D-半胱氨酸盐酸盐制备研究

D-半胱氨酸盐是手性药物,主要是第三代头孢抗生素药物—头孢米诺钠的重要中间体。本文介绍了D-半胱氨酸盐酸盐的研制。采用L-半胱氨酸盐酸盐为出发原料,经消旋、沉淀异构体、结晶分离、提纯的方法,制成达到日本味之素公司1997年公布的产品质量指标的D-半胱氨酸盐酸盐。     

生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。     

鼠李糖乳杆菌D-乳酸脱氢酶基因ldhD的敲除

聚乳酸由可再生原料L-乳酸合成,是目前应用的最环保的生物塑料之一。鼠李糖乳杆菌JCM1553中的L-乳酸和D-乳酸,它们是由代谢途径中的L-乳酸脱氢酶和D-乳酸脱氢酶分别催化丙酮酸而生成。L-乳酸的光学纯度对于L-乳酸的应用至关重要。因此,为了获取光学纯的L-乳酸,需要敲除该鼠李糖乳杆菌编码D-乳酸脱氢酶的基因ldhD以阻断相关的D-乳酸代谢途径。本研究采用pK18mobsacB自杀质粒运用重叠延伸PCR和同源重组技术成功构建得到重组鼠李糖乳杆菌菌株JCM1553-△ldhD。构建的缺失突变体JCM1553-△ldhD菌株没有引入外源基因,完全符合食品、药品安全要求,发酵液中检测到的L-乳酸含量为99.92%,光学纯度达到99.84%,显著优于野生型菌株。     

Substrate specificity and reaction mechanism of human glycoasparaginase. The N-glycosidic linkage of various glycoasparagines is cleaved through a reaction mechanism similar to L-asparaginase.

Human glycoasparaginase (N4-(beta-N-acetyl-D-glucosaminyl)-L-asparaginase, EC 3.5.1.26) hydrolyzes a series of compounds that contain L-asparagine residue with free alpha-amino and alpha-carboxyl groups. Substrates include high mannose and complex type glycoasparagines as well as those that lack the di-N-acetylchitobiose moiety, L-aspartic acid beta-methyl ester and L-aspartic acid beta-hydroxamate. The enzyme is inactive toward L-asparagine and L-glutamine and glycoasparagines containing substituted alpha-amino and/or alpha-carboxyl groups. In the presence of the acyl acceptor hydroxylamine, glycoasparaginase catalyzes the synthesis of L-aspartic acid beta-hydroxamate from aspartyl-glucosamine, L-aspartic acid beta-methyl ester, and L-aspartic acid. 13C NMR studies using 18O-labeled L-aspartic acid demonstrate that glycoasparaginase catalyzes an oxygen exchange between water and the carboxyl group at C-4 of L-aspartic acid. These results indicate that glycoasparaginase reacts as an exo-hydrolase toward the L-asparagine moiety of the substrates and the free alpha-amino and alpha-carboxyl groups are required for the enzyme reaction. The results are consistent with an L-asparaginase-like reaction pathway which involves a beta-aspartyl enzyme intermediate. Since glycoasparaginase is active toward a series of structurally different glycoasparagines, we suggest the revised systematic name of N4-(beta-glycosyl)-L-asparaginase for the enzyme.     

A study into the role of L-aspartic acid on the metabolism of L-malic acid and D-glucose by Oenococcus oeni

AIMS: The purpose of this work was to study the effect of L-aspartic acid concentration on bacterial growth, D-glucose fermentation and L-malic acid consumption of Oenococcus oeni NCFB 1707. METHODS AND RESULTS: Bacterial cultures were performed in synthetic media. Bacterial growth, D-glucose fermentation and L-malic acid consumption were reduced when L-aspartic acid concentration became excessive. This inhibitory effect of high concentrations of L-aspartic acid on bacterial growth was also observed with several Oenococcus oeni strains, except O. oeni BL01. The L-aspartic acid inhibitory effect on bacterial growth could be reduced by increasing the concentration of L-glutamic acid. L-glutamic acid transport was found to be competitively inhibited by L-aspartic acid. In addition, an excessive amount of L-aspartic acid modified D-glucose metabolism, with an overproduction of acetic acid and reduced ethanol production. CONCLUSION: Since L-glutamic acid is an essential amino acid for the bacterial strain used, the L-aspartic acid inhibitory effect on bacterial growth could be linked to its involvement in an antagonistic interaction with L-glutamic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: Such antagonistic interactions between amino acids in O. oeni strains could be another explanation for the difficulties of inducing malolactic fermentation in wines.     

New synthetic route for RGD tripeptide

A new route was employed to synthesize RGD. First, Gly-Asp dipeptide was synthesized by a novel chemical method in two steps, including chloroacetylation of L-aspartic acid and ammonolysis of chloroacetyl L-aspartic acid. Second, Nalpha-Z- L-Arginine was reacted with Gly-Asp to synthesize RGD by the N-carboxyanhydride method. Less protected amino acids were used in this synthesis. This method possessed advantages of low cost, simplicity, and rapidity with a reasonable yield of 62% calculated from arginine. In addition, compared with the above method, a conventional solid phase method was also used to synthesize RGD, the yield was 75% calculated from the first amino acid anchored to resin.     

Ca2+-dependent cyclic nucleotide phosphodiesterase is activated by poly(L-aspartic acid)

Ca2+-dependent cyclic nucleotide phosphodiesterase (Ca2+-PDE) activity was stimulated by poly(L-aspartic acid) but not by poly(L-glutamic acid), poly(L-arginine), poly(L-lysine), and poly(L-proline). This activation was Ca2+ independent and did not further enhance the activation of Ca2+-PDE by Ca2+-calmodulin (CaM). Poly(L-aspartic acid) produced an increase in the Vmax of the phosphodiesterase, associated with a decrease in the apparent Km for the substrate, such being similar to results obtained with Ca2+-CaM. Poly(L-aspartic acid) did not significantly stimulate myosin light chain kinase and other types of cyclic nucleotide phosphodiesterase. CaM antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), trifluoperazine, and chlorpromazine selectively antagonized activation of the enzyme by poly(L-aspartic acid). Kinetic analysis of W-7-induced inhibition of activation of phosphodiesterase by poly(L-aspartic acid) was in a competitive fashion, and the Ki value was 0.19 mM. On the other hand, prenylamine, another type of calmodulin antagonist that binds to CaM at sites different from the W-7 binding sites, did not inhibit the poly(L-aspartic acid)-induced activation of Ca2+-dependent cyclic nucleotide phosphodiesterase. These results imply that poly(L-aspartic acid) is a calcium-independent activator of Ca2+-dependent phosphodiesterase and that aspartic acids in the CaM molecule may play an important role in the activation of Ca2+-PDE.     

Interaction between L-aspartic acid and L-asparaginase from Escherichia coli: binding and inhibition studies

Experiments using equilibrium dialysis and fluorescence quenching provided direct evidence that approximately four moles of L-aspartic acid were bound per mole of tetrameric L-asparaginase from Escherichia coli, with a dissociation constant on the order of 60-160 microM. In addition, a set of weaker binding sites with a dissociation constant in the millimolar range were detected. Kinetic studies also revealed that L-aspartic acid inhibited L-asparaginase competitively, with an inhibition constant of 80 microM at micromolar concentrations of L-asparagine; at millimolar concentrations of the amide, an increase in maximal velocity but a decrease in affinity for L-asparagine were observed. L-Aspartic acid at millimolar levels again displayed competitive inhibition. These and other observations suggest that L-aspartic acid binds not only to the active site but also a second site with lower intrinsic affinity for it. The observed "substrate activation" is most likely attributable to the binding of a second molecule of L-asparagine rather than negative cooperativity among the tight sites of the subunits of this tetrameric enzyme. Further support for L-aspartic acid binding to the active site comes from experiments in which the enzyme, when exposed to various group-specific reagents suffered parallel loss of catalytic activity and in its ability to bind L-aspartic acid. Different commercial preparations of Escherichia coli L-asparaginase were found to contain approximately 2-4 moles of L-aspartic acid; these were incompletely removed by dialysis, but could be removed by transamination or decarboxylation. Efficiency of dialysis increased with increasing pH. Taken together, this set of results is consistent with the existence of a covalent beta-aspartyl enzyme intermediate.     

Interaction Between L-Aspartic Acid and L-Asparaginase from Escherichia Coli: Binding and Inhibition Studies

Abstract

Experiments using equilibrium dialysis and fluorescence quenching provided direct evidence that approximately four moles of L-aspartic acid were bound per mole of tetrameric L-asparaginase from Escherichia coli, with a dissociation constant on the order of 60-160 μM. In addition, a set of weaker binding sites with a dissociation constant in the millimolar range were detected. Kinetic studies also revealed that L-aspartic acid inhibited L-asparaginase competitively, with an inhibition constant of 80 μM at micromolar concentrations of L-asparagine; at millimolar concentrations of the amide, an increase in maximal velocity but a decrease in affinity for L-asparagine were observed. L-Aspartic acid at millimolar levels again displayed competitive inhibition. These and other observations suggest that L-aspartic acid binds not only to the active site but also a second site with lower intrinsic affinity for it. The observed “substrate activation” is most likely attributable to the binding of a second molecule of L-asparagine rather than negative cooperativity among the tight sites of the subunits of this tetrameric enzyme. Further support for L-aspartic acid binding to the active site comes from experiments in which the enzyme, when exposed to various group-specific reagents suffered parallel loss of catalytic activity and in its ability to bind L-aspartic acid. Different commercial preparations of Escherichia coli L-asparaginase were found to contain ～ 2-4 moles of L-aspartic acid; these were incompletely removed by dialysis, but could be removed by transamination or decarboxylation. Efficiency of dialysis increased with increasing pH. Taken together, this set of results is consistent with the existence of a covalent β-aspartyl enzyme intermediate.     

Chemo-enzymatic synthesis of tripeptide RGD diamide in organic solvents

The tripeptide BzArgGlyAsp(NH(2))(2) was synthesized by a combination of chemical and enzymatic methods in this study. First of all, GlyAsp(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, kinetically controlled synthesis of BzArgGlyAsp(NH(2))(2) catalyzed by trypsin in organic solvent was conducted. The optimum conditions are pH 8.0, 30 degrees C in ethanol/Tris-HCl buffer system (85:15, v/v) for 80 min in the maximum yield of 74.4%.     

Immobilized cells of recombinant Escherichia coli strain for continuous production of L-aspartic acid

For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40 degrees C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour.