恶臭假单胞菌TS1138转化生产L-胱氨酸的工艺研究

对以DL-2-氨基-?2-噻唑啉-4-羧酸(DL-2-amino-?2-thiazoline-4-carboxylic acid, DL-ATC)为底物原料, 经微生物酶法催化合成L-半胱氨酸, 并进一步氧化和分离纯化产物L-胱氨酸的生产工艺和条件进行了研究。建立了以恶臭假单胞菌TS1138 (Pseudomonas putida TS1138)全细胞为酶源, 反复多次催化底物合成L-半胱氨酸, 并以2.0%二甲基亚砜(DMSO)为氧化剂氧化生成L-胱氨酸, 进而通过001×7型阳离子交换树脂纯化胱氨酸的新工艺。采用高效液相色谱法考察该方法L-胱氨酸的总收率可以达到78.55%, 纯度为99.12%。该方法简单高效, 解决了酶稳定性差不能重复使用, 而固定化酶方法繁琐成本高的问题, 为我国L-半胱氨酸和L-胱氨酸的生产开辟一条新途径。     

恶臭假单胞菌TS1138转化生产L-胱氨酸的工艺研究

对以DL-2-氨基-△2-噻唑啉-4-羧酸(DL-2-amino-△2-thiazoline-4-carboxylic acid,DL-ATC)为底物原料,经微生物酶法催化合成L-半胱氨酸,并进一步氧化和分离纯化产物L-胱氨酸的生产工艺和条件进行了研究.建立了以恶臭假单胞菌TS1138(Pseudomonas putida TS1138)全细胞为酶源,反复多次催化底物合成L-半胱氨酸,并以2.0%二甲基亚砜(DMSO)为氧化剂氧化生成L-胱氨酸,进而通过001×7型阳离子交换树脂纯化胱氨酸的新工艺.采用高效液相色谱法考察该方法L-胱氨酸的总收率可以达到78.55%,纯度为99.12%.该方法简单高效,解决了酶稳定性差不能重复使用,而固定化酶方法繁琐成本高的问题,为我国L-半胱氨酸和L-胱氨酸的生产开辟一条新途径.     

由人发制备L-胱氨酸工艺的改进及某些机制探讨

<正> 本文介绍了由人发制备L-胱氨酸的生产工艺。为提高L-胱氨酸生产收率,对原工艺有些工序进行分析研究。作者为了能获得理想水解终点,因而作了不同盐酸浓度对L-胱氨酸收率影响曲线,认为用8.8N～9N盐酸浓度制备L-胱氨酸收率最佳;也做了不同时间水解人发对L-胱氨酸收率影响,对从人发中制备单一L-胱氨酸采用6.5小时水解时间,对L-胱氨酸收率最佳、最经济;水解浓缩液经中和后马上采用连续搅拌结晶;粗制时先采用液碱后用饱     

不同来源毛发酸水解液中胱氨酸含量的差异

<正> 以毛发为原料酸水解生产 L-胱氨酸已在我国许多单位进行,如何提高 L-胱氨酸的收得率,降低成本是生产中关键问题。L-胱氨酸的收得率与水解条件,中间提取条件是密切相关的,但在水解条件相同的情况下,不同来源的毛发酸水解液中 L-胱氨酸含量有无差别,对 L-胱氨酸收率有什么影响呢?我们1976年在广东省东莞县拷胶厂,对不同来源毛发酸水解液中 L-胱氨酸含量和产量进行了分析和试验。     

代谢过程中相关氨基酸对高密度发酵生产腺苷蛋氨酸的影响

对清酒酵母高密度发酵生产S-腺苷-L-蛋氨酸（SAM）代谢过程中的相关氨基酸进行了考察。分别考察了十二种氨基酸对生物量和SAM产量的影响。实验发现L-胱氨酸、L-半胱氨酸、L-赖氨酸、L-组氨酸和L-蛋氨酸对SAM的积累有利,其中L-赖氨酸和L-组氨酸可以提高生物量,进而提高SAM的产量;L-胱氨酸、L-半胱氨酸和L-蛋氨酸可以提高SAM的含量,但是会抑制生物量的增长。通过3种补加方式的比较,得到最优的补加方式为：L-赖氨酸和L-组氨酸在培养基中加入,L-胱氨酸,L-半胱氨酸和L-蛋氨酸采取在发酵过程前24h流加。通过正交实验确定补加量为：L-赖氨酸为1g／L,L-组氨酸为1g／L,L-胱氨酸为1．5g／L,L-半胱氨酸为1g／L,L-蛋氨酸为1g／L。将此结果应用于5L发酵罐培养,SAM最高产量为5．53g/L,生物量为128g／L。     

L-茶氨酸新型酶法制备及其催化工艺优化

L-茶氨酸是茶叶中游离氨基酸的主要组成部分,关于其良好的生理活性已有广泛报道。首次报道了来源于Cunnighamella echinulata 9980的L-氨基酰化酶用于高光学纯度的L-茶氨酸的酶法制备。该酶在pH 6.5,底物N-乙酰-DL-茶氨酸浓度为50 mM,且有40 mM CoCl2时催化效果较好。结果表明,在上述条件下,50℃作用12 h得L-茶氨酸22.5 mM,转化率90%。     

氨基酸的消旋研究

在纯乙酸溶剂中,以乙酸酐酰化消旋酪氨酸,消旋温度为90℃,L-酪氨酸和乙酸酐的摩尔比为12,每消旋1gL-酪氨酸时,溶剂乙酸的用量为5mL,效果较好。同样的方法应用于L-脯氨酸,L-苏氨酸,L-缬氨酸,L-赖氨酸,L-半胱氨酸等氨基酸,消旋率均可达到100%。     

PPK和GMAS共表达重组菌株的构建及其在L-茶氨酸合成中的应用

构建了共表达ATP再生和L-茶氨酸合成酶的重组大肠杆菌菌株,并将其应用于L-茶氨酸的合成中。合成多聚磷酸盐激酶(PPK)和γ-谷氨酰甲胺合成酶(GMAS)基因序列,分别利用p ETDuet-1和p ET-21a(+)载体,构建共表达重组质粒p ETDuet-ppk+gmas和p ET21a-ppk+gmas。将上述两种重组质粒转入大肠杆菌BL21(DE3)中,获得重组菌株TPG和APG。IPTG诱导表达后,SDS-PAGE结果表明,PPK和GMAS在两种重组菌中均可溶性表达。当用于催化L-茶氨酸合成时,来自APG的GMAS-PPK要优于TPG。利用APG所产的酶进行L-茶氨酸合成,在37℃、p H 7.0条件下,使用催化量ATP可实现L-茶氨酸的摩尔产率为86.0%。该结果一方面扩展了酶法ATP再生系统的应用,另一方面为生物催化合成L-茶氨酸提供了一种有效方法。     

北京猿人和丁村人的氨基酸年龄测定

本文试图用北京猿人和山西丁村人所在地层中伴生动物化石内的天门冬氨酸、异亮氨酸外消旋程度推算北京猿人和山西丁村人地点的年龄和山顶洞所经历的平均温度。     

L-茶氨酸对H_2O_2致L02细胞损伤的保护作用及其机制研究

研究L-茶氨酸对肝细胞损伤的保护作用及其机制。利用H2O2诱导的L02肝细胞损伤模型,分别用MTT法检测细胞存活率、测定LDH、流式细胞术检测细胞凋亡率、Western blot法检测Caspase-3和PARP蛋白表达及Bax/Bcl-2比值的变化,评价L-茶氨酸是否能保护H2O2诱导的肝细胞损伤。结果表明,L-茶氨酸能提高H2O2损伤的L02细胞存活率,减少LDH的渗漏,降低肝细胞凋亡,且L-茶氨酸通过抑制Caspase-3的激活和PARP的切割及Bax/Bcl-2比值的升高而发挥抗凋亡的作用。L-茶氨酸对肝细胞损伤有一定的治疗和保护作用。     

Age-dependent accumulation of protein residues which can be hydrolyzed to D-aspartic acid in human erythrocytes

We have measured the rate of accumulation of amino acid residues in human erythrocyte membrane and cytosolic proteins which give D-aspartic acid upon acid hydrolysis. These residues would include D-aspartic acid, D-asparagine, as well as the beta-transpeptidation product, D-isoaspartic acid. Measurements made using age (density) fractionated cells indicate that racemization at these residues occurs on membrane proteins with a t1% (the time required to convert 1% to the D configuration) of about 38.6 days. Fractionation of membrane components revealed a faster rate of racemization for intrinsic proteins than for extrinsic proteins. On the other hand, significant age-dependent racemization was not detected for cytosolic proteins, and the calculated t1% value for these proteins is at least 4 times larger. These results suggest that in the 120-day life span of an erythrocyte, significant racemization of membrane (but not cytosolic) proteins can occur. We have also determined that the rates of accumulation of these residues for erythrocyte membrane and cytosolic proteins incubated in vitro are similar to those observed in vivo. These observations are discussed in terms of the possible cellular metabolism of racemized proteins.     

从鸡毛中提取复合氨基酸的研究

运用正交实验研究了以鸡毛为原料硫酸水解生产复合氨基酸的水解条件。结果表明 :硫酸浓度为 8mol·L- 1 ,鸡毛质量 (g)与硫酸体积 (mL)之比为 1∶2 .5 ,水解时间 10h ,其水解率可达 5 5 .2 3%。得到的固体氨基酸质量分数为 95 .31%。     

Racemization of alpha-melanotropin

Racemization of the amino acid residues of alpha-melanotropin was measured after exposure of the peptide to alkali for various lengths of time. Rates of racemization were then compared to the rate of transformation by alkali of alpha-melanotropin into a hormone with prolonged melanotropic activity. When in vitro prolongation became maximal, serine, methionine, histidine, phenylalanine and arginine were racemized 50-70%, glutamic acid, tyrosine and tryptophan 30-40% and lysine, proline and valine 10% or less. Racemization of a particular amino acid residue in alpha-melanotropin could not be associated with induction of prolongation of activity. Rather, partial racemization at multiple sites in the molecule seems almost as effective as extensive or total racemization of a single residue in producing a hormone with prolonged biological effects.     

Geochemical evolution of amino acids in dentine of Pleistocene bears

A linear correlation was established between aspartic acid racemization ratio from cave bear dentine collagen and absolute dating. The high correlation coefficient obtained allowed age calculation through amino acid racemization. Aspartic acid and glutamic acid racemization kinetics have also been explored in dentine from a North American black bear (Ursus americanus Pallas). Three sample sets were prepared for kinetic heating experiments in nitrogen atmosphere: one water soaked, one with a water-saturated nitrogen atmosphere, and one without any moisture. It was possible to show that the presence of water is a factor controlling amino acid racemization rate. The aspartic acid in a heating experiment at 105 degrees C shows an "apparent kinetics reversal" which can be explained by a progressive hydrolysis of amino acid chains (proteins and polypeptides). Because of the low potential of collagen preservation over long periods of time, the apparent kinetics reversal phenomenon will not affect the dating of old material where no traces of collagen remain. An apparent kinetics reversal was not observed in glutamic acid, which racemizates more slowly.     

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography.

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluene-sulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.     

Topochemical studies on the wall of beech bark sclereids by enzymatic and acidic degradation

Summary Sclereids isolated from the bark of beech (Fagus sylvatica L.) were delignified and treated with 1.3% sulfuric acid or with purified enzymes, viz., avicelase, carboxymethylcellulase, xylanase as well as combinations of xylanase and avicelase. Monitoring of the degradation was performed by quantitative liquid chromatography. Sulfuric acid dissolves about 30% sugars, especially hemicelluloses after 12 hours treatment. The avicelase (cellulase) and carboxymethylcellulase treatment degraded cellulose only to a very small extent. The xylanase degraded xylan selectively from the delignified sclereids amounting to about 60% after 51 hours incubation. The combined action of xylanase and avicelase brought about a xylan degradation of about 70%. Addition of avicelase to the initially xylanase-treated material resulted in the degradation of cellulase up to 25%.Electron microscopy of the variously treated samples showed the micromorphological changes effected and gave an indication of the topochemical distribution of xylan and cellulose. Sulfuric acid treatment removed wall components from all the lamellae of the sclereid wall, showing no definite pattern. Xylanase effects an intense decrustation of wall material both at the lumen boundary as well as near to the middle lamella, whereby the pattern of degradation is irregular; the cellulose fibrils also become well exposed. The addition of avicelase to xylanase-treated sclereid holocellulose creates an increase in the degradation, which is especially localized in the lamellated wall near to the middle lamella/primary wall region and at the lumen boundary. There appears to be a total hydrolysis of both matrix and fibrillar substances, characteristically more in the lamellae with longitudinal bow-shaped fibrils. Based on these results it is concluded that there appears to be no definite differential distribution pattern of xylan in the two lamellae. The higher contrast in the lamellae with transversely oriented fibrils is interpreted as resulting from the packing density of cellulose fibrils.     

The influence of extrusion on loss and racemization of amino acids

Summary. The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-β-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 °C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 °C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 °C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L – D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize. Authors’ address: J. Csapó, Faculty of Animal Science, Institute of Chemistry, University of Kaposvár, Guba S. u. 40., H-7400 Kaposvár, Hungary     

Investigations of in vivo methionine racemization in mammalian tissues

The racemization rate of methionine at pH 7.4 and 100 degrees C was determined to be similar to that of aspartic acid. However, analyses of dentin and ocular lens nucleus samples from known age Rhesus monkeys showed that unlike aspartic acid, methionine does not undergo in vivo racemization in mammalian proteins.