固定化红酵母细胞合成L-苯丙氨酸研究

In this paper, bioconversion of trans-cinnamic acid(t-Ca)to L-phenylalanine (L-phe) has been investigated by using immobilized yeast cells with induced L-phe Ammonia-lyase(PAL, EC.4.3.1.5) as biocatalysts. The contents are the following. (1) Thirty strains of yeasts, including two genera (Rhodotorula, Sporobolomyces), six species (R. glutinis R. minuta,R.rubra,R.sineses,R.roseus and S.salmonicolor)were screened for their ability to converse the substrates, t-Ca and ammonia, to the product, L-phe, by using yeast cells as biocatalyst, and primary evaluation for PAL activity of the selected strains was investigated. From the results of the screening experiments, it was found that 22 strains were able to produce L-phe from t-Ca with the range of conversion yield from 2% to 67%. Studies on PAL formation time course during cultivation show that the maximum PAL activity of several different strains ranges from 2.3 to 14.4×10-3U/mg cell dry weight. The biomass of tested strains at their maximum enzyme activity is also greatly varied. (2)One of the selected strains, R. rubra as 2.166, was used for immobilized cells as biocatalysts to produce L-phe. The optimum conversion conditions and effective stablization agents were investigated. The results shown that polyacrylamide gel was chosen as a suitable matrix for immobilization of the yeast cells, and it can retain 88% of the PAL activity in the reverse direction at the following reactive conditions: [t-Ca]: 34mM. [NH4OH]: 6.OM.PH10.00, temperature: 30℃. (3) The effects of various kinds of effectors on the production of L-phe were also examined. Membrane permeabilizing agents can stimulate L-phe synthesis, but make the stability of PAL decline greatly. Polyalchoholic agents and glutamic acid were very effective for the stabilization of PAL. At the presence of glutamic acid (5%), the half life of L-phe productivity with the immobilized cells was extended to 192 hours, which was much higher than most of that having been reproted, while the half life of resting cells was only about 15 hours. (4) Use of initial velocity studies on the kinetics of enzyme-catalized reaction indicated that the apparent Km value was 13.0mM for the immobilized cells, and 4.8mM for the resting cells. Thermostability of the immobilized cells was better than the resting cells. Fluid bed bioreactor is more effective than batch bioreator in prolonging the thermostability of the biocatalysts. (5) CGA- 688 resin column chromatographic procedure was employed in the isolation and purification of L-phe, t-Ca and other substances from the reactire mixture. (6) Preparative-scale production of L-phe on a level of gram amount by immobilized cells from the culture broth of R. rubra AS2.166 allowed for the conversion yield with 30%. The characteristic physico-chemical criteria (including melting point, optical activity, elements analysis, IR, NMR) are the same with the standard L-phe. 本文报告了利用诱导的苯丙氨酸解氨酶 (PAL.EC.4.3.1.5)催化反式肉桂酸(t-Ca)氨加 成制备L-苯丙氨酸(L-phe)的研究，主要内容为：(1) 我们搜集了三十株酵母菌株，利用全细胞转化t-Ca生成L-phe的能力进行了直 接筛选，并对其PAL活性水平进行了初步评估研究。研究结果表明，其中22株酵母具有转化t-Ca生产L-phe的能力，它们包括 Rhodotorula glutinis,R.rubra, R.sineses 和Sporobolomyces roseus 的菌株，转化率在2-67%。细胞生长和PAL形成过程的研究 表明，不同菌株PAL最大活力在2.3-14.4×10-3U/mg 细胞干重，达到最大PAL活性时各株酵母的生长情况也极不一致。(2) 利用筛 选出的一株深红酵母R.rubra AS2.166 作为供试菌株，研究了细胞固定化条件下生物转化的最适条件及PAL在固定化条件下的稳定 性。结果表明以聚丙烯酰胺凝胶包埋法较为理想，能使细胞合成L-phe活力保持88%，最适t-Ca浓度为34mM，最适NH4OH浓度为6M,最 适PH10.0，最适温度45℃。(3) 多种效应物对L-phe 合成的影响研究表明：表面活性剂能刺激L-phe的合成，但使PAL稳定性下降。 多羟基化合物及Glu对PAL的稳定十分有效在有Glu存在下，能使固定化细胞合成L-phe的半寿期达192小时左右，高于大部分现已报 导的固定化结果。(4) 用初速度法研究了深红酵母AS2.166中PAL的酶促反应特征，测得固定化细胞对t-Ca的表观米氏常数Km为 13.0mM，全细胞为4.8mM，细胞固定后热稳定性提高。(5) 建立了适合低浓度分离纯化产物与底物的聚苯乙烯大孔树脂柱层析技术 ，能使L-phe与t-Ca及产物混合物中其它成分有效分开。(6) 利用固定化的R.rubra AS2.166细胞所做的制备实验能够使L-phe的产 率达到30%左右，其主要的理化指标(包括熔点、比旋光度、元素分析、IR、NMR等)与标准L-phe一致。

Dependence of the cross sections for Ir isotopes on the values of Qgg in the heavy ion collision

197Au were irradiated with 47 MeV/u 12C ions. Iridium was produced via the multinucleon transfer reactions in bombardments of 197Au with 12C. and was separated radiochemically from Au and the mixture of the reaction products. The γ radioactivities of Ir isotopes were measured by using a HPGe detector. The production cross sections of Ir isotopes were determined from activities of Ir isotopes at the end of bombardment and the other relative data. It has been found that the cross sections for neutron-rich iso...

RF system design and measurement of HIRFL-CSRe

An RF system for the CSRe (cooling storage experimental ring) is designed and manufactured domestically. The present paper mainly describes the RF system design in five main sections: ferrite ring, RF cavity, RF generator, low level system and cavity cooling. The cavity is based on a type of coaxial resonator which is shorted at the end with one gap and loaded with domestic ferrite rings. The RF generator is designed in the push-pull mode and the low level control system is based on a DSP+FGPA+DDS+USB inter...

Comparison doses of secondary neutron with the heavy ions in a 75-mev/n heavy ion beam

国科图

(12)~C~(6+)离子辐照诱发人类肝L02细胞hprt基因突变的研究

本文研究12C6+离子辐照人类肝细胞系L02细胞诱发hprt基因突变与剂量的效应关系,为正确评价重离子对人体正常组织细胞的辐射风险及危害提供基础数据和依据。分别用12C6+离子束(LET为30keV/μm)和X射线(LET为0.2keV/μm)对L02细胞进行0～6Gy照射后,用克隆形成法检测细胞的存活分数,另外在含有6-TG的培养基中克隆、筛选hprt突变细胞株,测定突变频率。结果表明:12C6+离子辐照后L02细胞的存活分数明显小于X射线照后。两种射线照射后,每106个存活细胞中突变克隆的个数随照射剂量增大而增大,受照细胞的突变频率也都在1Gy处最大。但相对于X射线,人类肝细胞系L02细胞对高LET重离子辐射更敏感,而且12C6+离子束诱发更多的存活细胞hprt基因突变。

SFC的改进

根据兰州重离子加速器的注入器SFC运行20年来所发现的问题,近几年进行了一次大的改造,使其真空度、磁场分布以及高频系统的状态都得到了明显的改善。半年多的调束和运行实践表明这次改造是十分成功的。

在πh_(11/2)νi_(13/2)扁椭球转动带中观测到旋称反转

利用160Gd(35Cl,5nγ)190Tl反应研究了双奇核190Tl的高自旋态,建立了由πh9/2?νi13/2扁椭球转动带和一个具有单粒子激发特征的级联组成的190Tl能级纲图.确定地指定了190Tl扁椭球转动带的自旋值,首次发现了190Tlπh9/2?νi13/2扁椭球转动带的低自旋旋称反转.基于双奇核Tl能级结构的相似性,重新指定了双奇核192~200Tlπh9/2?νi13/2扁椭球转动带能级自旋值,在这些扁椭形变核中均出现了低自旋旋称反转.考虑了p-n剩余相互作用的2-准粒子-转子模型能够定性地解释πh9/2?νi13/2扁椭球转动带出现的低自旋旋称反转现象.

类氦铀离子的双电子激发态辐射衰变特性

讨论了类氢铀离子与H2分子碰撞中,经KLL共振转移和激发过程形成类氦铀离子双电子激发态的机制,分析了实验测量到的X射线能谱和各种竞争过程对X射线能谱的贡献。对由于纯电子电子相互作用形成的双激发态的Kα辐射衰变的超级卫星线的角分布测量结果表明,角分布呈各向异性,电子态布居存在很强的定向排列。

非束缚核~9He的实验研究

简要介绍了目前有关非稳定核9He的实验和理论研究现状.现代壳模型理论计算表明9He的基态自旋宇称为1/2+,而不是1/2-,即存在自旋宇称翻转现象.到目前为止,实验上虽然观测到了9He的几个共振态,但还不能确认其基态的能量和自旋宇称值.利用新的实验技术———逆运动学弹性共振散射,对9He核进行研究,给出了共振态的自旋宇称可能的取值范围.在ISOL型次级束装置上的实验有望能给出9He核能级的精确测量.

加速~(12)C~(6+)离子照射诱发人肝癌细胞SMMC-7721的DNA双链断裂

重离子为高传能线密度(LET)辐射;;在引起肿瘤细胞失活上有更大的相对生物学效应。DNA的双链断裂在细胞辐射损伤中是极其重要的;;其最终导致细胞的失活。本实验研究检测细胞DNA的双链断裂(DSB);;以探讨细胞失活的机理。用脉冲场凝胶电泳方法结合溴乙锭染色的荧光扫描技术;;定量测定C诱导人肝癌126+细胞SMMC?7721DNA的DSB剂量效应关系。结果发现;;DNA片段释放百分比随吸收剂量的增加而增加。

陕西蓝田公王岭黄土古土壤序列的磁组构特征及其古环境意义

IEECAS SKLLQG

束流调制器在核物理实验研究中的应用

介绍了束流调制器在几个不同的核物理实验研究中的实验方法:显示出束流调制器已成为核物理实验研究中的重要的核仪器之一。

从14MeV中子辐照过的锇靶中分离钨

用14MeV中子轰击天然锇靶,取道~(192)Os(n,α)反应生成~(189)W。利用改进的锇蒸馏装置蒸馏OsO_4,完成锇与反应产物的分离;通过四苯砷铼沉淀除去铼。使用HPGe探测器测量了分离的钨样品的γ射线谱,该γ射线谱显示钨样品中杂质元素的去污是令人满意的。