新疆紫草细胞培养产生紫草宁衍生物的代谢调控

本文对新疆紫草培养细胞的生长及紫革宁衍生物形成的调节与控制进行了研究．结果表朗：在不同时期照射白光、蓝光，对紫草培养细胞生长及紫草宁衍生物的积累均有明显的抑制作用;而第三周照红光一天、第二周以及整个培养过程均照远红光、第19天照紫外光等，都对生产培养基中紫草宁衍生物的积累有促进作用;其中，远红光与紫外光的最佳照射时间长度都为30分钟，且细胞内苯丙氨酸解氨酶的活性与紫草宁衍生物的含量有明显的正相关性．研究了不同照光条件下，紫草培养细胞生长，紫草宁衍生物积累以及苯丙氨酸解氨酶活性变化的动态，基本上找出了它们的变化规律以及它们之间的相互关系． 通过试验发现，紫草细胞培养的适宜温度为25℃，适宜pH值为5.3—5.8：在培养基中加入O,l%-0.30%的活性炭、用磁水器处理培养基等，对紫草宁衍生物的积累有良好的促进作用。 提高培养基中的Cu2+，Mg2+，Zn2+．K+浓度，有利于紫草宁衍生物的积累，而在生产培养基中加入NH：，则显著抑制了紫草宁衍生物的产生;培养基中蔗糖浓度为50g／L时，对紫草细胞生长及紫草宁衍生物的积累最有利，而生产培养基中不加IAA，KT对紫草宁衍生物的积累有利．以密环菌为诱导子，促进了紫草细脆生长及紫草宁衍生物的积累。 此外，对Cu2+、密环菌发酵液处理条件下，紫革宁衍生物积累与苯丙氨 酸解氨酶活性的关系以及不同蔗糖浓度条件下，生长培养基中紫草细胞生长、 紫草宁衍生物的积累，细胞内可溶性糖含量变化的动态进行了研究．

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。