氮源對藜豆轉殖毛狀根生長及二次代謝產物之影響以改進噴霧滴流式反應器之性能

Effects of Nitrogen Source on Proliferation of Transformed Hairy Root and Secondary Metabolite Production for Promoting the Performance of Mist Trickling Bioreactor

周士農(Shih-Nung Chou)

指導教授 ： 黃世佑

國立臺灣大學/工學院/化學工程學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00337

摘要

藉由農桿菌(Agrobacterium rhizogenes)所誘導的轉殖毛狀根，除了具有高基因生化穩定性、高生長速率以及不須添加荷爾蒙即可培養等優點外，也能夠生產高經濟價值的二次代謝產物。本研究利用轉殖藜豆毛狀根生產二次代謝產物L-DOPA，並以250mL錐形瓶及9L噴霧滴流式生物反應器(mist trickling bioreactor, MTB)進行培養。基於培養末期氮源不足的問題，本研究將進一步地探討無機氮源(硫酸銨，硝酸鉀)中期添加的最適濃度以及如何藉由無機氮源與其它養分(蔗糖，ascorbic acid)的添加來達到提高生質量以及L-DOPA產率的目的。此外，對於有機氮源(Tyrosine，Glycine)的添加也將一併探討。錐形瓶培養結果顯示，無機氮源中期最適添加濃度方面，以添加2×B5濃度之硫酸銨以及硝酸鉀所得到的L-DOPA產量最多，並發現此硫酸銨濃度的添加會造成蔗糖不足的現象。若同時補充2×B5濃度之硫酸銨以及蔗糖，則會更提高L-DOPA的總產量(提高9.54%)。有機氮源中期添加0.33 M Glycine對於L-DOPA總產量的提高有利，而1.38mM Tyrosine則否。反應器培養結果顯示，多量接種會使根與根纏結叢密，阻抑根之生長，造成根的生質量減少。氮源添加方面，2倍(NH4)2SO4的添加對於培養液之pH、導電度、ORP以及蔗糖利用率的影響很大，而從rH的變化可知NH4+的加入會再一次地抑制NO3-代謝，並且也伴隨其它的氧化反應(如碳源代謝等)，造成培養液之氧化反應大於還原反應再次使得rH上升；而當NH4+濃度下降至一定時，NO3-代謝不再受到抑制，並且此時其它如碳源代謝等氧化反應減少，以致於培養液的還原反應大於氧化反應，再次使得rH下降。

關鍵字

轉殖毛狀根 ； L-DOPA ； 氮源 ； 噴霧滴流式生物反應器

並列摘要

The transformed hairy roots induced by Agrobacterium rhizogenes can have several advantages, including highly genetic stability, high growth rate, cultivation without hormones and production of high value of secondary metabolites. In this work the transformed Stizolobium hassioo hairy roots was cultivated in 250 ml of shake flasks as well as in a 9 L of mist trickling bioreactor (MTB) for producing L-DOPA. To overcome the shortage of nitrogen source in the later stage of culture, the optimal dosage and feeding time of inorganic nitrogen source as well as other nutrients were investigated. This is aimed at the enhancement of hairy root proliferation and L-DOPA production. In addition, the effects of addition of organic nitrogen source have also been studied.

In the shake flask study, the optimum concentration of the inorganic source fed at a mid-term stage was a 2-fold dosage of (NH4)2SO4 and KNO3 to that of B5 basal medium. The highest L-DOPA concentration in the culture medium was obtained in this dosage. It was found that the sucrose during the culture medium was exhausted. The overall productivity of L-DOPA can be increased further if the above-mentioned concentration of ammonium sulfate together with 0.75% sucrose was dosed at the mid-term stage of culture. The dosage of 0.33M glycine in a mid-term stage of culture enhanced the total L-DOPA productivity, nevertheless, the dosage of 1.38mM of tyrosine did not.

In the MTB study, less inoculum density promoted the hairy root proliferation owing to the larger growing space. For nitrogen source supplementation by feeding a 2-fold B5 ammonium sulfate greatly influenced the pH, conductivity, redox potential and sucrose consumption rate of the medium. Throughout an observation by rH, it was elucidated that ammonium sulfate feeding would inhibit the metabolism of nitrate as well as the other oxidation reaction (e.g. carbon source metabolisms) which resulted in the predominance of oxidation reaction over reduction reaction and increased the rH. As concentration of ammonium ions decreased to a critical value, the metabolism of nitrate triggered. Consequently, other metabolisms of carbon source were retarded resulted in the decrease of rH.

並列關鍵字

L-DOPA ； Nitrogen source ； Mist trickling bioreactor ； Transformed hairy root

參考文獻

Albrecht R.R. and Kohlenbach H.W. (1990), L-DOPA content, peroxidase activity, and response to hydrogen peroxide of Vicia faba L. and Vicia narbonensis L. in situ and vitro, Protoplasms, 154, 144-150

Bhadra R. and Shanks J.V. (1997), Transient studies of nutrient uptake,growth,and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus., Biotechnol. Bioeng., 55,527-534

Bloom A.J., Meyerhoff P.A., Taylor A.R. and Rost T.L. (2003), Root development and absorption of ammonium and nitrate from the rhizosphere, J. Plant Growth Regul, 21, 416-431

Boyer R. (1998), Concepts in Biochemistry, An International Thomson Publishing Company, California, p.p.593-628

Brain R.D., Freeberg J.A., Weiss C.V. and Briggs W.R. (1977), Blue light-induced absorbance changes in membrane fractions from corn and Neurospora, Plant Physiol., 59, 948-952