以雙水相系統進行聚羥基烷酯聚合酶之純化及生物聚酯高分子體外合成研究

An Integration System Applied to Recover Polyhydroxyalkanoates Polymerase from Recombinant Escherichia coli and PHA Synthesis in vitro Using Aqueous Two-Phase Systems Technique

葉俊毅(Chun-Yi Yeh)

指導教授 ： 藍祺偉

元智大學/工程學院/化學工程與材料科學學系/碩士(2012年)

摘要

本研究利用日本東京工業大學拓植實驗室(Tsuge Laboratory of Tokyo Institute of Technology in Japan)所提供的pET-15b::PhaCRe質體進行培養，接著利用雙水相系統分離目標酵素聚羥基烷酯聚合酶(PHA polymerase)，本研究共分為兩大部分：1.利用含有PhaCRe蛋白質的澄清菌液(Clarified feedstock)及混濁菌液(Unclarified feedstock)進行雙水相系統，以純化目標酵素並分析活性之最適化條件。雙水相系統測試條件包含選擇不同環境pH值、雙結點曲線(Binodal curve)、節線長度(Tie-line length, TLL)、分離速度(Centrifugation speed)、鹽類(Salt)、操作溫度(Operational temperature)、體積比(Volume ratio)、工作體積(Working volume)及系統中菌液負載濃度(Loading biomass)對PhaCRe蛋白質的影響，進行分離效果的探討。2.利用經最適化條件純化後的PhaCRe蛋白質及利用雙水相系統建立一個酵素反應平台來合成聚羥基烷酯(PHAs)，探討PhaCRe蛋白質催化基質(R)-3HB-CoA之酵素動力學模式(Enzyme kinetics model)。
目前最適化純化結果為將澄清菌液(Clarified feedstock)之pET-15b::PhaCRe經由30% (w/w) PEG 6000 / 8% (w/w) PO4 pH 8.7所組成的雙水相系統於4 oC下操作進行純化程序，其比活性可達到1.76 U mg-1、純化倍率可達到16.23倍及回收率可達到95.32%；將混濁菌液(Unclarified feedstock)之pET-15b::PhaCRe經由30% (w/w) PEG 6000 / 8% (w/w) PO4 pH 8.7所組成的雙水相系統於4 oC下操作進行純化程序，其比活性可達到1.82 U mg-1、純化倍率可達到16.81倍及回收率可達到96.42%。研究結果顯示ATPS是一高效率的蛋白純化及分離技術，可直接從混濁菌液中純化聚羥基烷酯聚合酶。而探討PhaCRe蛋白質之酵素動力學方面，由結果得知經雙水相系統純化後的PhaCRe蛋白質與基質之間的親和力變化雖然不大，但催化速率卻有提升，且酵素催化機制關係呈現”非競爭型”；再藉由實驗結果證實反應催化機制為三級反應，即兩個酵素分子與一個基質分子進行反應。以雙水相系統為酵素反應平台合成的聚羥基烷酯(PHAs)重量平均分子量為8.8 × 103、數目平均分子量為8.5 × 103、分子量分佈指數為1.04，由此結果顯示合成的聚合物分子量集中，易於後續發展應用。

關鍵字

聚羥基烷酯聚合酶 ； 雙水相系統 ； 比活性 ； 純化倍率 ； 回收率 ； 反應機制 ； 酵素動力學 ； 分子量分佈指數

並列摘要

In this study, the recombinant Escherichia coli pET-15b::PhaCRe obtained from Tsuge Laboratory in Tokyo Institute of Technology (Japan) was employed as expression host for PHA polymerase production. Firstly, the expressed enzyme was directly recovered by using aqueous two-phase system (ATPS). The clarified and unclarified feedstock containing PhaCRe protein was introduced to the ATPS for separation. The impacts of pH value, binodal curve, tie-line length (TLL), centrifugation speed, ionic strength, operational temperature, volume ratio of ATPS, Working volume and loading concentration of biomass upon recovery performance were investigated and discussed. Secondly, an integrated aqueous two-phase system contained purified PhaCRe protein was applied to enzymatic reaction of P(3HB) synthesis in vitro. Moreover, the enzyme kinetics models of PhaCRe protein catalyzed with substrate, (R)-3HB-CoA, was determined.
The results demonstrated that the optimal conditions for PhaCRe recovery from clarified feedstock are by applying ATPS consisted of 30% (w/w) PEG 6000 and 8% (w/w) PO4 at pH value of 8.7 and 4 oC. The specific activity and purification factor can achieve 1.76 U mg-1 and 16.23, respectively. The efficiency of recovery achieved 95.32%. The identical conditions were also found for PhaCRe protein separation from unclarified feedstock in ATPS. The specific activity compared to the value derived from clarified feedstock increased to 1.82 U mg-1 and the purification factor achieves 16.81. The 96.42% of recovery efficiency was fulfilled. The results illustrated that ATPS can be a promising technique introduced to recover PHA polymerase in one-step operation. In addition, the present study is the first report in purification of PHA polymerase using ATPS technique. In part of kinetics study of PhaCRe polymerase, the results showed that the Km between the purified PhaCRe protein and the (R)-3HB-CoA substrate was similar. However, the Vmax can be improved, and the “non-competitive” enzymatic mechanism was depicted. Determination of polymerization showed a reaction order of 3 where two enzyme molecules react with one substrate molecule. The synthesis P(3HB) polymer in vitro has Mw, Mn and PDI are 8.8 × 103, 8.5 × 103 and 1.04, respectively.

並列關鍵字

ATPS ； Specific activity ； Purification factor ； Recovery ； Reaction mechanism ； Enzyme kinetics ； Polydipersity index ； PHA polymerase

參考文獻

Agus, J., Kahar, P., Hyakutake, M., Tomizawa, S., Abe, H., Tsuge, T., Satoh, Y., and Tajima, K. (2010). Unusual change in molecular weight of polyhydroxyalkanoate (PHA) during cultivation of PHA-accumulating Escherichia coli. Polymer Degradation and Stability 95, 2250-2254.

Akbari, N., Daneshjoo, S., Akbari, J., and Khajeh, K. (2011). Isolation, characterization, and catalytic properties of a novel lipase which is activated in ionic liquids and organic solvents. Applied Biochemistry and Biotechnology 165, 785-794.

Albertsson, P. A. (1986). Partition of cell particles and macromolecules. 3rd edition. Wiley & Sons, New York.

Andrews, B. A., and Asenjo, J. A. (2010). Theoretical and experimental evaluation of hydrophobicity of proteins to predict their partitioning behavior in aqueous two phase systems: A review. Separation Science and Technology 45, 2165-2170.

Andrews, B. A., Schmidt, A. S., and Asenjo, J. A. (2005). Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge. Journal of Biotechnology and Bioengineering 90, 380-90.