Soluble Expression and Characterization of Recombinant Lactococcus Lactis Subsp. Cremoris Tifn6 L-Arginine Deiminase

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簡歷

基本資料

Project title

Code/計畫編號

MOST109-2320-B019-002

Translated Name/計畫中文名

重組Lactococcus lactis subsp. cremoris TIFN6 L-精胺酸脫亞胺酶之可溶性表現與特性探討

Project Coordinator/計畫主持人

Tsuei-Yun Fang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=13530922

Year

2020

Start date/計畫起

01-08-2020

Expected Completion/計畫迄

31-07-2021

Bugetid/研究經費

1150千元

ResearchField/研究領域

食品科技(農)

L-瓜胺酸(L-citrulline)具有許多醫療保健和製藥上的應用，可避免氧化損傷、維持心血管正常功能、保持運動時的內臟灌注並刺激肌肉蛋白質合成、改善性功能、預防癌症、預防中風和心臟病。因此L-瓜胺酸被廣泛用於製藥行業，全球需求量逐年增長。利用酵素法生產 L-瓜胺酸，是以L-精胺酸脫亞胺酶 (L-arginine deiminase, ADI)催化L-精胺酸的不可逆水解而生產。因為基質易取得、產物的高產率、簡單的製備過程和純化步驟; 酵素法比其他生產方式更簡單且環保。我們已將L. lactis subsp. cremoris TIFN6 ADI (LcADI)基因大量表現於大腸桿菌中(佔總蛋白之40%)，然而卻形成包涵體，造成90%以上的重組LcADI發生變性而不可溶，破菌後只能純化到非常少量的可溶性重組LcADI。本計畫第一年將以提升可溶性LcADI的表現量為目標: (1)利用蛋白質工程技術於LcADI之C 端融合入短胜肽ATS (DPDNEAYEMPSEEGYQDYEPEA, 淨負電荷10，含C端COO-)或T7A3 (LEDPERNKERKEAELEAETAEQ, 淨負電荷6) 序列; (2)進行突變使融合的短胜肽淨負電荷增加;(3)以較低溫度、化學伴護子或是與分子伴護蛋白共表現，期望提升可溶性LcADI的表現量。第二年將針對融合前與後之LcADI進行大量表現、純化、特性探討及酵素動力學分析。本計畫目的是提升可溶性LcADI的表現量並探討融合前與融合後酵素特性，由較便宜的L-精胺酸直接生產L-瓜胺酸而降低生產成本，最終增加L-瓜胺酸在各方面的應用。 L-Citrulline has many health care and pharmaceutical applications, such as avoiding oxidative damage, maintaining the normal cardiovascular function, maintaining splanchnic perfusion and stimulating muscle protein synthesis during exercise in athletes, improving sexual function, preventing cancer, stroke and heart diseases. Therefore, L-citrulline is widely used in pharmaceutical and has a large growth in the global fine chemical industries with the demand increasing annually.The enzymatic method for the production of L-citrulline by using L-arginine deiminase (ADI) to catalyze arginine hydrolysis is preferred, due to the availability of the substrate L-arginine, high yield of the product, simple preparation processes and purification steps. Additionally, the enzymatic production of L-citrulline is simpler and more environmentally friendly than other synthesis approaches.We have expressed the gene of Lactococcus lactis subsp. cremoris TIFN6 ADI (LcADI) in endotoxin-free Escherichia coli. Although the recombinant LcADI was highly expressed (about 40% of the total protein), more than 90% of the expressed protein was insoluble and formed inclusion body, and only a small part of the expressed LcADI was soluble and obtained from purification after cell disruption. In the first year of this proposal, we will aim to increase the soluble expression of recombinant LcADI in endotoxin-free E. coli ClearColi BL21(DE3) by three approaches: 1) using protein engineering technology to fuse short peptide ATS (DPDNEAYEMPSEEGYQDYEPEA, with negative charges of 10 including the C-terminal COO-) or T7A3 (with negative charges of 6) sequence to the C-terminus of LcADI ; 2) carrying out mutagenesis to increase the negative charges of the fused peptides; 3) using lower temperature, chemical chaperons, and co-expression with molecular chaperons. In the second year of this proposal, we will overexpress, purify, characterize the LcADI (with and without fusions), and investigate the enzyme’s kinetic. Producing L-citrulline directly from the much cheaper L-arginine by ADI can reduce the prodcution cost, and will finally accelerate L-citrulline applications in many different areas.

Keyword(s)

L-精胺酸脫亞胺酶
L-瓜胺酸
L-精胺酸
融合
突變
大腸桿菌
L-arginine deiminase
L-citrulline
L-arginine
fusion
mutagenesis
Escherichia coli

DSpace CRIS

Soluble Expression and Characterization of Recombinant Lactococcus Lactis Subsp. Cremoris Tifn6 L-Arginine Deiminase

基本資料

Description