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標題: 定點突變鏈黴菌屬 Streptomyces mobaraensis 轉麩氨醯胺酶的純化與活性
Purification and activity studies of two transglutaminase mutants from Streptomyces mobaraensis
作者: 溫宗翰
Zong-Han Wun
關鍵字: 轉麩氨醯胺酶;定點突變;酵素活性;transglutaminase;streptomyces mobaraensis;point mutatuon
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轉麩氨醯胺酶 (Transglutaminase, 簡稱 TGase;EC 為醯基轉移酵素 (acyltransferase),催化麩醯胺酸 (glutamine) 的 r-甲醯胺基 (r-carboxyamide) 與各種一級胺基 (如lysine) 形成共價鍵結。解析 Streptomyces mobaraensis TGase (smTGase) 結構顯示酵素近似圓盤狀 (disk-like),具有一裂縫在圓盤的邊緣,此裂縫為活性區域,催化核心的三個胺基酸 C64, D255 和 H274 即位於裂縫底部。點突變 smTGase 的研究顯示位於活性區域附近與酵素正、背面的部分胺基酸可能與酵素活性、受質辨識有關。本研究探討活性區域附近的胺基酸與酵素活性的關聯性。目前已構築突變株 F254A 和 L285A 並在大腸桿菌 BL21*(DE3)(pMT32C-smOVA)(pRK1037) 以共表達 TVMV 蛋白酶於胞內切除 pro region 的方式得到水溶性的 mature smTGase 。以 N-carbobenzoxy-glutaminyl-glycine (CBZ) 為受質測量酵素活性,得到野生株、F254A、L285A 在細胞粗萃液中的活性分別為 2.7, 0.3, 3 U/mg。野生株的粗萃液經 Ni-NTA 管柱純化時發現 pro region 會與 smTGase 共同析出而無法分離,用含 pH step-gradient 的緩衝溶液沖提可去除部分的 pro region,接著用 SP 陽離子交換樹脂去除殘餘的 pro region。純化突變株的過程中發現 pro region 在 Ni-NTA 純化中以不含 pH step-gradient 沖提可被去除,推測突變點會降低 pro region 與 mature smTGase 的親和性。純化後野生株、F254A、L285A 的活性分別為 25, 0.4, 4 U/mg。L285A 純化後酵素活性不變甚至下降,推測純化過程中有部分酵素失活,需要調整純化條件。F254A 在純化前後皆沒有活性,推測 F254 與酵素活性有關,或在純化過程中造成酵素失活。

Transglutaminase (TGase; EC is an acyltransferase which catalyzes the formation of a covalent bond between r-carboxamide group of glutamine residues and a variant primary amine, including the amino group of lysine. The structure analysis of mature smTGase from Streptomyces mobaraensis (sm) reveals that the shape is disk-like with a cleft at the edge of disk. This cleft is active area of enzyme. The catalytic triad of smTGase consists of C64, D255, and H274 which are located at the bottom of the cleft. The studies of mutations on the smTGase revealed that the amino acid which located around the active cleft and the front and the posterior region of smTGase may be related to substrate recognition and enzyme activity. We aim to investigate the relation between the amino acid which located at active cleft and enzyme activity. I have constructed the mutant smTGase, F254A and L285A and obtained soluble mature smTGase in E. coli strain, BL21*(DE3) (pMT32C-smOVA)(pRK1037) by co-expression TVMV protease which cleaves pro region. The enzyme activity was determined using N-carbobenzoxy-glutaminyl-glycine (CBZ) as a substrate. The enzyme activity of wild type and mutant strain, F254A and L285A in the crude extract values are 2.7, 0.3, and 3 U/mg. The crude extract of wild type was purified by using Ni-NTA column and the pro region was co-eluted with mature smTGase. Wash buffer containing pH step-gradient can partial remove the pro region. The residual pro region can be further purified by using SP cation column. The pro region of mutants can be removed by Ni-NTA column without pH step-gradient. This result may because of point mutations decrease the affinity between mature smTGase and pro region. The specific activity of the purified smTGase, wild type, F254A and L285A were determined to be 25, 0.4, 4 U/mg. Compared to crude extract, the specific activity of purified mutant, L285A was unchanged or decreased. This phenomenon may cause by purified processes, therefore refining the purified processes to obtain the active mutant is needed. The specific activity of the crude extract and purified mutant, F254A was closed to background level. This phenomenon may cause by purified processes or the amino acid, F254A which is important for smTGase activity.
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