Bacillus macerans環狀糊精葡萄糖苷基轉移酶之研究I. N-端甲硫胺酸殘基對環狀糊精葡萄糖苷基轉移酶產物特異性的影響II.利用Cytophaga sp.生澱粉水解酶基因的啟動子及訊息胜肽�

Abstract

環狀糊精是由環狀糊精葡萄糖苷基轉移酶(CGTase, EC 2.4.1.19)催化澱粉環化反應而得，在工業上的應用相當廣泛。本研究室先前構築之E. coli (W101F/L304W-pUG) 雙位置突變CGTase於反應初期主要之環化產物為β-CD，與B. macerans CGTase反應初期主要之環化產物為α-CD有明顯不同。而利用pHG表現系統，同時於B. subtilis與E. coli菌體中生產W101F/L304W-CGTase，並未發生產物特異性轉變。追究可能的原因為pUG-CGTase之N-端較pHG-CGTase多了一個Met殘基，而使CGTase之產物特異性改變，因此欲得到由B. subtilis表現的mCGTase及W101F/L304W-mCGTase，以探討N-端Met殘基對CGTase活性的影響。此外，本研究室亦曾於土壤中篩出一株具有生澱粉水解能力的菌株Cytophaga sp. ，將其生澱粉水解酶(raw starch digesting amylase, RSDA)基因選殖於B. subtilis中，發現RSDA可在B. subtilis中大量表現，並經證實RSDA的結構基因上游有一明確的 promoter及signal sequence，於是本實驗中賦予cgt此一可在B. subtilis中大量表現目標蛋白質的promoter及signal sequence，得到B. subtilis與E. coli雙向穿梭重組質體 — pHRG、pHRMG及W101F/L304W-pHRMG，其分別可表現CGTase、N-端帶有Met的CGTase (mCGTase)及W101F/L304W-mCGTase。 結果顯示rsda的promoter及signal sequence可使CGTase在E. coli中大量表現，但在B. subtilis中的表現量卻不高。另外，pHRG重組質體造成E. coli表現CGTase於胞外，而且在不同的E. coli菌株中，CGTase的表情況會有所差異。另外由純化的mCGTase及W101F/L304W-mCGTase進行環化產物及性質分析得知N-端Met對環化初期產物特異性並未造成可測得的影響，但卻使最適溫度範圍的降低。而W101F/L304W雙位置突變的確會影響CGTase各種反應活性，或許加上E. colii與B. subtilis對蛋白質的折疊機制不同，進而使得CGTase環化反應初期產物有所不同。

Cyclodextrins(CDs) are product by cyclization reaction catalyzed with cyclodextrin glucosyltransferase(EC 2.4.1.19, CGTase) on starch. This material had broad application in various industries. In our previous study, a significant change in product specificity was observed in Escherichia coli W101F/L304W-CGTase with an extra Met residue in N-terminal. But this phenomenon was not reproductive in Bacillus subtilis and E. coli W101F/L304W-CGTase without N-terminal Met residue. The effect of extra Met residue in N-terminal on the biochemical properties of CGTase may be elucidated by constrain of cgt with an extra Met residue in N-terminal. Besides, it was found that Cytophaga sp. raw starch digesting amylase (RSDA) gene could be expressed to a high level in B. Subtilis. In order to improve the expression of CGTase in B. subtilis, we used the promoter and signal sequence of rsda to replace the elements in cgt. We constructed three B. subtilis and E. coli shuttle expression systems — pHRG, pHRMG and W101F/L304W-pHRMG and expected that the transformants could over express CGTase, CGTase with an extra N-terminal Met (mCGTase) and W101F/L304W-mCGTase in B. subtilis, respectively. The promoter and signal sequence of rsda resulted in significant increase expression of CGTase in E. coli than in B. subtilis. In addition, E. coli (pHRG) could express CGTase directly into culture medium. The recombinant plasmid pHRG showed different expression model in different hosting E. coli strain. And it was found that CGTase with an extra Met residue in N-terminal showed no significant difference on product specificity, but decrease optimal temperature range of coupling activity. Either W101F/L304W-mCGTase from E. coli or B. subtilis showed change in the coupling activity, hydrolysis activity and substrate affinity. It was suggested that both W101F/L304W mutation and different folding mechanism between E. coli and B. subtilis would have cooperative effect on the product specificity of CGTase.