| 摘要: | 果糖轉移酶 (FT)能水解葡萄糖與果糖之間的糖苷鍵，將果糖轉移至另一蔗糖上，形成蔗果三糖 ( GF2, 1-kestose)、蔗果四糖 ( GF3, nystose)、蔗果五糖 ( GF4, 1F-β-fructofuranosyl nystose)，進而形成聚果糖。本實驗室過去已將 Aspergillus japonicus 的果糖轉移酶 (AjFT)利用大腸桿菌BL21(DE3)表達，並完成結晶結構解析，顯示其結構具有兩個區塊：N 端的結構為5個β折板所形成的螺旋槳結構，而 C 端則為由β折板所形成的三明治結構。結構顯示其對受質的次結合位可分為-1、+1、+2及+3次結合位，分別由若干胺基酸位置組成。其中Asp60、Asp191及Glu292組成-1結合位，為酵素活性中心。
本研究的第一部分是針對受質次結合位做定點突變，檢測對於活性或產物的影響；已獲得rFTe-L141E (序列第141位置Leu突變成Glu)、 -H144K、-N290D、 -Q327R、 -Y404W、 -E405V、 -Q406S等突變體；結果顯示所有突變體產出的四糖量皆比WT少，其中以-H144K、-H144S及-H332K的四糖量最少，以產三糖為主；而-G81D、-L141R、-H332K及-E405K的三糖產量皆比WT多；-G81D、-L141R、-Q406R及-L141R/E405S則是可產出微量的六糖，暗示這些突變體有新的結合位產生；rFTe-N290D及-E318A的活性消失，顯示其位點對於活性的重要性；而rFTe-Y404W無法被表達，暗示其位點在酵素結構的重要。
從Aspergillus japonicus的果糖轉移酶已確認有不均勻的醣基化修飾，可能與酵素的結構穩定性有關；本研究的第二部分是在可能的醣基化位置做定點突變檢測酵素的穩定性；在熱穩定的檢測方面，rFTp-WT與多數的rFTp-mutants皆與AjFT類似，而rFTp-N545D的熱穩定性降低，於70℃時便沒有活性；在SDS的影響之下，rFTp-WT與多數的rFTp-mutants其活性都維持在90~100%之間，然而rFTp-N38D的活性卻明顯下降至30%左右；這些結果顯示，N545和N38可能的醣基化修飾，在結構穩定上扮演重要的角色。
Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan.
The fructosyltransferase from Aspergillus japonicas ( AjFT) was expressed in E. coli BL21(DE3), and its structure was revealed by X-ray crystallization in our previous studies. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade β-propeller fold linked to a C-terminal β-sandwich domain. Structures of AjFT-substrate complexes reveal complete four substrate-binding subsites (-1 to +3) in the catalytic pocket. D60, D191, and E292 govern the binding of the terminal fructose at the -1 subsite and the catalytic reaction.
The first part of this study is to test the site-directed mutagenesis at each amino acid that forming subsite, on the enzyme activity or product. Several mutants were obtained, including rFTe-L141E( 141 Leu mutated to Glu), -H144K, -N290D, -Q327R, -Y404W, -E405V, and -Q406S, etc. The results show that the the GF3 content in the product of all mutants were less than that of WT. The mutants -H144K, -H144S, and –H332K are of the least GF3 production, while the mutants -G81D, -L141R, -H332K, and –E405K produce more GF2 than that of WT. The mutants -G81D, -L141R, -Q406R, and -L141R/E405S can produce GF5, indicated the mutation introduces a new binding subsite. The rFTe-N290D and -E318A lost their overall activity, indicating the importance of these position on the activity. The rFTe-Y404W mutant was unable to be expressed, indicated the structual importance of this position.
Fructosyltransferase from the Aspergillus japonicas contains uneven glycosylation, that may be related to their structual stability. The second part of this study is to study the effect of mutagenesis of some possible glycosylation site on the activity and stability. The rFTp-WT and most mutants show similar thermal stability as FT purified from Aspergillus japonicus. The N545D mutant show decreased thermal stability. In the presence of 3% SDS, rFTp-WT and most mutant showed maintained activity (90-100%). However, N38D mutant showed significant decreased activity to around 30%. These data indicated that the N545 and N38 play important role in the structural stability that may be related to glycosylation. |
