臺灣博碩士論文加值系統

幾丁聚醣水解酵素(chitosanase)是煙麴菌(Aspergillus fumigatus)中擁有極高工業應用性的酵素，屬於黴菌幾丁聚醣水解酵素所屬的GH-75家族。煙麴菌的幾丁聚醣水解酵素基因被克隆出且置於大腸桿菌中表現並定性。水解產物利用核磁共振光譜以及質譜定序方法，證實此酵素是以反轉機制水解以及切GlcNAc-GlcN和GlcN-GlcN鍵而不切GlcNAc-GlcNAc和GlcN-GlcNAc linkages鍵。重組的酵素在大腸桿菌中以內含體(inclusion body)形式表現，利用5M尿素可救回35%的活性。另外，此酵素也在另一訊息胜肽表現系統中以胞內可溶蛋白質形式表現。將GH-75家族中五條幾丁聚醣水解酵素序列作比對，篩選十個保留度最高的胺基酸做定點突變。因為D160N與E169Q明顯失去活性且其他突變點仍保有40%以上活性。而利用circular dichroism證明不是因為二級結構的改變造成。另外，由理論計算方式也的到相同預測結果。而表面電漿共振儀證明D160N與wild type對於幾丁聚醣的三醣與四醣有相同的親和特性。這些結果證明Asp160與Glu169在此酵素中扮演重要的催化角色。最後，由多方資料整合歸納出Asp160是catalytic base而Glu169 是proton donor。

A powerful chitosanase for the preparation of chitooligosaccharide was previously purified from Aspergillus fumigatus. The corresponding gene was also cloned and the enzyme was further classified into glycosyl hydrolase family 75. The recombinant chitosanase was over-expressed in E. coli with a form of inclusion body, which was rescued by treating with 5 M urea and subsequently purified by cation-exchanged chromatography. Alternatively, the recombinant enzymes were also expressed in a pRSET_SP system containing a signal peptide. The recombinant chitosanases were found to produce as soluble protein intracellularly. A time-course 1H-NMR experiment on the enzymatic formation of chitooligosaccharides revealed that the mechanism of the enzyme involved an inversion of an anomeric configuration. Through analysis of the products and their corresponding methylated derivatives with LC/MS/MS, the pattern of enzymatic hydrolysis of the GlcNAc-GlcN and GlcN-GlcN linkages in chitosan were unequivocally determined, whereas the GlcNAc-GlcNAc and GlcN-GlcNAc linkages were not digestible. Site-directed mutagenic studies on the ten conserved carboxylic amino acids of the family were performed. Among them, the mutants of D160N and E169Q lost all activity, whereas the other mutants retained > 40 % activity of the wild-type chitosanase. Measurements of circular dichroism of D160N, E169Q, wild-type enzyme and other active mutants yielded similar spectra, indicating that activity loss of the two mutants was not due to the change of protein structure. Surface plasma resonance (SPR) studies revealed that the binding properties of D160N and the wild type enzyme with either chitotetramer or chitotrimer are comparable. We conclude that Asp160 and Glu169 are the two essential residues of A. fumigatus chitosanase.

Abstract (in cChinese)---------------------------------------------------------Ⅰ
Abstract (in English)----------------------------------------------------------Ⅱ
Content---------------------------------------------------------------------------Ⅲ
Chart content-------------------------------------------------------------------Ⅴ
Chapter 1 Introduction
1-1 Chitosan oligosaccharides-----------------------------------------------1
1-2 Chitosanase----------------------------------------------------------------5
1-3 Aspergillus fumigatus---------------------------------------------------14
1-4 Escherichia coli expression system of recombinant chitosanase
from Aspergillus fumigatus--------------------------------------------16
1-5 The objective of this study---------------------------------------------19
Chapter 2 Material and method
2-0 General description------------------------------------------------------20
2-1 Site-directed mutagenesis-----------------------------------------------22
2-2 Expression and purification of chitosanase---------------------------25
2-3 Enzyme activity test by DNS-------------------------------------------30
2-4 Mass analysis-------------------------------------------------------------31
2-5 Investigation of secondary structure-----------------------------------33
2-6 Investigation of anomeric configuration by NMR-------------------35
2-7 Characterization of enzymes by SPR----------------------------------36
2-8 Chemical label------------------------------------------------------------43
Chapter 3 Results and discussion
PartⅠProduction of enzyme
3-1 Site-directed mutagenesis-----------------------------------------------47
3-2 Inclusion body expression system-------------------------------------52
3-3 The pRSET_SP (PSP) expression system ----------------------------59
PartⅡCharacterization of enzyme
3-4 Analysis of activity and structure--------------------------------------68
3-5 Investigation of anomeric configuration by NMR ------------------71
3-6 Identification of hydrolyte pattern by LC/MS/MS ------------------74
3-7 Investigation of the binding properties of chitosanases by SPR---78
3-8 Prediction of Aspergillus chitosanase topology----------------------80
3-9 Chemical label------------------------------------------------------------84

Chapter 4 Conclusion------------------------------------------------------87
Chapter 5 Reference--------------------------------------------------------90
Appendix------------------------------------------------------------------------97

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