臺灣博碩士論文加值系統

海藻糖(Trehalose) 是新一代崛起的多功能醣類，存在於自然界的許多物種內，其在生物體內的功用除了可作為儲備能源與碳源外，還能幫助生物抵抗惡劣環境。海藻糖的應用範圍很廣，除了當作甜味劑外，因其具有穩定生物巨分子的能力，所以也可用來幫助保存食品、化妝品或是醫藥材料。海藻糖合成酶是生物體內用來生成海藻糖的一種途徑，能直接將麥芽糖轉化成海藻糖；本研究之新型海藻糖合成酶是從一株極度耐酸、適中溫的古生菌Picrophilus torridus基因組序列中找到，利用overlap extension PCR方式合成並在大腸桿菌中大量表達之基因重組酵素，尾端接有六個組胺酸 (Histidine)幫助純化。此酵素最適作用溫度與pH值分別是45 °C與pH 6.0，但到60 °C 與pH 5.0時仍具有極高的活性與穩定性，比目前已發表的海藻糖合成酶具有較廣的pH耐受度。在熱安定性方面，此酵素的熱變性反應屬於一級反應，在60 °C的情況下半衰期約為16小時。另外，此酵素對於麥芽糖的催化效率(kcat/KM)是對海藻糖的2.5倍，顯示其傾向於以麥芽糖當受質，催化產生海藻糖；而此酵素所催化的最大轉化率具有不受受質濃度影響，且與溫度成反比的特性，在pH 6.0， 20 °C的情況下可達到71 %的最大轉化率。在1 mM 濃度的Ag+, Hg2+, Al3+ 或SDS存在的情況之下酵素的活性會被完全抑制。此外，若與其他同屬α-澱粉酶家族(α-amylase family)的酵素做序列比對，可發現此海藻糖合成酶與其他α-澱粉酶家族的酵素都有三個保留的羧酸型胺基酸(carboxylic acid amino acids) (Asp203, Glu245 與Asp311)與兩個保留的組胺酸(His106 與 His310)，由定位點突變的結果可發現這五個胺基酸對此酵素活性有極大的影響，推測此海藻糖合成酶可能利用與其他α-澱粉酶家族酵素類似的機制進行反應。本研究發現之新型海藻糖合成酶因同時具有耐熱與耐酸的能力，相信其在未來工業生產方面將具有極大的潛力。

Trehalose is a newly emerging multi-functional disaccharide. It serves a variety of functions in organisms and protects organisms to survive harsh environments. In addition to be a sweetener, the ability of trehalose to stabilize macromolecules makes it a useful compound to preserve biomaterials in the food, cosmetic, and pharmaceutical industries. Trehalose synthase (TSase) is one of the pathways employed by organisms to synthesize trehalose. It catalyzes the reversible conversion of maltose into trehalose by intramolecular transglucosylation. In the present work, a new TSase gene identified from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and expressed in Escherichia coli. The recombinant P. torridus TSase (PTTS) with a C-terminal (His)6-tag was then purified by Ni-column to an electrophoretically homogeneous state. Activity analyses, using maltose as substrate, showed an optimum pH and temperature of 6.0 and 45 °C, respectively. However, the recombinant PTTS still maintained high activity up to pH 5.0 and 60 °C. A 2.5-fold higher catalytic efficiency (kcat/KM) for maltose than trehalose was shown in the kinetics analysis, indicating maltose as the preferred substrate for PTTS. The thermal inactivation of the recombinant PTTS showed the first order kinetics. The half-life time of the recombinant PTTS at 60 °C was about 16 h. The maximum conversion rate of maltose into trehalose by the enzyme tended to increase at lower temperatures, and reached about 71% at 20 °C. The activity of the enzyme was inhibited by Ag+, Hg2+, Al3+, and SDS. In addition, three active sites (Asp203, Glu245, Asp311) and two substrate-binding sites (His106 and His310) were deduced from the sequence alignment and confirmed by site-directed mutagenesis. As these residues are all conserved in other α-amylase family enzymes and have been found to be involved in catalysis, trehalose synthase might use a similar hydrolysis mechanism as other α-amylase family enzymes to cleave the α-1,4-bond of maltose. Since this newly found TSase is thermostable and more acid-resistant than any others reported, it is potentially useful for industrial applications.

Abbreviations..…………………………………………………………………... Ⅰ
Abstract (in Chinese) ....………………………………………………………… Ⅲ
Abstract..………………………………………………………………………… Ⅳ
Introduction.…………………………………………………………………..... 1
Distribution of trehalose.………………………………………………..…. 1
Physical and chemical properties of trehalose.…………………………..… 1
Functions of trehalose.…………………………………………………..…. 2
Applications of trehalose.…………………..………………………….…... 4
Trehalose biosynthesis.……………………………………………….……. 5
Turnover or hydrolysis of trehalose ….……………………………….…… 6
Manufacture of trehalose.…………………………………………….……. 7
Trehalose synthase ……………………………………………………..….. 8
Picrophilus torridus ………………………………………………….……. 8
Thesis aim………………………………………………………………….…… 9
Materials and Methods. ……………………………………………………..… 10
Materials …………………………………………………………………... 10
Bacterial strains and plasmid ..…………………………………………….. 10
Synthesis of trehalose synthase gene ……………………………………… 10
DNA gel extraction ……………………………………………………….. 11
Preparation of competent cells …………………………………………….. 12
Transformation ………………………………………………………….…. 12
Isolation of plasmid DNA …………………………………………………. 13
Construction of wild-type PTTS gene …………………………………….. 14
Purification of the recombinant PTTS ………………………………….…. 14
Purification of the wild-type PTTS ………………………..…………….… 15
Protein assay …………………………………………………………….… 16
Preparation of the purification table …………………………………….… 16
Enzyme assay …………………………………………………………….... 17
Kinetic parameters analysis …………………………………………….…. 18
Thermostability ………………………………………………………….… 18
Carbohydrates analysis …………..………………………………………... 20
NMR spectroscopy ..………………………………………………………. 20
Construction of PTTS mutants ……………………………………………. 21
Results ……………………………………………………………………….…. 22
Expression of the recombinant PTTS ………...…………………………… 22
Purification of the recombinant PTTS …………………………………….. 22
Biochemical properties of the recombinant PTTS ………………………… 22
Effects of pH and temperature on the activity and stability
of the recombinant PTTS ……………………………………..……… 22
Kinetics analysis ………………………………………………….….. 23
Thermostability ………………………………………………………. 24
Effects of metal ions and reagents on PTTS activity ………………… 24
Substrate specificity of the recombinant PTTS ……………………… 25
Effects of substrate concentration and temperature on the
maximum yield of trehalose ……………………………………...……...... 25
Purification of the Wild-Type PTTS …………………………………….… 26
Sequence alignment and site-directed mutagenesis analysis ……………… 26
Discussion …..……………………………………………………………….…. 28
References ….....……………………………………………………………….. 34
Tables and Figures ……….………………………………………………….… 41
Table 1. Comparison between six reported TSases …….………….…….. 41
Table 2. Summary of the purification procedures of the recombinant
PTTS from E. coli. …….……………………………………….. 42
Table 3. Kinetic parameters of PTTS ..……………….………………….. 43
Table 4. Thermodynamic parameters for the inactivation of the
recombinant PTTS at different temperatures .………………….. 44
Table 5. Effects of metal Ions and reagents on the activity of PTTS .….... 45
Table 6. Summary of the purification procedures of the wild-type
PTTS from E. coli. ……………………………………………… 46
Table 7. Specific activity of Wild-type PTTS and its mutant enzymes ….. 47
Figure 1. Three biosynthetic pathways of trehalose .…………………..…. 48
Figure 2. The result of carbohydrate analysis by high performance
liquid chromatography ……….…………………………….…… 49
Figure 3. 12% SDS-PAGE analysis for the purification of
the recombinant PTTS .…………………………………………. 50
Figure 4. Effects of pH on the activity and stability of PTTS ….…………. 51
Figure 5. Effects of temperature on the activity and stability of PTTS ..…. 52
Figure 6. Kinetics of thermoinactivation for the recombinant PTTS
at 60˚C, 65˚C, 70˚C, 75˚C, 80˚C, respectively ……………..……. 53
Figure 7. Arrhenius plot for thermal inactivation of the recombinant PTTS.54
Figure 8. The 2D HSQC NMR analysis result of the unknown product
converted from sucrose by PTTS ……………………….………. 55
Figure 9. Effects of substrate concentration on the maximum yield of
trehalose by PTTS ………………………………………………. 57
Figure 10. Effects of temperature on the maximum yield of trehalose
by PTTS. ……………………………………………………..…. 58
Figure 11. 12% SDS-PAGE analysis for the purification of
wild-type PTTS ……………………………………………...…. 59
Figure 12. Amino acids sequence alignment result .……………………….. 60
Figure 13. 12% SDS-PAGE analysis of the purified recombinant PTTS
and its mutant proteins ..………………………………………… 61
Figure 14. Possible catalysis mechanism of PTTS on maltose .………..…... 62
Appendix
Appendix 1. Construction of PTTS ………………………………………….. 63
Appendix 1-1. Designing of 36 oligonucleotides ……………….…… 63
Appendix 1-2. Overlap Extension PCR……………………………… 66
Appendix 1-3. Scheme for the Plasmid Construction ……..………… 67
Appendix 1-4. DNA sequence of the synthesized PTTS gene ………. 68 Appendix 1-5. The Amino Acid Sequence of the Recombinant PTTS. 69
Appendix 2. Vector Map ……………………………………………………… 70
Appendix 3. E. coli Strain Information ………………………………………. 71
Appendix 4. Carbohydrate Structure …………………………………………. 72
Appendix 5. Media & Reagents……………………………………………….. 73

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