臺灣博碩士論文加值系統

1.Bacillus pumilus NCCU的木聚素水解酵素蛋白純化、物化性質分析、蛋白質體鑑定和基因轉殖

摘要

Bacillus pumilus NCCU,空氣中漂流的微生物,會分泌木聚素水解酵素到培養液中,利用硫酸銨將此酵素沈澱下來並用離子交換樹脂(mono-S)進一步純化此酵素。此純化的木聚素水解酵素的分子量為26 kDa,且在含有木聚素的 zymogram gel 中表現出活性。在37 ℃下此酵素對於木聚素的水解作用在 pH 值 5.0到9.0 之間活性最佳。在 pH 值為7的情況下,酵素作用最佳溫度為50 ℃且在50 ℃時此酵素的半衰期為40分鐘。此純化的酵素在50 ℃, pH 值為7情況下和受質 birchwood xylan 作用得到的 Km 值為2.49 mg,Vmax 值為 1.29 μmole min-1 μg-1,由於純化前的木聚素水解酵素在 zymogram gel 上出現兩個有木聚素水解活性的蛋白質,而這兩個蛋白質與SDS-PAGE上的蛋白質做比對後,將對到的蛋白質由SDS-PAGE 上的切出並利用蛋白質體方法分析。分子量為26 kDa的木聚素水解酵素比對到一個由 Bacillus pumilus 所分泌的 endo-1,4-β-xylanase [xyn A, Accession No. CAA25278]酵素,另外一個分子量比較高的蛋白質比對到Bacillus subtilis [Accession No. CAA97612] and Bacillus amyloliquefaciens FZB42 [Accession No. CAE11246]的 ynfF木聚素水解酵素。根據蛋白質體分析結果,以所比對到的 Bacillus pumilus的 xyn A的胜肽序列來合成 N- 端變質密碼子引子(degenerate primer),並依據此類木聚素水解酵素於 Bacilli 族群中保留的胜肽序列合成 C- 端變質密碼子引子。由這兩個引子在聚合酶連鎖反應下得到的214 bp長的 DNA產物的核酸序列和轉譯的胺基酸序列更確定26 kDa的木聚素水解酵素跟Bacillus pumilus 所分泌的 xyn A 幾乎一樣,根據 xyn A核酸序列合成N-和C-端特定的引子,利用聚合酶連鎖反應得到包含核糖體結合位置的序列的整段DNA片段。這個26 kDa的木聚素水解酵素含有687個核酸,並可譯出一個含有228個胺基酸的蛋白,且與已知的B. pumilus 上的xyn A 基因高度相似。

2.Bacillus megaterium NCCU的金屬蛋白水解酶物化性質分析、蛋白質體鑑定和基因轉殖
摘要
Bacillus megaterium NCCU, 在空氣漂流的微生物,會分泌蛋白分解酶到培養液中,利用硫酸銨將此酵素沈澱下來以便做蛋白分解酶活性實驗。金屬離子,鈣、鈉、鎂、鋅和錳離子會增加蛋白分解酶活性二到三倍,然而三價鐵離子會強烈抑制酵素活性。至於鎳離子只會稍微抑制酵素活性,而鈷和鉀離子對蛋白分解酶的活性沒有顯著影響。在含有 gelatin 的 zymogram gel上 有五條清澈的活性區帶,而在含有 casein 的 zymogram gel上 則有四條。有一條最清澈的活性區帶同時出現在兩種 zymogram gel,分子量大約是37 kDa。為了分析此蛋白質,將在SDS-PAGE上相對位置的蛋白切出並利用 in-gel digstion 和串聯質譜儀分析。分析的結果發現這個蛋白質符合B. megaterium的 Bacillolysin (中性蛋白分解酶) [Accession No. Q00891]。根據這個蛋白質的核酸序列合成N- 和C- 端特定的引子,利用聚合酶連鎖反應取得包含核糖體結合位置的序列整段DNA片段。這個蛋白分解酶含有1709個核酸,並可譯出一個含562個氨基酸的蛋白質,且與B. megaterium的nprM 基因高度相似。

1.Purification, characterization and proteomic identification of a xylanase and cloning of its gene from Bacillus pumilus NCCU.

Abstract

Bacillus pumilus NCCU, an air-borne microorganism, secreted xylanase into media. A 26 kDa xylanase was precipitated by ammonium sulfate fractionation and purified to homogeneity using a mono-S ion exchanger which showed enzymatic activity on xylan-zymogram. This xylanase showed broad optimal pH of 5.0 to 9.0 toward hydrolysis of birchwood xylan at 37 ℃. The optimal temperature for the enzyme activity was 50 ℃ at pH 7 and preincubation of this xylanase at 50 ℃displayed a half-life of 40 min. For birchwood xylan, this purified xylanase gave a Km value of 2.49 mg and a Vmax of 1.29 μmole min-1 μg-1 when the reaction was carried out at 50 ℃ and pH 7. For identification two protein bands on SDS-PAGE corresponding to the active xylanases on zymogram gel were incised and analyzed by proteomic approach. This 26 kDa xylanase matched to an endo-1,4-β-xylanase [xynA, Accession No. CAA25278] from Bacillus pumilus and the protein with higher molecular weight of 42 kDa matched to a ynfF xylanase of Bacillus subtilis [Accession No. CAA97612] and that of Bacillus amyloliquefaciens FZB42 [Accession No. CAE11246]. Based on a matched peptide on xynA of B. pumilus, an N-terminal degenerate primer was synthesized and a C-terminal degenerate primer was designed according to a C-terminal conserved sequence among closely relative xylanases of Bacilli family. The sequence of the PCR products from these two degenerate primers further confirmed this 26 kDa-xylanase indeed is highly similar with xynA xylanase of B. pumilus. According to the published DNA sequences of closely relative xylanases in NCBI database, the N- and C- termini specific primers corresponding to this xynA gene were synthesized and used in a PCR reaction to obtain a DNA fragment containing whole open-reading frame (ORF) including the ribosome binding site. This protein consists of 687 nucleotides and encoded 228 amino acid residues and highly homologous with known xyn A genes of B. pumilus around the world.

2.Characterization and proteomic identification of a metalloprotease and cloning of its gene from Bacillus megaterium NCCU.
Abstract

Bacillus megaterium NCCU, an air-borne microorganism, secreted proteases into media that were precipitated by ammonium sulfate for protease activity assay. In the presence of 1 mM metal ions Ca2+, Na+, Mg2+, Zn2+, Mn2 the activity of crude protease increased two to three folds, whereas strong inhibition of enzyme activity was observed in the presence of Fe3+. The nickel ion slightly inhibited the enzyme activity but Co2+ and K+ did not obviously influence the protease activity. On a gelatin zymogram gel five clear bands were shown and four clear bands presented on a casein zymogram gel. The clearest bands appeared on both zymogram gels showed the same molecular mass of 37 kDa. In order to identify this protease, it was incised and analyzed by in-gel trypsin digestion and ESI-QUAD-TOF. The MS/MS data of this 37 kDa protein matched to a Bacillolysin (neutral protease) from B. megaterium [Accession No. Q00891]. According to the DNA sequence of Bacillolysin [Accession No. X75070] in NCBI database, the N- and C- termini specific primers corresponding to this gene were synthesized and used in a PCR reaction to obtain the DNA fragment containing whole open-reading frame (ORF) including the ribosome binding site. This protein consists of 1736 nucleotides and encoded 562 amino acid residues and highly homologous with known nprM gene of B. megaterium [Accessiom No. X75070].

TABLE OF CONTENTS

SECTION Ι: Xylanase
Introduction 1
Environmental Microorganisms 1
Xylan 1
Xylanase 2
Regulation of Xylanase Gene 3
Classification of Xylanase 4
Structure of Xylanases 5
Catalytic Sites and Mechanism for Xylanases 6
Application of Xylanases 6

Materials and Methods 8
Screening Environmental Microorganisms Produce Xylanase 8
Growth of B. pumilus NCCU and Production of Xylanase 9
Measurement of Xylanase Activity by DNS Reagent 9
The Standard Curve for Unit Determination of Xylanase 10
Ammonium Sulfate Precipitation 10
Protein Precipitation by TCA Method 10
Xylanase Purification-FPLC 11
Measurement of Protein Concentration by Bio-Rad protein assay 12
SDS-Polyacrylamide Electrophoresis 12
Zymogram for Xylanase Activity 14
Effect of pH on Xylanase Activity 14
Effect of Temperature on Xylanase Activity 15
The Thermostability of Xylanase at 50 ℃ 15
Determination of Enzyme Kinetics 15
Chromosomal DNA Purification by Commercial Kit (UltraCleanTM Microbial
DNA Isolation Kit) 16

Preparation of Bacterial Chromosomal DNA by Enzymatic Method 17
Identification of Bacteria by 16S rDNA Sequence 18
Agarose Gel Electrophoresis 18
DNA Electroelution 19
DNA Ligation 20
Competent Cell Preparation-Rubidium Chloride Method for E.coli XL10-Gold 20
Competent E.coli Preparation for Electroporation (E.coli strain- XL1-Blue
MRF’) 22

Chemical Transformation (E.coli strain XL-10 GOLD) 23
Transformation by Electroporation- Bio-Rad Gene Pulser 24
Plasmid Mini-Preparation 25
Proteomic Analysis 26
Cloning of Bacillus pumilus NCCU Xylanase gene 28

Result 29
Microorganism Screening and Identification 29
Purification of Xylanase 29
Effect of pH on Enzyme Activity 30
Effect of Temperature on Enzyme Activity and Thermostability 30
Kinetics Parameters 31
Identification of Xylanases by Proteomic Approach 31
Cloning of Bacillus pumilus NCCU Xylanase gene 32
Comparison of Amino Acid Sequences of B. pumilus NCCU Xylanase with 33
those of Bacilli Family

Discussion 34

Reference 37




SECTION ΙI: Protease
Introduction 41
Protease 41
Classification of Protease 41
Metalloprotease 42
Mechanism of Action of Metalloprotease 42
Application of Protease 43

Materials and Methods 45
Screening Environmental Microorganisms Produce Protease 45
Growth of B. megaterium NCCU and Production of Protease 45
Measurement of Protease Activity by Azocasein Hydrolysis 46
Effect of metal ions on protease activity 46
Zymogram 46
Identification of Bacteria by 16S rDNA 47
Proteomic Analysis 47
Cloning of Bacillus megaterium NCCU protease gene 48

Results 49
Microorganism Screening and Identification 49
Effect of Metal Ions on Protease Activity 49
Zymogram Analysis 50
Identification of Protease by Proteomic Approach 50
Cloning the Protease gene of Bacillus megaterium NCCU 51
Comparison of Amino Acid Sequences of B. megaterium NCCU Protease 52
with those of Bacilli Family

Discussion 53

Reference 55

Table 57
Table 1. List of primers in this thesis 57
Table 2. Purification steps of 26 kDa xylanases isolated from Bacillus pumilus NCCU
58
Figure 59
Fig. 1. Structure of xylan and the sites of its attack by xylanolytic enzymes 59
Fig. 2. Hypothetical model for xylanase gene regulation in bacteria 60
Fig. 3. The represent active three-dimensional structures of families 11 and 10 xylanaes 61
Fig. 4. Reaction mechanism by Bacillus circulans xylanase (1XNB) 62
Fig. 5. The parameters used in MASCOT MS/MS ion search program 63
Fig. 6. Schematic illustration of the proposed mechanism of catalysis for
thermolysin 64
Fig. 7. The microorganisms exhibited xylanase activity on xylan-TB plate 65
Fig. 8. Comparison of nucleotide sequence of 16S rDNA from microorganism 66
NCCU 7 with published data
Fig. 9. Electrophoretic analysis of crude and purified xylanases from
B. pumilus NCCU 67
Fig. 10. The enzymatic activity of the purified 26 kDa xylanase measured 68
under different pH conditions
Fig. 11. Influence of the temperature on the activity of purified 26 kDa xylanase 69
Fig. 12. Thermostability of purified 26 kDa xylanase 70
Fig. 13. Double reciprocal plot for determining the Vmax and Km values 71
of xylanase
Fig. 14. The MS/MS data of the purified xylanase with lower molecular weight 72
(26 kDa) secreted by B. pumilus
Fig. 15. The MS/MS data of the xylanase with higher molecular weight 73
(42 kDa) secreted by B. pumilus
Fig. 16. Comparison of nucleotide sequence of the specific DNA fragment with
endo-1,4-xylanase of Bacillus pumilus 74
Fig. 17. Comparison of deduced amino acids of the specific DNA fragment with endo-1,4-xylanase of Bacillus pumilus 74
Fig. 18. Nucleotide sequence of xylanase (xynA) gene and its deduced 75
amino acid sequence from Bacillus pumilus NCCU
Fig. 19. Alignment of the deduced amino acid sequence of xynA of 76
Bacillus pumilus NCCU with xylanases of Bacillus family
Fig. 20. The microorganisms exhibited protease activity on skim milk-TB plate 77
Fig. 21. Aligment of 537 bp nucleotide sequence of 16S rDNA from 78
mcroorganism NCCU 4 with same region of 16 S rDNA from
Bacillus megaterium
Fig. 22. Effect of metal ions on the protease activity produced from 79
B. megateriums NCCU
Fig. 23. Electrophoretic analysis of crude proteases from
B. megaterium NCCU 80

Appendix
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