臺灣博碩士論文加值系統

本實驗室由土壤中篩選出具有分解膠原蛋白能力的菌株Bacillus amyloliquefaciens 23-7A，經脫脂黃豆醱酵培養後，依據分解受質的種類 (FALGPA、Caein及Gelatin)，總共純化到8種外泌性蛋白酶，分別命名為PepFBA酵素、C酵素群(C1, C2A, C2B酵素)及G酵素群 (G1~G4)。根據PepFBA酵素N端及部分胜肽序列，設計出的引子進行PCR。以PCR的產物做為探針，再從染色體基因庫中選殖出完整的基因。PepFBA酵素的基因全長為2,013 bp，可轉譯出670個胺基酸 (77,049 Da)的pre-pro蛋白。當含23個胺基酸的signal peptide被切除後，pro-PepFBA會在胞外進行另一次剪切，將7個胺基酸長度的pro序列切除。經兩次剪切後，PepFBA的分子量為73,647 Da，與利用SDS-PAGE求得的分子量接近 (69,000 Da)。在蛋白序列中，具有結合鋅離子的His-Glu-X-X-His motif序列，這與PepFBA會被金屬螫合劑所抑制的特性相符。以NCBI Blast軟體進行蛋白序列的比對，PepFBA與Bacillus subtilis PepF及Bacillus licheniformis Pz-peptidase具有85%及87%的相似性。這兩個酵素都是屬於oligopeptidase M3 family，PepFBA的酵素性質與這個家族的其他oligopeptidases相似，即僅能分解分子量小的胜肽鏈 (介於含5~21胺基酸)，對於完整的蛋白無法作用。PepFBA在分解受質時，C端至少要有3個胺基酸，而受質的P1位置的胺基酸種類非常多變，幾乎可以是任何胺基酸。PepFBA在菌體所扮演的角色，可能是和訊息傳導分子 ”Phr” 的生成有關。Phr是含5個胺基酸的胜肽鏈，是調控Bacillus subtilis 產孢及competence的訊息傳導分子。但另外，PepFBA也可以促進膠原蛋白胜肽的分解，不排除它亦參與在胞外胜肽鏈的分解，以利菌體吸收利用。

另外在B. amyloliquefaciens 23-7A外泌的8個酵素中，只有C1及G1酵素可以分解膠原蛋白。C1酵素是分子量40 kDa的metallopeptidase，活性會受到EDTA及1,10-phenanthroline抑制。而G1酵素則是45 kDa的serine protease，活性受到PMSF的抑制。透過脂類受質的分析，可以了解C1酵素在P1位置的胺基酸性偏好Asp，G1酵素是Leu。依據蛋白序列分析，C1與G1酵素分別與B. subtilis pectate lyase及bacillopeptidase F相似。如同PepFBA，這兩個酵素在醱酵的初期就開始分泌，推測它們主要是參與在蛋白質的分解，以提供菌體生長的營養需求。

A collagenase-producing strain was screened from soil and identified as Bacillus amyloliquefaciens 23-7A. Eight different secreted proteases were purified and classified as PepFBA, C group (C1, C2A, and C2B), and G group (G1, G2, G3, and G4) proteases according to their abilities to hydrolyze FALGPA (2-furylacryloyl-Leu-Gly-Pro-Ala), Casein, and Gelatin. From the N-terminal and internal peptide sequences of PepFBA, a pair of degenerate primer were designed for PCR operation by using genomic DNA as template. The amplified PCR product was used as a probe to get the full length of PepFBA gene from genomic library. The PepFBA gene was 2,013 bp long and could be translated as a pre-pro protein containing 670 amino acid residues. This pre-pro protein would carry two processings to remove a 23-residue long signal peptide while was transported outward and a 7-residue long pro-sequence extracellularly. After processing, the molecular weight of PepFBA was 73,647 Da that was close to the mass detected by SDS-PAGE (69,000). A zinc-binding motif, His-Glu-X-X-His, could be found in the protein sequence and this matched the characteristic that PepFBA was inhibited by metal-chelating agents. PepFBA had 85% and 87% identity in protein sequence with Bacillus licheniformis Pz-peptidase and Bacillus subtilis PepF which both belong to oligopeptidase M3 family. The enzymatic properties of PepFBA had typical enzymatic characteristics of this family that could only digest oligopeptides. PepFBA could hydrolyze the substrates that comprise 5 and 21 residues with a rather wide specificity and at least 3 residues were needed in the C-terminal part. PepFBA may involve in active Phr formation, the pentapeptides that are regarded as signal molecules to regulate the sporulation and competence development of B. subtilis. However, because of the ability to accelerate collagen peptides degradation, it shouldn’t be ruled out that PepFBA played a role in hydrolyzing peptides into shorter form that could be utilized by cell.

Among the 8 purified proteases, only C1 and G1 proteases could hydrolyze intact collagen. C1 protease was a metallopeptidase with molecular weight 40 kDa. EDTA and 1,10-phenanthroline would inhibit its activity. As for the G1 protease, it was a serine protease with molecular weight of 45 kDa. From the analyzed results of ester substrates, C1 protease preferred Asp in position P1 of the cleaved bond and G1 protease was Leu. They, C1 and G1 protease, both had the similarities in peptide sequences with B. subtilis pectate lyase and bacillopeptidase F, respectively. It is speculated that these two proteases might hydrolyze proteins for nutrient requirement. However, further experiments are needed.

縮寫 ………………………………………………………………I
中文摘要 …………………………………………………………II-III
英文摘要 …………………………………………………………IV-V
壹、前言 …………………………………………………………1-16
1. 蛋白酶簡介 ………………………………………………1-3
2. Oligopeptidase M3 family ……………………………3-9
3. Bacillus subtilis的訊息傳遞系統 ……………………10-13
4. 微生物之膠原蛋白酶 (bacterial collagenase) ……14-16
貳、研究目的 ………………………………………………… 17-18
參、材料與方法 ……………………………………………… 19-36
1. 菌株來源 …………………………………………………19
2. 菌株培養 …………………………………………………19
3. 生長曲線分析--菌數與孢子的計量 ……………………19
4. 酵素純化 …………………………………………………20-23
5. 酵素活性分析 ……………………………………………23-27
6. PepFBA酵素內鋅離子含量分析 ………………………… 27
7. 蛋白質含量的測定 ………………………………………28
8. SDS-PAGE分析 ……………………………………………28
9. 蛋白定序 …………………………………………………29-30
10. 蛋白質分子量的測定 ………………………………… 30
11. DNA萃取 ……………………………………………… 30-31
12. 染色體基因庫的建立 ………………………………… 31-36
18. PepFBA促進膠原蛋白胜肽的分解 …………………… 36
肆、結果 ……………………………………………………… 37-50
1. 各酵素的生產與生長曲線 …………………………… 37
2. 各酵素的純化 ………………………………………… 38-39
3. PepFBA、C1與G酵素的基本性質 ……………………… 39-42
4. 各酵素之蛋白定序 …………………………………… 42-44
5. 基因庫的構築：PepFBA酵素基因選殖與比對 ……… 44-46
6. PepFBA的切位分析 …………………………………… 46-49
7. PepFBA鋅離子含量 …………………………………… 49
8. PepFBA促進膠原蛋白胜肽的分解 …………………… 49-50
伍、討論 …………………………………………………… 51-61
1. B. amyloliquefaciens 23-7A的蛋白酶生產 ……… 51-52
2. PepFBA蛋白序列比對 ……………………………… 52-53
3. PepFBA之切位分析與比較 …………………………… 53-55
4. PepFBA的鋅含量及鋅離子的活性抑制 ……………… 55-56
5. PepFBA可能的生理功能 ……………………………… 56-59
6. G1酵素與B. subtilis bacillopeptidase F ……… 59-60
7. C1酵素與B. subtilis pectate lyase (Q34819) … 60-61
8. 未來方向 ……………………………………………… 61
陸、參考文獻 ……………………………………………… 62-72
圖表 ………………………………………………………… 73-107

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