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題 名 | 豬體脂肪蓄積與調節 |
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作 者 | 劉昌宇; | 書刊名 | 現代養豬 |
卷 期 | 20:6=234 1998.12[民87.12] |
頁 次 | 頁39-43 |
分類號 | 437.654 |
關鍵詞 | 豬體; 脂肪蓄積; 調節; |
語 文 | 中文(Chinese) |
中文摘要 | 以B. macerans 之染色體DNA為模板,利用聚合�t鏈反應(PCR)合成含有 ribosome binding site、signal sequence之環狀糊精葡萄糖�羺臕鉦濂t( CGTase)基因 ,並將其選殖於一雙向載體--pHY300PLK,分別轉形至E.coli XL1-Blue及B. subtilis DB430,所得之質體稱為pHG。插入的基因片段無明顯的promoter區域,但pHG能分別在E. coli XL1-Blue及B.subtilis DB430中,表現CGTase蛋白質,因此推測pHY300PLK上有一段未 知的promoter區堿。培養B.subtilis(pHG)於不同的培養基中,發現在2YT培養基培養至24 小時,菌液會具有最高CGTase酵素活性表現。將粗酵素液以β-CD親合性層析管柱及DEAE Sepharose CL 6B離子交換層析管柱等步驟進行CGTase的純化分離。將純化所得的酵素進行N 端胺基酸序列分析,結果與文獻之B. macerans CGTase N端胺基酸序列相同,此外由酵素活 性分析之結果,顯示出B.subtilis 所生產之CGTase與B.macerans之CGTase具有相似的催化 活性。在此系統表現之E344D、E344Y兩定點突變CGTase其偶合反應及澱粉水解反應的活性較 wild type CGTase低,而前者對pH的耐受性亦有下降的趨勢且對澱粉分子之親和力亦有改變 。 |
英文摘要 | A 2.1 kb DNA fragment which encodes the cgt gene (with ribosome binding site and signal sequence)of B. macerans was synthesized by means of polymerase chain reactions (PCR) and cloned into a shuttle vector-pHY300PLK. There was no promoter sequence in the inserted cgt gene, but the recombination plasmid, pHG, could express CGTase in B. subtilis DB430. It was speculated that an unknown promoter sequence was located at the upstream of the cgt gene on pHG. A higher amount of CGTase was produced as B. subtilis carrying pHG was grown on 2YT medium than on 2xSG or LB medium. B. subtilis CGTase was purified through β-CD affinity chromatography and DEAE Sepharose CL 6B ion exchange chromatography. The N-terminal amino acid sequence of the CGTase expressed from B. subtilis was found to be consistent with that of the B. macerans CGTase. The B. subtilis CGTase also has enzyme activities similar to those of the B. macerans CGTase. The results revealed that B. subtilis harboring pHG produces the authentic CGTase of B. macerans. Two point-mutated cgt genes, E334D and E344Y, were also introduced onto this vector system. E334D and E344Y CGTase produced from transformated B. subtilis showed reduced starch digestion and coupling activities compared to the wild type enzyme. The relative enzymatic activity of E344D CGTase decreased in both acidic and alkaline pH ranges. These mutated enzymes also showed different affinity for starch molecules as revealed by electrophoresis. |
本系統中英文摘要資訊取自各篇刊載內容。