利用土壤多源基因庫篩選新穎酵素之研究

Abstract

土壤中之微生物具有高度多樣性以及含有許多未知待開發之產物，因此，利用建構好之土壤多源基因庫來開發新型酵素、化合物、抗生素或是菌種，已經是今日的趨勢。本篇研究主要擬利用本研究室已建構好之土壤多源基因庫篩選目標產物，利用功能性篩選以及基因序列篩選方法進行。本篇研究第一部分為檢驗前人利用聚合酶連鎖反應方式，由土壤多源基因庫篩選出來的ORF4基因序列，疑似具有N-醯化胺基酸消旋酶，經過次選殖及酵素呈色法試驗，結果，證實此疑似具有N-醯化胺基酸消旋酶活性之基因片段無法表現該酵素活性。第二部份為利用前人建構的土壤多源基因庫，以pH 8.5的脫脂牛乳瓊脂培養基進行功能性篩選鹼性蛋白酶，同時亦利用DNA雜交方法篩選蛋白酶。經DNA雜交實驗及培養基篩選的結果並未能成功篩選出具有鹼性蛋白酶活性之菌株。將這批土壤多源基因庫抽取其BAC質體經限制酶截切後，以電泳分析發現此土壤多源基因庫具有高歧異度，仍可供日後篩選其他目標產物之用。

Since environmental microorganisms holds a great of diversity and are available for developing multiple unknown products, environmental metagenomes can be also applied in different fields, such as medical and chemical industries nowadays. Metagenome is thought as a powerful tool to exploit chemical molecules, antibiotics, enzymes, and bacterial species. Screening of soil-based libraries can be based either on metabolic activity (function-based approach) or on nucleotide sequence (sequence-based approach). There are two sections in this study. First part, a putative N-acylamino acid racemase naaar gene, orf4 gene are amplified by PCR and cloned into E. coli BL21(DE3). Analysis of the cell extracts from this clone, the NAAAR activity was unable observed. Second part, the alkaline serine protease was screened by using this soil metagenome library. Both of function-based screening, that was assayed on pH 8.5 skim milk plates, and DNA-DNA hybridization. It didn’t succeed to screen out bacteria with alkaline serine protease through function-based and sequence-based screenings. After extraction and digestion of plasmids from the soil metagenome library, it was found that this metagenome library provides a great of diversity for screening other target products in the future.