具脂肪分解酵素活性之耐高溫微生物

Abstract

脂肪酶在現今酵素工業上的應用越來越廣泛，其應用範圍包括再生能源、紡織工業、皮革工業、清潔劑工業、油脂化學工業、造紙工業、飼料業、製藥工業、環保工業、食品工業等，由於酵素在催化反應時容易受到溫度和反應環境的酸鹼值影響其活性，藉由耐高溫微生物所產生的酵素於催化反應時往往具有特定溫度和pH値的容忍範圍，因此具有相當高的工業價值，近年來也逐漸取代嗜溫酵素的地位。堆肥在堆肥化（發酵）的過程中溫度通常會上升至 70oC 以上，故可藉由從生物肥料中分離出耐高溫微生物，用以產生各種耐高溫酵素。 本研究，於宜蘭五結垃圾場和台中市環保局廚餘堆肥樣品中共分離出 23 株具脂肪酶活性菌株， 23 株菌株中經過三油酸甘油酯的洋菜培養基於 50℃ 培養 5 天後，發現有 2 株菌的脂肪酶活性較強，分別為分離株 B61 和 M63；以本實驗室先前自台大女九餐廳蔬果廚餘堆肥中所篩選出的耐高溫脂解菌 Brevibacillus borstelensis SH168作為本實驗對照菌株，將分離株 B61、M63 及 Brevibacillus borstelensis SH168 3 株菌株於含有三酪酸甘油酯液態培養基培養並測定其脂肪酶活性，發現分離菌株之活性皆比 Brevibacillus borstelensis SH168 高出 2~3 倍，認為分離株 B61、M63 適於產生脂肪酶的菌株，分離株 B61 在 pH 5~9 皆可生長，生長溫度介於 20 oC ~ 60oC；分離株 M63 在 pH 6~10 可生長，生長溫度介於 30 oC ~ 50oC； 2 株分離菌株以 16S rRNA定序結果分離株 B61 為 Bacillus circulans，分離株 M63 為 Bacillus 屬。

The applications of lipase are more and more extensively, include bioenergy, textile industry, leather industry, detergents, oil chemical industry, pulp and paper industry, food industry, biomedical applications, biosensors, pesticides, cosmetics and perfume industry, environmental management etc. The enzyme activity is easily affected by temperature and pH. The enzymes of thermo-tolerant microbes have the better tolerance of temperature and pH, so the enzymes of thermo-tolerant microbes are considerably worth for all kinds of industry, recently the enzymes of thermo-tolerant microbes take the place of the enzyme of mesophilic microbes. When the compost is composting, the temperature will rise above 70oC. So the thermo-tolerant microbes can be isolated from the compost. In this study, isolate 23 microbes with lipase activity from Wu-Jie scrap heap at Ilan and Taichung Environmental Protection Section. When the microbes incubator in 50oC, there are isolates B61 and M63 that lipase activity are better than others. Brevibacillus borstelensis SH168 isolated from NTU resterant food waste compost is used as control check. Isolates B61, M63 and Brevibacillus borstelensis SH168 determine the lipase activity by the LB with tributyrin. The lipase activity of isolates B61 and M63 are 2~3 fold higher than Brevibacillus borstelensis SH168. So isolates B61 and M63 are more useful in producing lipase. Isolate B61 can grow at pH 5~9, and at temperature 20oC ~ 60oC; isolate M63 can grow at pH 6~10, and at temperature 30oC ~ 50oC. Isolates B61 and M63 were identified by 16S rRNA. Isolate B61 is Bacillus circulans, and isolate M63 is Bacillus species.