膠原蛋白酶產(chǎn)生菌的篩選及酶的分離純化

劉麗莉，馬美湖，余秀芳，王文濤

1 華中農(nóng)業(yè)大學(xué)食品科技學(xué)院，武漢 430070 2 河南科技大學(xué)食品與生物工程學(xué)院，洛陽 471003

膠原蛋白酶產(chǎn)生菌的篩選及酶的分離純化

劉麗莉1,2，馬美湖1，余秀芳1，王文濤1

1 華中農(nóng)業(yè)大學(xué)食品科技學(xué)院，武漢 430070 2 河南科技大學(xué)食品與生物工程學(xué)院，洛陽 471003

從堆積骨骼的土樣中篩選出高產(chǎn)膠原蛋白酶的MBL13菌株，經(jīng)鑒定為蠟樣芽孢桿菌Bacillus cereus。對其所產(chǎn)的膠原蛋白酶BCC進(jìn)行分離純化，并進(jìn)行酶學(xué)性質(zhì)的研究。從菌株的發(fā)酵液中純化出分子量約為38.0 kDa BCC酶。酶反應(yīng)的最適溫度為40℃，最適pH為8.0。在50℃以下穩(wěn)定，60℃保溫1 h酶活僅保留10%；在pH 7.0～8.5活性較穩(wěn)定；金屬離子Ca2+、Zn2+、Mg2+對酶有激活作用，金屬離子Cu2+對酶有顯著的抑制作用。EDTA和EGTA能抑制該酶，表明BCC酶為一種金屬蛋白酶。酶的底物特異性表明該酶為骨膠原蛋白酶，且對Ⅰ型骨膠原蛋白水解能力極顯著高于Ⅱ型膠原蛋白和Ⅲ型膠原蛋白。將純化的BCC酶應(yīng)用于骨膠原蛋白的水解可以得到不同鏈長的多肽，其水解能力高于標(biāo)準(zhǔn)品膠原酶Ⅰ型。本研究為工業(yè)酶提供了新的菌株和新型膠原蛋白酶，為膠原蛋白酶的開發(fā)提供了重要的理論依據(jù)。

膠原蛋白酶，篩選，純化，性質(zhì)

Abstract:We isolated the strain MBL13 with high collagenase productivity from the soil of piled up animal bones.It was identified as Bacillus cereus.We purified and characterized Bacillus cereus collagenase(BCC).The molecular weight of BCC was 38.0 kDa and the optimum temperature and pH for the enzyme activity were 40°C and 8.0 respectively.The enzyme was stable when the temperature was below 50°C, but only retained 10% activity when kept at 60°C for 1 h.The enzyme activity was stable between pH 7.0–8.5.Some metal ions such as Ca2+, Zn2+, Mg2+enhanced the enzyme activity, and Cu2+brought the obvious inhibition.In addition, EDTA and EGTA could inhibit the enzyme activity.We suggested that the purified enzyme was a member of the metalloproteases.Based on the experiment of substrate specificity, we found that the purified enzyme was bone collagenolytic protease, and had a much stronger capacity of hydrolysis for type I collagen than that for type II collagen and type III collagen.By BCC hydrolyzing bone collagen, we obtained polypeptides with different chain lengths.The comparative test indicated that the hydrolysis capacity of BBC was higher than that of standard type I collagenase.The results introduced a new strain and a novel collagenolytic protease for industrial enzyme.

Keywords:collagenase, screening, purification, property

隨著我國養(yǎng)殖業(yè)迅猛發(fā)展，屠宰所產(chǎn)生的副產(chǎn)物是不可忽視的。骨骼是豐富的蛋白質(zhì)資源，主要成份是膠原蛋白，骨膠原蛋白是膠原蛋白的一種。現(xiàn)今利用高新技術(shù)如酶技術(shù)對龐大的骨資源進(jìn)行深加工，開發(fā)新型的功能性骨膠原多肽具有十分重要的經(jīng)濟(jì)價值成為研究的熱點(diǎn)[1-3]。……