云南松毛虫肠道中产脂肪酶菌的研究

采用微生物筛选培养和酶学方法,研究了云南松毛虫肠道内产脂肪酶菌群及产酶特性,为松毛虫的综合开发利用提供理论依据。结果表明,从云南松毛虫幼虫肠道内共分离筛选获得193株产脂肪酶的菌株,涉及3个菌属:82株假单胞菌属(Pseudomonas)、67株芽孢杆菌属(Bacillus)和44株克雷伯氏菌属(Klebsiella);其中,假单胞菌属为优势菌群,占总菌株数量的42.49%;供试的9株菌株表现出不同的产脂肪酶活性,并从中筛选得到了1株高产脂肪酶菌株P-50,最适作用温度为35℃,pH值为8,经发酵至60 h时生长量达到最大,在48 h左右时酶活性达到最高;P-50初步确定为中温产碱性脂肪酶菌。

不同生境产脂肪酶菌株的筛选及多样性

为发掘不同特性脂肪酶微生物资源,采用纯培养方法从青海高寒草地、雅安山区、成都平原分离得到产脂肪酶菌株54株.通过对菌株的DNA进行ERIC-PCR指纹图谱分析,按聚类树划分为5个操作分类单元.16S rDNA序列测定和聚类分析显示,分离菌株分布于芽孢杆菌属(Bacillus)、克雷伯氏菌属(Klebsiella)、假单胞菌属(Pseudomonas)、葡萄球菌属(Staphylococcus)、肠杆菌属(Enterobacter).多样性分析表明,与青海高寒草地、雅安山区相比,成都平原产脂肪酶菌株遗传多样性更为丰富.在54株菌中都出现了300 bp和1 300 bp两个特异且稳定出现的条带,这两个特异条带可能是产脂肪酶菌株的SCAR标记条带.

产脂肪酶重组菌SRI-01随机突变及诱导表达条件的研究

采用大引物全质粒法(MEGAWHOP)随机突变,快速有效地对研究室前期构建的一株脂肪酶重组菌株SRI-01进行分子改造,通过对易错聚合酶链反应(ep-PCR)获得的2 300个突变株进行96孔板高通量筛选,得到一株优良突变株SRM08。通过单因素试验,优化诱导前的菌体生物量、诱导剂异丙基硫代半乳糖苷(IPTG)的浓度、Ca Cl2的浓度、诱导温度。通过比较3种不同破碎缓冲液浓度和缓冲液p H对脂肪酶活力的影响,选择最优的破碎缓冲液。确定突变株SRM08最佳诱导表达条件为菌液生物量OD600 nm值为0.7,IPTG的浓度为0.1 mmol/L,Ca Cl2浓度为4 mmol/L,诱导温度为25℃,采用5 mmol/L磷酸钠缓冲液为破碎缓冲液,p H值为7.0。在此优化条件下,脂肪酶活达到88 508 U/L,为原重组菌株SRI-01的2.84倍。

Selection of Strains Producing Lipase with Transesterification Activity and Its Characterization

The interest for lipase production is due to the ability of this enzyme to catalyze some reactions, such as the transesterification. Although industrial biodiesel is produced chemically, there are several problems associated with this technology that can be prevented through the use of lipases. The present work aimed to select microorganisms with potential for production of lipase with transesterification activity. The lipase from Burkholderia cepacia was the one with the most promising results for this type of reaction, showing results of hydrolytic activity at 37 ℃and pH 8.0. The pH and volume of crude enzyme extract that showed favorable for synthesis ofbiodiesel is at about pH 6.0 and 3.75 mL, respectively, which represents approximately 42% of water in the system, ensuring the conversion of nearly 60% to biodiesel.