金龟子绿僵菌原生质体的制备和再生及其羟化酶活性研究

Isolation and regeneration of Metarhizium anisopliae protoplasts and their hydroxylase activity

对金龟子绿僵菌（Metarhizium anisopliae）原生质体的制备和再生的影响因素进行实验，并在此基础上考察了甾体底物对原生质体羟化酶的诱导作用。结果表明，原生质体制备的合适条件是：42h的茵丝体用纤维素酶（10mg／mL）和蜗牛酶（5mg／mL）的混合酶在含有0．8mol／L甘露醇的pH5．8磷酸缓冲液中，28℃震荡（80r／min）酶解3h，原生质体产量可达到6．12×10^7／mL，在含有0．6mol／LKCl的双层马铃薯培养基上再生率达到7．79％。经过6h底物诱导的菌丝体制备的原生质体细胞色素P450的表达量比没经过诱导的菌丝体制备的原生质体高约40％，证明该茵羟化酶系统的可诱导性。由于没有细胞壁的阻碍经过底物诱导的原生质体能够高效的将底物转化为产物，且副产物相对较少。

The conditions for protoplast isolation and regeneration from Metarhizium anisopliae as well as the inducement of steroid substrates on hydroxylase in protoplasts were investigated. The optimum conditions for protoplast isolation were also determined. Myeelia of Metarhizium anisopliae cultured for 42 h were digested with cellulase （10 mg/mL） and snailase （5 mg/mL） in phosphate buffer （pH 5.8） containing 0.8 mol/L mannitol as the osmotic stabilizer for 3 h at 28℃, and the yield of protoplasts was 6.12 × 10^7/mL. The optimum protoplast regeneration efficiency （7.79 % ） was obtained in two-layer 0.6 mol/L KCI containing PDA （Potato-Dextrose Agar） medium. The cytoehrome P450 content in protoplasts released from mycelia cultured in the presence of AD （Androst-4-ene-3,17-dione） was about 40 % higher than that in protoplasts released from mycelia without AD treatment, indicating that the steroid hydroxylase could be induced by steroid substrates, and they could hydroxylate steroid to its lla-hydroxylated products effectively with relatively less by-products in comparision with intact mycelia, due to a facilitated substrate aocess to the enzyme reaction site.