植酸酶高产菌株的诱变选育和融合筛选

【作者】 彭益强；

【导师】 贺淹才；

【作者基本信息】 华侨大学 ， 化学工程， 2002， 硕士

【摘要】 本文以多轮紫外诱变为主线技术筛选植酸酶的黑曲霉高产菌，分析比较植酸酶酶活定义和植酸酶酶活测定方法并修正其测量波长，考查黑曲霉原生质体制备的影响因素，并在此基础上，用原生质体紫外复合诱变和原生质体融合技术筛选植酸酶的黑曲霉产生菌。 首先用粗略制备的原生质体经紫外诱变筛选到一株酶活为2250u／ml的实验出发菌；制备成菌悬液，紫外灯下照射诱变，红光下涂抹筛选平板，恒温培养2～3d；按透明圈大小进行预初筛，挑选径圈比大的菌落接斜面，恒温培养3d；按一株一瓶的方式进行初筛；从中选取酶活较大的3～5株，按一株2～3瓶的方式进行复筛；挑取酶活大且稳定的1～2株进入下一代诱变筛选。经过5代如此反复诱变选育，筛选到一株酶活稳定在12200～12800u／ml的黑曲霉菌株Asp-5-12，酶活力提高了5.7倍。 分析比较了目前文献上所见到的四种植酸酶酶活定义和四种植酸酶酶活测定方法，并修正了测量波长。确定本实验用的植酸酶酶活定义为：55℃，pH4.6的条件下，每分钟从0.1％的植酸钠溶液中释放1nmol的无机磷为一个酶活单位（u）。确定本实验用的植酸酶酶活测定方法为丙酮法，测量波长修正为420nm。 影响黑曲霉菌株制备原生质体的因素主要有：菌丝体菌龄、酶浓度、酶组成、酶解温度、酶解时间、稳定剂和离子Ca²⁺等。分别采用单因子法和正交实验法考查，认为16～18hr菌龄的菌丝体，32℃,用含0.2％Ca²⁺的PBA高渗缓冲液配制的1％纤维素酶与1％蜗牛酶的混合酶酶解3hr，得到的原生质体数最多。平板再生时用蔗糖高渗液稀释涂抹，有利再生。 采用原生质体紫外复合诱变技术加大诱变力度。从中筛选出一株可能更适合于作动物饲料添加剂的植酸酶产生菌，与一般报道不同的是，它的第一酶活峰在pH2.6处，正好适于在单胃动物的胃酸环境（pH1.5～3.5）内作用。 本文还初步进行了用原生质体融合技术筛选高产植酸酶的黑曲霉菌株的实验研究。采用灭活原生质体融合技术，既简化了实验，又实现了遗传物质单向传递。实验结果，原生质体融合率为0.403％，可深入研究提高融合率的条件。更多还原

【Abstract】 High active phytase producing fungs-Aspergillus niger were selected by mutiple UV mutation, the definitions of phytase activity were analysised and the measure wavelength of the enzyme was modified, the factors that influence the preparation of protoplast were investigated. Base this, use protoplast-UV mutation and protoplast fusion to filter phytase produce Asp. niger.One Asp. niger with phytase activity about 2250 u/ml which selected by protoplast-UV mutation was used as original, prepared it into fungu-suspend-liquid, through UV mutation,daub on the filter-substract. pre-primary-selection was according to the lucency-circle, primary-selection was one fungus inoculate one flask to ferment, secondary-selection was using several high phytase activity fungus through one fungus inoculate 2-3 flasks to ferment. Then one or two high phytase activity fungus of the secondary selection were used in the next mutation cycle. Such mutation done for 5 circles, Asp-5-12, its phytase activity among 12200-12800 u/ml,improve 5.7 multpile from the original one, were selected out.The 4 definitions and 4 measure ways of phytase activity were analyzed, and their measure wavelength were also modified. In this study the definition is under the conditions of 55,pH4.6,from 0.1 % phytic acid sodium salt,release 1 nmol free phosphorus in one mintiue is one enzyme activity unit and the acetone method is the suitable measure way of the research was also decided. The measure wavelength was modified to 420 nm as preciseA protoplasting technique has been developed for the Aspergillus niger. The fungus age, enzyme system, osmotic stabilizer, Ca2+ concentration influenced on the formation of the protoplast have been confirmed.The result show that 16-18hr fermention, 32 1.0 % cellulase plus 1.0 % snailase confected by PBA(phosphate blend ammonium chloride) contant 0.2 % Ca2+, acted 3 hr is optimal for the protoplast production of Aspergillus. niger.To improve the mutation efficiency, protoplast irradiation by UV was done. Phy-19, which produce phytase that fit for the mono-gastric animal was sought out among these mutated fungus . Differ from others report, first enzyme activity apex of the enzyme located at pH2.6.Since the gastric-acid pH is among 1.5-3.5, the phytase that produced by Phy-19 maybe more fit for using as feed additive.The fusion fungus that produce phytase were also selected by protaplast-fusion methode. The fusion-experiment may be predigested before protaplast-fusion, it can simplify the fusion orperation,and enable the genetic substance single-direction tranfer.The fusion ratio is 0.403 %, This means the fusion was successd but more optimal condtions need further study.更多还原