一株产植酸酶菌株的选育
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摘要
植酸酶的研究已经有近百年的历史，到目前为止，国内外对植酸酶的研究已经深入到了饲料、食品、医药等多个领域，并且都取得了一定成果。美国、芬兰、荷兰和德国等国家已经推出了多种植酸酶制剂；国内的一些学者在植酸酶的研究上也取得了一定成果，然而，在应用的过程中依然存在很多问题。因此，有必要进一步选育新的产植酸酶菌株，最终获得具有实际应用价值的生产菌株。本研究主要做了以下工作：
1．从6份土壤样品和1份酒糟样品中筛选出61株产植酸酶的菌株，挑选一株透明圈直径与菌落直径比值最大的菌株为出发菌株，对其进行形态学鉴定和18S rRNA基因序列分析，结果表明该菌株属于散囊菌目(Eurotiales)，发菌科(Trichocomaceae)，蓝状菌属(Tarlaromyces)的黄蓝状菌(Tarlaromyces flavus)，其18S rRNA基因与标准菌株相似性为100％。
2．对所选取的菌株进行产酶条件优化和酶学性质研究。最适培养基组成为：葡萄糖6％；胰蛋白胨0.2％；(NH_4)_2SO_4 0.5％；KCl 0.05％；MgSO_4·7H_2O 0.05％；FeSO_4·7H_2O 0.004％；MnSO_4·H_2O 0.004％。最适发酵产酶条件为：培养温度32℃；发酵培养基最适pH5.5；装液量50ml(500ml锥形瓶)：接种量2％；摇床转速80r／min；培养时间8d。对酶学性质进行研究的结果为：最适反应温度55℃；最适反应pH5.5；最适反应时间10min。最终酶活达到294U／ml。
The research of phytase has been last for one hundred years. So far, it has beensuccessfully applied in many fields, such as the feedstuff, the grocery and themedicine. The America, Finland, Holland and Germany et al have been recommendedmany phytase preparations. Domestic scholars also have some achievements onphytase. But there are also some problems on the applications. So, it's necessarily toselect new strains which produce phytase, and finally obtain phytase which havepractical value. The main work this study have done as follows:
1. Totally, 61 phytase-producing strains have been isolated from 6 soil samplesand 1 distillers'grains sample. Among them, one has the maximum diameter ratio ofits clear zone to its colony. This swain was identified as Tarlaromyces flavus accordingto its morphological properties and the analysis of its 18S rRNA gene sequence. Thesimilarity of its 18S rRNA gene and the standard strain is 100％.
2. Studied on the enzyme producing condition and enzymological properties. Theoptimum medium was composed of 6％glucose, 0.2％peptone, 0.5％(NH_4)_2SO_4,0.05％KCl, 0.05％MgSO_4·TH_2O, 0.004％FeSO_4·7H_2O, 0.004％MnSO_4·H_2O. Theoptimum producing condition was: cultivate temperature was 32℃; optimum pH ofthe fermentation medium was 5.5; medium quantity was 50mL(500mL conical flask);inoculation proportion was 2％; rotate speed of the shaker was 80r/min; fermentationtime was 8d. The result of study on enzymological properties was: optimum reactiontemperature was 55℃; optimum reaction pH was 5.5; optimum reaction time was10min; and the final enzyme activity was 294U/ml.
1．从6份土壤样品和1份酒糟样品中筛选出61株产植酸酶的菌株，挑选一株透明圈直径与菌落直径比值最大的菌株为出发菌株，对其进行形态学鉴定和18S rRNA基因序列分析，结果表明该菌株属于散囊菌目(Eurotiales)，发菌科(Trichocomaceae)，蓝状菌属(Tarlaromyces)的黄蓝状菌(Tarlaromyces flavus)，其18S rRNA基因与标准菌株相似性为100％。
2．对所选取的菌株进行产酶条件优化和酶学性质研究。最适培养基组成为：葡萄糖6％；胰蛋白胨0.2％；(NH_4)_2SO_4 0.5％；KCl 0.05％；MgSO_4·7H_2O 0.05％；FeSO_4·7H_2O 0.004％；MnSO_4·H_2O 0.004％。最适发酵产酶条件为：培养温度32℃；发酵培养基最适pH5.5；装液量50ml(500ml锥形瓶)：接种量2％；摇床转速80r／min；培养时间8d。对酶学性质进行研究的结果为：最适反应温度55℃；最适反应pH5.5；最适反应时间10min。最终酶活达到294U／ml。
The research of phytase has been last for one hundred years. So far, it has beensuccessfully applied in many fields, such as the feedstuff, the grocery and themedicine. The America, Finland, Holland and Germany et al have been recommendedmany phytase preparations. Domestic scholars also have some achievements onphytase. But there are also some problems on the applications. So, it's necessarily toselect new strains which produce phytase, and finally obtain phytase which havepractical value. The main work this study have done as follows:
1. Totally, 61 phytase-producing strains have been isolated from 6 soil samplesand 1 distillers'grains sample. Among them, one has the maximum diameter ratio ofits clear zone to its colony. This swain was identified as Tarlaromyces flavus accordingto its morphological properties and the analysis of its 18S rRNA gene sequence. Thesimilarity of its 18S rRNA gene and the standard strain is 100％.
2. Studied on the enzyme producing condition and enzymological properties. Theoptimum medium was composed of 6％glucose, 0.2％peptone, 0.5％(NH_4)_2SO_4,0.05％KCl, 0.05％MgSO_4·TH_2O, 0.004％FeSO_4·7H_2O, 0.004％MnSO_4·H_2O. Theoptimum producing condition was: cultivate temperature was 32℃; optimum pH ofthe fermentation medium was 5.5; medium quantity was 50mL(500mL conical flask);inoculation proportion was 2％; rotate speed of the shaker was 80r/min; fermentationtime was 8d. The result of study on enzymological properties was: optimum reactiontemperature was 55℃; optimum reaction pH was 5.5; optimum reaction time was10min; and the final enzyme activity was 294U/ml.
引文
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2．毕士峰，张毅．一种新的食品添加剂一植酸酶[J]．食品科学，2000，21(8)：9-10．
3．陈述，李多川，刘开启，等．黄蓝状菌一种几丁质酶的纯化、性质及抗菌活性[J]．中国森林病虫，2003，22(3)：6-9．
4．丁宏标，刘建平，李富伟，等．植酸酶的基因工程研究与工业化生产应用[J]．中国饲料，2003，21：28-31．
5．董生杰，张苓花，陈海昌等．产植酸酶菌株的筛选[J]．大连轻工业学院学报，1999，18(4)：283-287．
6．郭明元，刘广峰，冯娟．1株军曹鱼病原弧菌的鉴定及其系统发育树分析[J]．中国水产科学，2006，13(5)：823-828．
7．韩利刚，袁毅，王罡．丝状真菌组织DNA的提取[J]．生物技术，1999，9(6)：38-41．
8．韩延明，杨风，周安国，等．微生物植酸酶或麦麸对断奶到肥育阶段猪的生产性能和骨骼发育的影响[J]．畜牧兽医学报，1996，27(3)：207-211．
9．韩延明．猪饲料中利用植峻酶的研究进展[J]．四川农业大学学报，1994，12(4)：520-523．
10．黄培堂等译．分子克隆实验指南(第三版)[M]．北京：科学出版社，2002：463-486．
11．黄遵锡．基因工程酵母产植酸酶的应用性质研究[J]．饲料研究，2000，14(6)：11-14．
12．余雪冰．毕赤氏酵母植酸酶工程菌高密度培养条件的研究[J]．生物技术，2000，6(3)：26-29．
13．江均平．热稳定的曲霉植酸酶[J]．微生物学报．1996，36(6)：476-478．
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