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1、蛋白酶產(chǎn)生菌的篩選.鑒定及酶學(xué)性質(zhì)研究■農(nóng)學(xué)論文蛋白酶產(chǎn)生菌的篩選、鑒定及酶學(xué)性質(zhì)研究李嬋娟1,王婿2，楊潔2（1.華中農(nóng)業(yè)大學(xué)楚天學(xué)院，武漢430205;2.湖北省農(nóng)業(yè)科學(xué)院農(nóng)業(yè)質(zhì)量標(biāo)準(zhǔn)與檢測(cè)技術(shù)硏究所，武漢430064）摘要:采用干酪素平板透明圈法從垃圾附近的土壤中分離得到1株產(chǎn)蛋白酶的優(yōu)良菌株，通過(guò)測(cè)定其16SrDNA基因序列，鑒定該菌為銅綠假單胞菌(Pseudomonasaeruginosa^以酪蛋白為底物對(duì)該產(chǎn)蛋白酶粗酶液進(jìn)行了酶學(xué)性質(zhì)研究，結(jié)果表明，該酶最適反應(yīng)pH為8.5,為堿性蛋白酶；在pH6.0~9.0的緩沖液中于4°C放置12h后仍有90%以上的酶活
2、性，說(shuō)明該酶在中性和堿性條件下非常穩(wěn)定。該酶最適反應(yīng)溫度為55°C,在70°C和80°C處理30min后仍分別保有68%和45%的殘余酶活性，從而表明該酶具有較好的熱穩(wěn)走性。Mg2+和Fe2+對(duì)該酶活性幾乎沒(méi)有影響，K+和Mn2+對(duì)該酶活性有輕微的抑制作用zEDTA對(duì)該酶活性有明顯的抑制作用；Cu2+對(duì)該酶活性也有26%的抑制作用,只有Ca2+對(duì)該酶活性有10%左右的促進(jìn)作用。關(guān)鍵詞：蛋白酶產(chǎn)生；分離；鑒定；酶學(xué)性質(zhì)中圖分類號(hào):Q814.1文獻(xiàn)標(biāo)識(shí)碼:A文章編號(hào)：0439?8114(2015)19?4794-04D01:10.14088/j.cnki.issn0439-
3、8114.2015.19.035Iso1ationandIdentificationofAProtease-ProducingBacteriaandCharacterizationofItsProteaseLIChan?juanl,WANGJing2,YANGJie2(1.ChutianCollege,HuazhongAgricu1tura1University,Wuhan430205zChina;2.InstituteofQualityStandardandTestingTechnologyforAgro?Products,HubeiAcademyofAgricult
4、uralSciences,Wuhan430064,China)Abstract:Abacteriumproducinghighproteasewasisolatedfromsoilaroundthedumpbymethodofcaseinplate.ThestrainwasidentifiedasPseudomonasaeruginosabymeasuringits16SrDNAsequence.Thecharacteristicsofthecrudeproteaseproducedbythisstrainwerestudiedbyusingcaseinasthesub
5、strate.TheresultsshowedthatthecrudeproteasedisplayedthemaximumactivityatpH8.5,therefore,thisenzymewasanalkalicprotease.Thecrudeenzymestillhad90%ofthehighestactivityafterovernightincubationat4°C,pH6.0~9.0,indicatingthattheenzymewasverystableunderneutra1oralkalineconditions.Theoptimumtempe
6、ratureoftheenzymewas55°C.Afterincubationat70°Cand80°Cfor30min,itremained68%and45%ofthehighestactivity,respectively,sotheproteasewasconsideredtohaveasuperiorthermalstability.Theactivityoftheproteasemaybedependentondivalentmetalions,becauseitwasnotaffectedbyMg2+orFe2+andwasslightlyinhibite
7、dbyK+andMn2+,butwasobviouslyinhibitedbyEDTA,strong1yinhibitedbyCu2+,reducingby26%ofitsactivity,andonlyCa2+cou1dincreasedtheactivityby10%.Keywords:protease-producingbacteria;isolation;identification;enzymaticproperty收稿日期：2015?06?15基金項(xiàng)目：湖北省農(nóng)業(yè)科技創(chuàng)新中心資助項(xiàng)目（2014?620-000?001）作者簡(jiǎn)介
