水产科学
水產科學
수산과학
FISHERIES SCIENCE
2009年
12期
763-766
,共4页
凡纳滨对虾%蛋白酶%纯化%生化特性
凡納濱對蝦%蛋白酶%純化%生化特性
범납빈대하%단백매%순화%생화특성
Litopenaeus vannamei%protease%purification%biochemical property
采用Tris-HCl缓冲液抽提、Sephadex G-100凝胶过滤层析、DEAE-Sepharose F.F.阴离子交换层析和SDS-PAGE等方法,分离和纯化了凡纳滨对虾蛋白酶,研究了其生化特性.试验结果表明,该蛋白酶粗提物经层析后,得到聚丙烯酰胺凝胶电泳纯的一酶组分.该蛋白酶的比活力从128.35 U/mg增至1835.65 U/mg,提高了14.3倍,产率达31.9%.SDS-PAGE显示该蛋白酶只含一条谱带,相对分子量为25 kD.以酪蛋白为底物,该酶的最适pH为7.0,最适温度为60 ℃, 在50 ℃以下,比较稳定,放置1 h后活性仍超过60%,而超过50 ℃时蛋白酶活性急剧下降,60 ℃放置1 h后,酶活性只残留4.7%.10 mmol/L EDTA、Fe~(2+)、Ba~(2+)和Zn~(2+)对该蛋白酶有较强的抑制作用,抑制率分别为84%、53%、43%和38%,10 mmol/L Mg~(2+)和Cu~(2+)对蛋白酶活性有轻微抑制作用,而10 mmol/L Ca2+能显著促进蛋白酶活性,据此推测凡纳滨对虾蛋白酶可能为一种金属蛋白酶.
採用Tris-HCl緩遲液抽提、Sephadex G-100凝膠過濾層析、DEAE-Sepharose F.F.陰離子交換層析和SDS-PAGE等方法,分離和純化瞭凡納濱對蝦蛋白酶,研究瞭其生化特性.試驗結果錶明,該蛋白酶粗提物經層析後,得到聚丙烯酰胺凝膠電泳純的一酶組分.該蛋白酶的比活力從128.35 U/mg增至1835.65 U/mg,提高瞭14.3倍,產率達31.9%.SDS-PAGE顯示該蛋白酶隻含一條譜帶,相對分子量為25 kD.以酪蛋白為底物,該酶的最適pH為7.0,最適溫度為60 ℃, 在50 ℃以下,比較穩定,放置1 h後活性仍超過60%,而超過50 ℃時蛋白酶活性急劇下降,60 ℃放置1 h後,酶活性隻殘留4.7%.10 mmol/L EDTA、Fe~(2+)、Ba~(2+)和Zn~(2+)對該蛋白酶有較彊的抑製作用,抑製率分彆為84%、53%、43%和38%,10 mmol/L Mg~(2+)和Cu~(2+)對蛋白酶活性有輕微抑製作用,而10 mmol/L Ca2+能顯著促進蛋白酶活性,據此推測凡納濱對蝦蛋白酶可能為一種金屬蛋白酶.
채용Tris-HCl완충액추제、Sephadex G-100응효과려층석、DEAE-Sepharose F.F.음리자교환층석화SDS-PAGE등방법,분리화순화료범납빈대하단백매,연구료기생화특성.시험결과표명,해단백매조제물경층석후,득도취병희선알응효전영순적일매조분.해단백매적비활력종128.35 U/mg증지1835.65 U/mg,제고료14.3배,산솔체31.9%.SDS-PAGE현시해단백매지함일조보대,상대분자량위25 kD.이락단백위저물,해매적최괄pH위7.0,최괄온도위60 ℃, 재50 ℃이하,비교은정,방치1 h후활성잉초과60%,이초과50 ℃시단백매활성급극하강,60 ℃방치1 h후,매활성지잔류4.7%.10 mmol/L EDTA、Fe~(2+)、Ba~(2+)화Zn~(2+)대해단백매유교강적억제작용,억제솔분별위84%、53%、43%화38%,10 mmol/L Mg~(2+)화Cu~(2+)대단백매활성유경미억제작용,이10 mmol/L Ca2+능현저촉진단백매활성,거차추측범납빈대하단백매가능위일충금속단백매.
The protease was extracted and purified from heads of white leg shrimp Litopenaeus vannamei by Tris-HCl buffer solution, gel-filtertion with Sephadex G-100, and ion-exchange with DEAE-Sepharose F.F. The enzymological nature was studied with biochemical and electrophoretic techniques. The enzyme was purified to an electrophoretically homogenous state, reaching 14.3-fold purification with a yield of 31.9%. The extracted protease showed a single band in its relative molecular weight at 25 kD based on SDS-PAGE.The optimum pH was 7.0 for the enzymatic reaction and the optimum temperature was 60 ℃ for casein as the substrate This protease showed a good thermal stability below 50 ℃, and more than 60% of the activity still remained when the protease was cultured at 50 ℃ for 1 h. But the enzyme became inactivated rapidly at the temperature above 60 ℃, and only 4.7% of protease activity remained at 60 ℃ for 1 h. The protease activity was found to be inhibited by 10 mmol/L of EDTA(at a rate of 84%), and Fe~(2+)(53%), Ba~(2+)(43%)and Zn~(2+)(38%). However, 10 mmol/L of Ca~(2+) increased the enzyme activity,while 10 mmol/L of Mg~(2+) and Cu~(2+) had less effect on the protease activity. It is suggested that the protease from the shrimp be a type of metalloenzyme.
