CN101633898B - 高温地衣芽孢杆菌及其产高温淀粉酶 - Google Patents
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Abstract
本发明公开了产生高温淀粉酶的地衣芽孢杆菌,和利用此菌株得到高温酸性淀粉酶。该菌株能在38-70℃下生长,最适生长温度为55℃。对高温酸性淀粉酶水解可溶性淀粉产物进行了TLC分析,完全水解可溶性淀粉终产物有麦芽糖、麦芽三糖和一些寡糖,表明粗酶液中含有淀粉酶。同时对其粗酶液进行了Native-PAGE分析和基本酶学性质分析,显示粗酶液中含有两种组分的淀粉酶,酶的最适左右温度为65℃,80℃下反应30min仍有85%以上的酶活,最适作用pH为4.5-5.5。本发明得到的淀粉酶作用温度高、作用pH低、在淀粉工业中的液化和糖化领域蕴藏一定的应用潜力。
Description
技术领域
本发明属应用微生物领域。具体地,涉及地衣芽孢杆菌(Bacilluslicheniformis)Tamy6菌株,以及从中获得的高温酸性淀粉酶。
背景技术
高温酶由于能更适合苛刻的工业生产过程,所以在工业和生物技术领域得到广泛的应用。
淀粉酶是目前应用最多的一种酶,它在淀粉工业中具有最广泛的应用,能水解和消化淀粉。淀粉酶(E.C.3.2.1.2)是一种外切型淀粉酶,它作用于淀粉时从非还原性末端依次切开α-1,4键,生成麦芽糖,与此同时将C1的构型由α-型转变成β-型。淀粉酶不能水解支链淀粉的淀粉酶是目前应用最多的一种酶,它在淀粉工业中具有最广泛的应用,能水解和消化淀粉。淀粉酶(E.C.3.2.1.2)是一种外切型淀粉酶,它作用于淀粉时从非还原性末端依次切开α-1,4键,生成麦芽糖,与此同时将C1的构型由α-型转变成β-型。淀粉酶不能水解支链淀粉的α-l,6键,也不能跨过分支点α-1,6键而切开内部的α-1,4键,故水解支链淀粉是完全的。
目前淀粉加工过程不仅需要耐高温的淀粉酶,而且需要将pH值调到5.8-6.5来进行液化,然后再将它降为4.2-4.5进行糖化。这两步pH值的调节增大了化学试剂的消耗,同时也需要在最后的终产物处理中利用离子交换将多余的盐分除去。如果能够有一种能在低pH值下作用的淀粉酶将降低这些消耗,简化过程,减少副产物的产生。因此筛选出能耐高温,且最适催化pH在5左右的淀粉酶的菌株,其产生的淀粉酶能较好地符合工业要求,对生产有一定的参考价值,值得做进一步的研究。
发明内容
本发明旨在提供一种能在高温下产生具有较高酶活性的高温酸性淀粉酶菌株。
本发明的另一目的在于提供一种从地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株中获得的高温酸性淀粉酶。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株,菌种保藏号为CGMCCNo.2642,其具有产生高温淀粉酶的能力。
所述的地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株,其菌落为白色,表面干燥,边缘圆整;细孢为短杆状,革兰氏阳性菌;温度范围是38-70℃,最适生长温度为55℃。
本发明同时提供从地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株中获得的高温酸性淀粉酶。
从地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株中获得的高温酸性淀粉酶的酶学性质为:最适反应温度为65℃,在80℃下反应30min仍有85%以上的酶活,在98℃下,有40%的酶活,最适pH为4.5-5.5,在pH为9时也有较高的活性。
从地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株中获得的高温酸性淀粉酶,Native-PAGE结果显示粗酶液中含有两种成分的淀粉酶。
本发明突变体地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株已经于2008年8月26日在北京中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为:CGMCC No.2642。
上述方案中的地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株,分离自云南省腾冲热海。菌株筛选固体培养基为(g/L):(NH4)2SO4,1.3;蛋白胨,1;可溶性淀粉,10;KH2PO4,0.276;MgSO4·7H2O,0.25;酵母提取物,1;Na2MoO4·2H2O,0.000025;CuSO4,0.000016;MnSO4·H2O,0.0022;H3BO3,0.0005;ZnSO4·7H2O,0.0005;CoCl2·6H2O,0.000046;CaSO4·H2O,0.06;FeSO4,0.099;琼脂,20;pH7.5。
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株,菌落为白色,表面干燥,边缘圆整;细孢为短杆状,革兰氏阳性菌;温度范围是38-70℃,最适生长温 度为55℃。
经形态学和生理生化特征鉴定,结合16S rRNA基因序列分析,将分离的高温细菌鉴定为地衣芽孢杆菌种的一个菌株,命名为Bacillus licheniformis sp.Tamy6。
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株所产高温酸性淀粉酶水解可溶性淀粉的产物TLC结果分析如下:
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株所产的高温酶水解可溶性淀粉的产物中不含有葡萄糖,水解的最终产物中含有麦芽糖,麦芽三糖和一些寡糖,说明地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株所产为高温淀粉酶,完全水解可溶性淀粉终产物有麦芽糖、麦芽三糖和一些寡糖。
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株所产高温酶Native-PAGE分析结果如下:
在Native-PAGE胶上能看到两条带,说明地衣芽孢杆菌(Bacilluslicheniformis)Tamy6菌株的发酵液中含有两种组分的高温淀粉酶。
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株所产高温酸性淀粉酶最适温度为65℃,在80℃下仍有85%以上的酶活,在98℃下,大约还剩下40%左右的酶活。最适pH为5左右,在pH为9时也有较高的活性。
附图说明
图1为Tamy6菌株产生的高温酸性淀粉酶水解可溶性淀粉的产物TLC结果,其中1:Marker(从上至下:葡萄糖、麦芽糖、麦芽三糖);2:菌株Tamy6粗酶液消化的可溶性淀粉;4:未消化的可溶性淀粉;
图2为Tamy6菌株产生的高温酸性淀粉酶Native-PAGE结果,其中1:Tamy6菌株所产粗酶液考马斯R250染色结果;2:Tamy6菌株所产粗酶液淀粉活性染色结果;
图3为Tamy6菌株产生的高温酸性淀粉酶最适催化温度曲线;
图4为Tamy6菌株产生的高温酸性淀粉酶最适催化pH曲线;
具体实施方式
下面结合本发明的实施例来进一步说明本发明的实质性内容,但本发明的内容并不局限于此。
实施例1:
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株的分离筛选及鉴定。
地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株,分离自云南省腾冲热海。腾冲热海的泥土在菌株筛选液体培养基中55℃,短时间摇瓶培养,涂布在菌株筛选固体培养基上,55℃培养1~2天,挑出菌株点种于菌株筛选固体培养基上,55℃培养3~4天后碘液着色,观察透明圈大小,筛选出产淀粉酶的Tamy6菌株。菌株筛选液体培养基为(g/L):(NH4)2SO4,1.3;蛋白胨,1;KH2PO4,0.276;MgSO4·7H2O,0.25;酵母提取物,1;Na2MoO4·2H2O,0.000025;CuSO4,0.000016;MnSO4·H2O,0.0022;H3BO3,0.0005;ZnSO4·7H2O,0.0005;CoCl2·6H2O,0.000046;CaSO4·H2O,0.06;FeSO4,0.099;pH7.5。菌株筛选固体培养基为(g/L):(NH4)2SO4,1.3;蛋白胨,1;可溶性淀粉,10;KH2PO4,0.276;MgSO4·7H2O,0.25;酵母提取物,1;Na2MoO4·2H2O,0.000025;CuSO4,0.000016;MnSO4·H2O,0.0022;H3BO3,0.0005;ZnSO4·7H2O,0.0005;CoCl2·6H2O,0.000046;CaSO4·H2O,0.06;FeSO4,0.099;琼脂,20;pH7.5。
采用细菌系统鉴定方法对地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株进行鉴定。该菌的形态学特征如下:菌落为白色,表面干燥,边缘圆整;细孢为短杆状,革兰氏阳性菌;温度范围是38-70℃,最适生长温度为55℃。
经形态学和生理生化特征鉴定,结合16S rRNA基因序列分析,经形态学和生理生化特征鉴定,结合16S rRNA基因序列分析,将分离的高温细菌鉴定为地衣芽孢 杆菌种的一个菌株,命名为Bacillus licheniformis sp.Tamy6。
实施例2:
将实施例1的地衣芽孢杆菌(Bacillus licheniformis sp.)Tamy6菌株接种于50ml菌株筛选液体培养基中,55℃摇床培养24h。按1∶10的比例接种至500ml菌株筛选液体培养基中,55℃摇床培养4天。发酵液于4℃,6500rpm离心10min,收集发酵上清液,再用滤纸抽滤两次。用10,000MWCO膜进行超滤浓缩至50ml,4℃保藏浓缩液。
实施例3:
实施例2浓缩液对可溶性淀粉的水解程度:
(1)将硅胶G板115℃烘箱活化30min
(2)点样:取硅胶薄板,在距底边1.5cm水平线上间隔均匀的点样,其中分别点上Marker(含有葡萄糖、麦芽糖和麦芽三糖)、经过16h水解1%可溶性淀粉的样品消化液,点样点用吹风机吹干。
(3)展层:层析缸内倒入深度为1cm左右的展层剂(正丁醇、冰乙酸、水,5∶3∶2,V/V/V),将薄层板放入层析缸进行展层,当展层剂前沿离薄板顶端1-2cm时,将薄板取出,凉干,进行二次展层。二次展层后将薄板放入65℃烘箱30min。
(4)显色:喷洒显色剂(4g二苯胺,4ml苯胺及20ml85%磷酸溶于200ml丙酮),然后于85℃烘箱中烘烤10-20min。
结果如图1所示,表明Tamy6菌株产生淀粉酶。
实施例4:
实施例2浓缩液的Native-PAGE:
Native-PAGE垂直凝胶电泳:按Woo Jin Lim法进行(Woo Jin Lim,2003)。
在凝胶上进行两份相同样品(浓缩发酵液进行0.22μm滤膜过滤除菌)的电泳,电泳后将凝胶切成两半,一半用于淀粉酶的活性染色,另一半样品用于考马斯 亮蓝染色。
①Native-PAGE:不连续电泳中,分离胶浓度为12%(分离胶中加入终浓度为0.1%的淀粉溶液),浓缩胶浓度为5%。电泳条件:加样15μl后,电压调为130V,在4℃下进行,电泳时间为3h。
②电泳结束后,将一半凝胶浸泡在30%Tris-Tcl缓冲液中,55℃温育6h,用碘液染色。根据碘-淀粉显色的性质,被淀粉酶水解后的周围会有明显的水解圈产生。
③凝胶染色、脱色:将另一半凝胶进行染色、脱色。其步骤为:
a.凝胶置于塑料盒中,加入考马斯亮蓝染色液(浸没凝胶),在脱色摇床上染色30min。
b.倒去考马斯亮蓝染液,用去离子水漂洗凝胶,然后加入100ml考马斯亮蓝脱色液,在脱色摇床上染色30min,倒去脱色液,重新加入新的脱色液,脱色过液。
c.当凝胶上的蛋白条带清晰,基本无底色时,停止脱色,倒去脱色液,用去离子水漂洗凝胶。
实施例5:
实施例2浓缩液特性:
实施例2浓缩液中淀粉酶活性的测定:酶活力测定方法为DNS测还原糖的方法进行淀粉酶活性测定(Rick W,1974)。高温淀粉酶的水解活力单位定义为:在一定温度下,每分钟水解可溶性淀粉产生1μg葡萄糖所需的酶量。
蛋白质浓度的测定:采用LOWRY法进行测定(LOWRY.OH,1951)。
①实施例2浓缩液的最适酶活温度:
将酶液加到磷酸盐缓冲液(50mM磷酸盐缓冲液,pH 7.2)中,在底物浓度为1%可溶性淀粉的条件下,测定Tamy6菌株所产淀粉酶在13-98℃范围内不同温度下的酶活。结果如图3所示,表明该高温淀粉酶的最适酶活温度为65℃。
②实施例2浓缩液的最适酶活pH:
用pH3.0至pH10.0的不同的缓冲液稀释酶液,分别加入相应pH的1%的可溶性淀粉,在结果如图4所示,表明Tamy6菌株所产的高温酸性淀粉酶在pH5.0时酶活最高。
Claims (4)
1.地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株,菌种保藏号为CGMCC No.2642,其具有产生高温淀粉酶的能力;该菌株的形态学特征是:菌落为白色,表面干燥,边缘圆整;细孢为短杆状,革兰氏阳性菌;温度范围是38-70℃,最适生长温度为55℃。
2.一种从权利要求1地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株中获得的高温酸性淀粉酶,其特征在于制备方法如下:
将地衣芽孢杆菌(Bacillus licheniformis sp.)Tamy6菌株接种于50ml菌株筛选液体培养基中,55℃摇床培养24h;按1∶10的比例接种至500ml菌株筛选液体培养基中,55℃摇床培养4天;发酵液于4℃,6500rpm离心10min,收集发酵上清液,再用滤纸抽滤两次;用10,000MWCO膜进行超滤浓缩至50ml,4℃保藏浓缩液;
所述菌株筛选液体培养基为(g/L):(NH4)2SO4,1.3;蛋白胨,1;KH2PO4,0.276;MgSO4·7H2O,0.25;酵母提取物,1;Na2MoO4·2H2O,0.000025;CuSO4,0.000016;MnSO4·H2O,0.0022;H3BO3,0.0005;ZnSO4·7H2O,0.0005;CoCl2·6H2O,0.000046;CaSO4·H2O,0.06;FeSO4,0.099;pH7.5。
3.根据权利要求2的高温酸性淀粉酶,其特征在于酶学性质为:最适反应温度为65℃,在80℃下反应30min仍有85%以上的酶活,在98℃下,有40%的酶活,最适pH为4.5-5.5,在pH为9时也有活性。
4.根据权利要求2所述的高温酸性淀粉酶,其特征在于Native-PAGE结果显示粗酶液中含有两种成分的淀粉酶。
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