CN109679937A - 一种具有高比酶活力的生淀粉水解酶、其编码基因及其应用 - Google Patents
一种具有高比酶活力的生淀粉水解酶、其编码基因及其应用 Download PDFInfo
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- CN109679937A CN109679937A CN201910010979.3A CN201910010979A CN109679937A CN 109679937 A CN109679937 A CN 109679937A CN 201910010979 A CN201910010979 A CN 201910010979A CN 109679937 A CN109679937 A CN 109679937A
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Abstract
本发明公开了一种具有高比酶活力的生淀粉水解酶、其编码基因及其应用,其中生淀粉水解酶名称为AmyZ1,其氨基酸序列为如下序列中的一种:(1)序列表中的SEQ ID No:2;(2)序列表中的SEQ ID No:2经取代、缺失或添加一个或几个氨基残基且编码相同功能蛋白质的氨基酸序列。本发明生淀粉水解酶来自海洋细菌Pontibacillus sp.ZY,具有高比酶活力,其对生淀粉及可溶性淀粉均具有高比酶活;且可于较低温度条件下,高效水解20‑30%的高浓度生淀粉,具有潜在应用价值。
Description
技术领域
本发明涉及一种具有高比酶活力的生淀粉水解酶、其编码基因及其应用,属于生物技术领域。
背景技术
α-淀粉酶(α-amylase,EC 3.2.1.1)可作用于淀粉、多糖及寡糖内部的α-1,4-葡萄糖苷键,产生α-异头物构型保留的低聚麦芽糖和葡糖糖。α-淀粉酶在淀粉转换成低聚糖的过程中起着非常重要的作用,因此对于以淀粉作为基本能量来源的有机体来说是至关重要的。另外,α-淀粉酶是苷水解酶类中一类重要的工业用酶,其被广泛应用于食品加工、污水处理、制药工业、酿酒工业、新型生物能源应用方面。
淀粉是一种廉价的能源物质,已广泛用于工业生产中。传统的淀粉降解主要包括淀粉糊化和淀粉酶解两个步骤。天然的生淀粉颗粒不能被普通的淀粉降解酶直接降解,只有通过高温糊化破坏生淀粉颗粒的结构,促使生淀粉变为熟淀粉,淀粉长链才能被淀粉降解酶有效切断。酶促淀粉转化的传统过程需要高能量输入并导致淀粉基产品的成本增加。为了节省能量和成本,能够在较低温度下水解高浓度生淀粉的α-淀粉酶引起了越来越多的关注。在已发现的α-淀粉酶中,只有不足10%的酶具有降解生淀粉的能力。此类酶直接作用不经糊化的生淀粉,可有效的简化现代发酵工业中的生淀粉的前期处理过程。至今,已从Bacillus sp.,Aspergillus sp.,Geobacillus sp.和Rhizopus sp.等种属微生物中发现了具备生淀粉水解能力的α-淀粉酶,能有效的降解小麦、玉米、马铃薯等生淀粉,但水解生淀粉的反应多在60℃以上高温条件下进行,或者通过延长反应时间以提高水解率。本发明AmyZ1淀粉酶能够在30~35℃水解20~30%底物浓度的生淀粉,反应4h后,大米、玉米、小麦等的水解率可达到37%以上,十分高效节能,具有潜在应用价值。
发明内容
本发明是为避免上述现有技术所存在的不足之处,提供了一种具有高比酶活力的生淀粉水解酶、其编码基因及其应用。
本发明具有高比酶活力的生淀粉水解酶,名称为AmyZ1,为海洋细菌来源,其原始来源于中国南海永兴岛针叶藻海底沉积物样品中筛选的Pontibacillus sp.ZY菌株,其氨基酸序列为如下序列中的一种:
(1)序列表中的SEQ ID No:2;
(2)序列表中的SEQ ID No:2经取代、缺失或添加一个或几个氨基残基且编码相同功能蛋白质的氨基酸序列。
编码所述生淀粉水解酶的基因,其核苷酸序列为如下序列中的一种:
(1)序列表中的SEQ ID No:1;
(2)序列表中的SEQ ID No:1经取代、缺失或添加一个或几个核苷酸且编码相同功能蛋白质的核苷酸序列;
(3)编码序列表中SEQ ID No:2蛋白质序列的多核苷酸序列。
含有所述生淀粉水解酶基因并能产生所述生淀粉水解酶的菌株,名称为Escherichia coli BL21(DE3)/pET22b(+)-amyZ1,由中国典型培养物保藏中心CCTCC保藏,保藏编号CCTCC NO:M 2018893,保藏日期为2018年12月13日。
本发明生淀粉水解酶的重组表达菌株的构建方法,包括如下步骤:
以包含具有高比酶活力的生淀粉水解酶基因的Pontibacillus sp.ZY细菌基因组DNA为模板,以P1和P2为引物进行PCR扩增,得到PCR扩增产物,并与pEASY-T3质粒连接,将连接产物转化大肠杆菌Trans1-T1感受态细胞,筛选阳性克隆,进行序列分析;挑选序列正确的克隆提取质粒,获得含有生淀粉水解酶基因的pEASY-T3重组质粒,用Nde I和Xho I双酶切所得的pEASY-T3重组质粒和表达质粒载体,然后用T4DNA连接酶将酶切后的amyZ1与表达质粒载体连接,得到连接产物;将得到的连接产物转化宿主菌,筛选阳性克隆,即可得到含有本发明生淀粉水解酶基因的工程菌株。
所述引物为:
P1:5’-CATATGYTNGGNATNWSNTTYGTNYTN-3’
P2:5’-CTCGAGYTTYTGYTTRTANACNSWNACNSW-3’
所述表达质粒载体指pCold、pET15、pET22或pET28等。
所述宿主菌是指E.coli BL21(DE3)、E.coli DH5α、E.coli JM109或E.coliRosetta等。
下面以表达质粒载体pET22b(+)、E.coli BL21(DE3)为例,介绍含有本发明海洋细菌淀粉酶基因的重组表达菌株的构建方法,具体包括以下步骤:
1、以包含海洋细菌来源、具有高比酶活力的生淀粉水解酶基因的Pontibacillussp.ZY细菌基因组DNA为模板,以P1和P2为引物进行PCR扩增,得到PCR扩增产物,所述引物为:
P1:5’-CATATGYTNGGNATNWSNTTYGTNYTN-3’
P2:5’-CTCGAGYTTYTGYTTRTANACNSWNACNSW-3’
2、将步骤1所得PCR扩增产物和pEASY-T3质粒连接,得连接产物;将连接产物转化大肠杆菌Trans1-T1感受态细胞,筛选阳性克隆,进行序列分析;挑选序列正确的克隆提取质粒,获得含有本发明生淀粉水解酶基因的pEASY-T3重组质粒;
3、用Nde I和Xho I双酶切步骤2所得的pEASY-T3重组质粒和pET22b(+)质粒,然后用T4DNA连接酶将酶切后的amyZ1与pET22b(+)质粒连接,得到连接产物;连接产物转化大肠杆菌BL21(DE3)感受态细胞,筛选阳性克隆,得到含有本发明生淀粉水解酶基因的工程菌株Escherichia coli BL21(DE3)/pET22b(+)-amyZ1。
本发明生淀粉水解酶的应用,是用于生淀粉的水解,其对生淀粉及可溶性淀粉均具有高比酶活;且可于较低温度条件下,高效水解20-30%的高浓度生淀粉。
本发明生淀粉水解酶的应用,是在所述生淀粉水解酶的存在下对生淀粉进行水解。水解体系为pH7.0的磷酸盐缓冲液中加入终浓度为1mM的CaCl2,随后加入生淀粉,以及所述生淀粉水解酶,30-35℃、150rpm摇床水浴下进行水解反应。
水解温度优选为30℃,水解时间为2-4小时。
所述生淀粉包括大米、玉米、小麦等中的一种或几种,添加浓度为20-30%(w/v)。
所述生淀粉水解酶的添加比例为1-5U/mg生淀粉。
与现有技术相比,本发明的有益效果体现在:
1、本发明海洋细菌淀粉酶基因来自于海洋细菌Pontibacillus sp.ZY;
2、本发明海洋细菌淀粉酶在pH5.5-7.5范围内具有良好的稳定性,30℃条件下半衰期为15小时;
3、本发明具有高比酶活力的生淀粉水解酶可于低温下,高效水解20-30%的高浓度生淀粉,反应体系在30-35℃、150rpm摇床水浴锅中反应4h,大米、玉米、小麦的水解率均到达37%以上,因此具有潜在应用价值。
附图说明
图1为本发明AmyZ1PCR扩增产物的电泳图谱。其中M为分子量标准;1为AmyZ1PCR扩增产物。
图2为本发明表达载体pET22b(+)/AmyZ1质粒的鉴定电泳图谱。图2中M为分子量标准;2、3为经过Nde I/Xho I双酶切的pET22b(+)/AmyZ1;4、5为经过Nde I/Xho I双酶切的pET22b(+)。
图3为以大米为底物时测定的本发明AmyZ1催化的最适温度、最适pH。图3中a为最适pH;b为最适温度。
图4为以大米为底物时测定的不同浓度CaCl2对本发明AmyZ1酶活及稳定性的影响。图4中a为不同浓度CaCl2对AmyZ1酶活的影响;b为在30℃下,0.5mMCaCl2和1mMCaCl2对AmyZ1蛋白稳定性的影响。
具体实施方式
下列实施例中的实施方法,如无特别说明,均为常规方法。
(一)含有本发明海洋细菌来源具有高比酶活力的生淀粉水解酶基因的表达菌株的构建1、含有本发明海洋细菌来源具有高比酶活力的生淀粉水解酶基因的阳性克隆菌株的筛选及全长基因的获得
取中国南海永兴岛针叶藻海底沉积物1g,与50mL TYS液体培养基中,30℃,120rpm,活化2h。取上清稀释不同的倍数涂布于含有2%(w/v)的可溶性淀粉的TYS固体平板上,倒置与16℃、28℃、37℃培养箱培养24h后观察平板上菌落生长情况。在长出菌落的平板上加入碘液进行颜色反应,菌落周围出现水解透明圈的为淀粉酶初筛阳性克隆。按照菌落形态的不同和阳性反应圈的大小,选择生长快、透明圈直径与菌落直径之比较大者为初选菌株。用灭菌的牙签挑取阳性克隆于10μL无菌水中,搅拌均匀,用接种环蘸取菌液,于新鲜的培养基上划线纯化。挑取纯化后的单克隆于5mL TYS液体培养基中30℃,200rpm,培养24h。用移液枪吸取0.1ml稀释液涂布于2%(w/v)的可溶性淀粉的TYS固体平板上,37℃恒温箱中培养2天。在平板上加入加入碘液进行颜色反应,菌落周围仍会出现透明水解圈的即为淀粉酶复筛阳性克隆。保种:吸取850μL菌液,与150μL灭过菌的甘油(装于1.5mL EP管中)混合均匀,-70℃保存。
按照试剂盒(购自上海生物工程有限公司)提供的说明抽取产淀粉酶阳性克隆菌株基因组,扩增16S rRNA。细菌16S rRNA基因的扩增采用通用引物序列,以阳性菌株基因组为模板。细菌16S rRNA基因通用引物序列:
正向引物(27F):5’AGAGTTTGATCCTGGCTCAG 3’
反向引物(1492R):5’ACGGCTACCTTGTTACGACTT3’
PCR扩增程序如下:第一阶段变性94℃,5min;第二阶段变性94℃,30sec,退火50℃,30sec,延伸72℃,120sec,共进行30个循环;第三阶段延伸72℃,10min。得到的PCR产物用1%琼脂糖电泳检测,在1600bp左右出现条带的初步认为是阳性克隆。将所得PCR扩增产物和pEASY-T3质粒连接,得连接产物;将连接产物转化大肠杆菌Trans1-T1感受态细胞,将转化产物均匀涂于含Amp抗性及IPTG和X-gal的筛选平板上,37℃过夜培养。根据蓝白斑筛选方法,挑白色克隆于5mL含Amp抗性的LB培养基的试管中,37℃培养8h。按照Axygen质粒小量制备试剂盒的说明书抽提菌液质粒,以质粒为模板进行PCR,鉴定是否阳性克隆。阳性重组子质粒送往上海生工测序,得到16S rRNA基因序列,运用NCBI中的Blast程序与数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)中的细菌16S rRNA基因序列进行相似性比对;根据文献报道,若未知菌株的16S rRNA基因序列与数据库中的已知序列相似性大于99%,则初步认为该未知菌株与数据库中的已知菌株同属。经序列比对后确定产淀粉阳性菌株与Pontibacillus sp.菌属的同源性较高,将产淀粉阳性菌株命名为Pontibacillussp.ZY。然后,根据数据库该种属基因组注释信息,找到相关淀粉酶基因注释序列,根据此序列设计简并引物,以Pontibacillus sp.ZY菌株基因组为模板,PCR扩增AmyZ1基因全长。
2、海洋细菌来源具有高比酶活力的生淀粉水解酶基因的扩增
设计一对简并引物引物P1和P2,其序列为:
P1:5’-CATATGYTNGGNATNWSNTTYGTNYTN-3’
P2:5’-CTCGAGYTTYTGYTTRTANACNSWNACNSW-3’
在引物的5′和3′分别引入Nde I和Xho I酶切位点,以步骤1抽提的Pontibacillussp.ZY菌株基因组为模板,扩增海洋细菌淀粉酶基因。PCR反应程序如下:第一阶段变性94℃,5min;第二阶段变性94℃,30sec,退火52℃,30sec,延伸72℃,2min,共进行30个循环;第三阶段延伸72℃,10min。得到的PCR产物用1%琼脂糖电泳检测,结果见图1。得到的海洋细菌淀粉酶的核苷酸序列如SEQ ID No:1所示,其氨基酸序列如SEQ ID No:2所示。
3、表达载体的构建
将步骤2中得到PCR扩增产物,与pEASY-T3质粒连接,建立如下酶切体系:25ngpEASY-T3质粒载体,50ng PCR扩增产物,补水至5μL,25℃连接5min。连接产物热激转化大肠杆菌Trans1-T1感受态细胞,将转化产物均匀涂于含Amp抗性及IPTG和X-gal的筛选平板上,37℃过夜培养。根据蓝白斑筛选方法,挑白色克隆于5mL含Amp抗性的LB培养基的试管中,37℃培养8h。按照质粒小量制备试剂盒的说明书抽提菌液质粒,以质粒为模板进行PCR鉴定是否阳性克隆,获得含有淀粉酶基因的pEASY-T3重组质粒。阳性重组子质粒送往上海生工测序,从测序结果中获得amyZ1淀粉酶基因序列;用Nde I和Xho I双酶切所得的pEASY-T3重组质粒和pET22b(+)载体,然后用T4DNA连接酶将酶切后的AmyZ1与表达质粒载体连接,建立如下酶切体系:25ng pET22b(+)载体,50ng AmyZ1酶切片段,3μL10×ligation buffer,1μLT4DNA连接酶,补水至30μL,16℃连接8h,得到连接产物;连接产物转化宿主菌,得到的转化子测序验证是否突变,挑选序列正确的转化子得到含有本发明基因AmyZ1的工程菌株Escherichia coli BL21(DE3)/pET22b(+)-amyZ1。
本发明菌株名称为Escherichia coli BL21(DE3)/pET22b(+)-amyZ1,由中国典型培养物保藏中心CCTCC保藏,保藏编号CCTCC NO:M 2018893,保藏日期为2018年12月13日。
(二)含本发明海洋细菌来源具有高比酶活力的生淀粉水解酶基因工程菌的表达与蛋白纯化将(一)中获得的基因的工程菌株Escherichia coli BL21(DE3)/pET22b(+)-amyZ1接种于含有氨苄青霉素的200ml LB液体培养基中,放置于37℃、200rpm条件下培养至OD600=0.6(UNICO UV2102紫外可见分光光度计,以培养LB培养基为空白);加入终浓度为0.2mM的IPTG进行诱导,并于37℃、200rpm条件下继续培养4小时;然后8000rpm、4℃离心收集菌体,用Tris-HCl缓冲液(pH8.0,50mM)重悬破碎菌体,4℃,1000rpm离心去上清。沉淀用Tris-HCl缓冲液清洗一次,离心去上清。然后用30mL Tris-HCl缓冲液(pH8.0,50mM,含8M尿素)重悬沉淀,混匀。室温静置3h变性。用预冷的水将尿素稀释至1M,加入终浓度为10mM的CaCl2,4℃静置复性10h。将复性液装入透析袋,放入已预冷且加入终浓度为1mM CaCl2的Tris-HCl(pH7.0,50mM)缓冲液中,透析24h。将透析好的复性蛋白液取出即为纯化好的AmyZ1蛋白,得到的蛋白经过检测达到SDS-PAGE纯度。
以大米生淀粉为底物,纯化的海洋细菌来源具有高比酶活力的生淀粉水解酶AmyZ1最适pH为7.0,酶在pH5.5~7.5范围内有较高的稳定性,能够维持原酶活力的90%以上。以可溶性淀粉为底物,海洋细菌淀粉酶AmyZ1的最适pH为6.5。AmyZ1在15℃~55℃范围内均能显示催化活性,其最适温度为35℃,30℃时酶的半衰期为15h。
(三)含本发明海洋细菌来源具有高比酶活力的生淀粉水解酶蛋白对不同类型淀粉底物比活力检测
在pH7.0的磷酸盐缓冲液中加入终浓度为1mM的CaCl2,适当稀释的酶液30μL,加入300μL 2%(w/v)的生淀粉底物,pH7.0的Na2HPO4-KH2PO4缓冲液补齐到600μL。35℃反应10min后加入300μL DNS置于冰上5min,在于沸水浴中煮15min,取反应管于冰上5min,再将反应管12000rpm离心3min。最后在OD540下测定读数。定义单位时间产生1μmol麦芽糖所需要的酶量为1U。
自然界中陆地来源生淀粉主要有A、B、C三种,分别代表谷物类(大米,小麦等),根莲类(马铃薯,土豆等)和豆类(豌豆,黄豆等)。表1汇总分别以A型淀粉(大米、玉米、大麦、小麦)、B型淀粉(土豆)、C型淀粉(豌豆)以及可溶性淀粉、直链淀粉、支链淀粉为底物时,AmyZ1对其水解能力的比较。由表1数据可知AmyZ1淀粉酶对不同类型淀粉底物均具有较高比酶活。且AmyZ1淀粉酶在以生淀粉为底物时更偏向于水解A型淀粉,其中对大米生淀粉底物比酶活为12621.28±196.42U/mg,是目前已报道生淀粉酶中比酶活最高的。
表1不同底物酶活
(四)含本发明海洋细菌来源具有高比酶活力的生淀粉水解酶蛋白对高浓度生淀粉水解能力检测
在pH7.0的磷酸盐缓冲液中加入终浓度为1mM的CaCl2,分别加入20%或30%(w/v)浓度的大米、玉米、小麦,以及1500U AmyZ1蛋白,以pH7.0的磷酸盐缓冲液补充反应体系至5mL。将制备好的反应体系于30~35℃,150rpm摇床水浴锅中进行水解反应。间隔适当的时间取样,用DNS法测定水解体系中还原糖含量,计算水解率。结果显示,水解0.5h,水解率为迅速上升阶段,2-4h时水解基本到达平台期,水解率上升不明显,而此时大米、玉米、小麦的水解率可达37%以上。因此,与已报道淀粉酶相比本发明AmyZ1淀粉酶水解反应具有快速,高效性。
SEQ ID No:1
ATGTTGGGGATTAGTTTCGTATTCATTGTGTCGATGTTTCTACCTACAAGTCATGTGAATGCGGCAAGCAAGAATGGGACCATGATGCAGTATTTTGAGTGGTATTTGCCGAATAGTGGTACTCACTGGAACAATTTGAAGAATGATGCTGGACATCTCAATGATTTGGGGATTACAGCTGTGTGGATTCCGCCGGCTTATAAAGGGACCTCTCAGGATGATGTTGGATATGGGGCATACGATTTATACGATCTAGGGGAATTTAATCAAAAAGGAACCGTACGTACGAAATATGGAACGAAAGCACAATTGAAAAATGCGATTCAAACCTTACAGCAGCAAGGGATTCAAGTATACGGGGACGTGGTTATGAATCATAAAGGCGGTGCGGATTTCACAGAGCAAGTGACCGCTGTAGAAGTGAATTCAGGCAACCGAAATGTGCAAACATCTGGGGCGTATACAATTGAGGCGTGGACGGGATTCAACTTTCCAGGGCGTGGAGATGCGTATTCAAGCTTTGATTGGCACTGGTATCACTTCGACGGGACAGACTGGGACCAATCGAGAGGTCTTAACCGAATTTATCAGTTCCAAGGTACAGGGAAGGCTTGGGACTATGAAGTAGATACAGAGAATGGGAACTATGATTACTTAATGTTCGCGGATGTGGATTACGACCACCCTGATGTAGTAAACGAGATGAAGAACTGGGGGTCTTGGTACACGAATGAGTTAAACCTAGATGGATTCCGACTCGACGCAGTCAAGCACATAAAGCATAGCTTCTTAAAGGATTGGGTGAGCCATGTACGTAGTACGACTGGAAAGGAAATGTTCACAGTCGCAGAATATTGGAAGAACGATGTGGGAGAACTTCAGGATTATTTAAGTGACGTGGGGTACAACCACTCCCTCTTTGATGCGCCCCTTCATTACAACTTCTATGATGCATCAAGGTCAAGTGGAAACTACGACATGCGTAATTTGTTAAACGGGACGCTTGTAGCTAGTCAACCAACAAAAGCTGTCACACTAGTTGAGAATCATGATTCTCAACCCGGTCAAGCGCTGGAGTCAGTTGTTCAGTCTTGGTTCAAGCCTCTTGCCTATGCGTTTATTCTGACAAGGGAGGCGGGCTATCCATCTGTGTTCTACGGTGATTATTATGGAACAAAGGGGAACAGCGGATATGAGATTCCGAATTTAAGTCAGAAGTTAGATCCACTTCTAGAAGCTCGGGAAACGTATGCATACGGAATTCAACACGATTACTTAGATCATCAAGATTTAGTAGGCTGGACACGTGAAGGAGACAGTGCTCATGCAAATTCCGGACTTGCTACTTTGATTACAGACCGAAATGGCGGTTCGAAATGGATGTATGTAGGGAAACGTAATGCAGGAGAAACATGGGTCGACATGACAGGAAACCGTTCCAACCAAGTCGTCATTAACAAAGACGGATGGGGAGAATTCTTCGTGAACGGCGGGTCTGTCTCGGTATATAAACAAAAGTAA
SEQ ID No:2
MLGISFVFIVSMFLPTSHVNAASKNGTMMQYFEWYLPNSGTHWNNLKNDAGHLNDLGITAVWIPPAYKGTSQDDVGYGAYDLYDLGEFNQKGTVRTKYGTKAQLKNAIQTLQQQGIQVYGDVVMNHKGGADFTEQVTAVEVNSGNRNVQTSGAYTIEAWTGFNFPGRGDAYSSFDWHWYHFDGTDWDQSRGLNRIYQFQGTGKAWDYEVDTENGNYDYLMFADVDYDHPDVVNEMKNWGSWYTNELNLDGFRLDAVKHIKHSFLKDWVSHVRSTTGKEMFTVAEYWKNDVGELQDYLSDVGYNHSLFDAPLHYNFYDASRSSGNYDMRNLLNGTLVASQPTKAVTLVENHDSQPGQALESVVQSWFKPLAYAFILTREAGYPSVFYGDYYGTKGNSGYEIPNLSQKLDPLLEARETYAYGIQHDYLDHQDLVGWTREGDSAHANSGLATLITDRNGGSKWMYVGKRNAGETWVDMTGNRSNQVVINKDGWGEFFVNGGSVSVYKQK
SEQUENCE LISTING
<110> 安徽大学
<120> 一种具有高比酶活力的生淀粉水解酶、其编码基因及其应用
<130>
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 1521
<212> DNA
<213> Pontibacillus sp.
<400> 1
atgttgggga ttagtttcgt attcattgtg tcgatgtttc tacctacaag tcatgtgaat 60
gcggcaagca agaatgggac catgatgcag tattttgagt ggtatttgcc gaatagtggt 120
actcactgga acaatttgaa gaatgatgct ggacatctca atgatttggg gattacagct 180
gtgtggattc cgccggctta taaagggacc tctcaggatg atgttggata tggggcatac 240
gatttatacg atctagggga atttaatcaa aaaggaaccg tacgtacgaa atatggaacg 300
aaagcacaat tgaaaaatgc gattcaaacc ttacagcagc aagggattca agtatacggg 360
gacgtggtta tgaatcataa aggcggtgcg gatttcacag agcaagtgac cgctgtagaa 420
gtgaattcag gcaaccgaaa tgtgcaaaca tctggggcgt atacaattga ggcgtggacg 480
ggattcaact ttccagggcg tggagatgcg tattcaagct ttgattggca ctggtatcac 540
ttcgacggga cagactggga ccaatcgaga ggtcttaacc gaatttatca gttccaaggt 600
acagggaagg cttgggacta tgaagtagat acagagaatg ggaactatga ttacttaatg 660
ttcgcggatg tggattacga ccaccctgat gtagtaaacg agatgaagaa ctgggggtct 720
tggtacacga atgagttaaa cctagatgga ttccgactcg acgcagtcaa gcacataaag 780
catagcttct taaaggattg ggtgagccat gtacgtagta cgactggaaa ggaaatgttc 840
acagtcgcag aatattggaa gaacgatgtg ggagaacttc aggattattt aagtgacgtg 900
gggtacaacc actccctctt tgatgcgccc cttcattaca acttctatga tgcatcaagg 960
tcaagtggaa actacgacat gcgtaatttg ttaaacggga cgcttgtagc tagtcaacca 1020
acaaaagctg tcacactagt tgagaatcat gattctcaac ccggtcaagc gctggagtca 1080
gttgttcagt cttggttcaa gcctcttgcc tatgcgttta ttctgacaag ggaggcgggc 1140
tatccatctg tgttctacgg tgattattat ggaacaaagg ggaacagcgg atatgagatt 1200
ccgaatttaa gtcagaagtt agatccactt ctagaagctc gggaaacgta tgcatacgga 1260
attcaacacg attacttaga tcatcaagat ttagtaggct ggacacgtga aggagacagt 1320
gctcatgcaa attccggact tgctactttg attacagacc gaaatggcgg ttcgaaatgg 1380
atgtatgtag ggaaacgtaa tgcaggagaa acatgggtcg acatgacagg aaaccgttcc 1440
aaccaagtcg tcattaacaa agacggatgg ggagaattct tcgtgaacgg cgggtctgtc 1500
tcggtatata aacaaaagta a 1521
<210> 2
<211> 506
<212> PRT
<213> Pontibacillus sp.
<400> 2
Met Leu Gly Ile Ser Phe Val Phe Ile Val Ser Met Phe Leu Pro Thr
1 5 10 15
Ser His Val Asn Ala Ala Ser Lys Asn Gly Thr Met Met Gln Tyr Phe
20 25 30
Glu Trp Tyr Leu Pro Asn Ser Gly Thr His Trp Asn Asn Leu Lys Asn
35 40 45
Asp Ala Gly His Leu Asn Asp Leu Gly Ile Thr Ala Val Trp Ile Pro
50 55 60
Pro Ala Tyr Lys Gly Thr Ser Gln Asp Asp Val Gly Tyr Gly Ala Tyr
65 70 75 80
Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
85 90 95
Lys Tyr Gly Thr Lys Ala Gln Leu Lys Asn Ala Ile Gln Thr Leu Gln
100 105 110
Gln Gln Gly Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly
115 120 125
Gly Ala Asp Phe Thr Glu Gln Val Thr Ala Val Glu Val Asn Ser Gly
130 135 140
Asn Arg Asn Val Gln Thr Ser Gly Ala Tyr Thr Ile Glu Ala Trp Thr
145 150 155 160
Gly Phe Asn Phe Pro Gly Arg Gly Asp Ala Tyr Ser Ser Phe Asp Trp
165 170 175
His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gly Leu
180 185 190
Asn Arg Ile Tyr Gln Phe Gln Gly Thr Gly Lys Ala Trp Asp Tyr Glu
195 200 205
Val Asp Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Val
210 215 220
Asp Tyr Asp His Pro Asp Val Val Asn Glu Met Lys Asn Trp Gly Ser
225 230 235 240
Trp Tyr Thr Asn Glu Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val
245 250 255
Lys His Ile Lys His Ser Phe Leu Lys Asp Trp Val Ser His Val Arg
260 265 270
Ser Thr Thr Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Lys Asn
275 280 285
Asp Val Gly Glu Leu Gln Asp Tyr Leu Ser Asp Val Gly Tyr Asn His
290 295 300
Ser Leu Phe Asp Ala Pro Leu His Tyr Asn Phe Tyr Asp Ala Ser Arg
305 310 315 320
Ser Ser Gly Asn Tyr Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val
325 330 335
Ala Ser Gln Pro Thr Lys Ala Val Thr Leu Val Glu Asn His Asp Ser
340 345 350
Gln Pro Gly Gln Ala Leu Glu Ser Val Val Gln Ser Trp Phe Lys Pro
355 360 365
Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala Gly Tyr Pro Ser Val
370 375 380
Phe Tyr Gly Asp Tyr Tyr Gly Thr Lys Gly Asn Ser Gly Tyr Glu Ile
385 390 395 400
Pro Asn Leu Ser Gln Lys Leu Asp Pro Leu Leu Glu Ala Arg Glu Thr
405 410 415
Tyr Ala Tyr Gly Ile Gln His Asp Tyr Leu Asp His Gln Asp Leu Val
420 425 430
Gly Trp Thr Arg Glu Gly Asp Ser Ala His Ala Asn Ser Gly Leu Ala
435 440 445
Thr Leu Ile Thr Asp Arg Asn Gly Gly Ser Lys Trp Met Tyr Val Gly
450 455 460
Lys Arg Asn Ala Gly Glu Thr Trp Val Asp Met Thr Gly Asn Arg Ser
465 470 475 480
Asn Gln Val Val Ile Asn Lys Asp Gly Trp Gly Glu Phe Phe Val Asn
485 490 495
Gly Gly Ser Val Ser Val Tyr Lys Gln Lys
500 505
Claims (10)
1.一种具有高比酶活力的生淀粉水解酶,其特征在于:
所述生淀粉水解酶,名称为AmyZ1,其氨基酸序列为如下序列中的一种:
(1)序列表中的SEQ ID No:2;
(2)序列表中的SEQ ID No:2经取代、缺失或添加一个或几个氨基残基且编码相同功能蛋白质的氨基酸序列。
2.编码权利要求1所述的生淀粉水解酶的基因,其特征在于其核苷酸序列为如下序列中的一种:
(1)序列表中的SEQ ID No:1;
(2)序列表中的SEQ ID No:1经取代、缺失或添加一个或几个核苷酸且编码相同功能蛋白质的核苷酸序列;
(3)编码序列表中SEQ ID No:2蛋白质序列的多核苷酸序列。
3.含有权利要求1所述的生淀粉水解酶基因并能产生所述生淀粉水解酶的重组表达菌株,其特征在于:所述重组表达菌株的名称为Escherichia coli BL21(DE3)/pET22b(+)-amyZ1,由中国典型培养物保藏中心CCTCC保藏,保藏编号CCTCC NO:M 2018893,保藏日期为2018年12月13日。
4.权利要求3所述的重组表达菌株的构建方法,其特征在于包括如下步骤:
以包含具有高比酶活力的生淀粉水解酶基因的Pontibacillus sp.ZY细菌基因组DNA为模板,以P1和P2为引物进行PCR扩增,得到PCR扩增产物,并与pEASY-T3质粒连接,将连接产物转化大肠杆菌Trans1-T1感受态细胞,筛选阳性克隆,进行序列分析;挑选序列正确的克隆提取质粒,获得含有生淀粉水解酶基因的pEASY-T3重组质粒,用Nde I和Xho I双酶切所得的pEASY-T3重组质粒和表达质粒载体,然后用T4 DNA连接酶将酶切后的amyZ1与表达质粒载体连接,得到连接产物;将得到的连接产物转化宿主菌,筛选阳性克隆,即可得到含有本发明生淀粉水解酶基因的工程菌株。
5.根据权利要求4所述的方法,其特征在于:
所述引物为:
P1:5’-CATATGYTNGGNATNWSNTTYGTNYTN-3’
P2:5’-CTCGAGYTTYTGYTTRTANACNSWNACNSW-3’。
6.根据权利要求4所述的方法,其特征在于:
所述表达质粒载体指pCold、pET15、pET22或pET28;
所述宿主菌是指E.coli BL21(DE3)、E.coli DH5α、E.coli JM109或E.coli Rosetta。
7.权利要求1所述的生淀粉水解酶的应用,其特征在于:在所述生淀粉水解酶的存在下对生淀粉进行水解。
8.根据权利要求7所述的应用,其特征在于:
水解体系为pH7.0的磷酸盐缓冲液中加入终浓度为1mM的CaCl2,随后加入生淀粉,以及所述生淀粉水解酶,于30-35℃、150rpm摇床水浴下进行水解反应。
9.根据权利要求8所述的应用,其特征在于:
所述生淀粉包括大米、玉米、小麦等中的一种或几种,添加浓度为20-30%。
10.根据权利要求8所述的应用,其特征在于:
所述生淀粉水解酶的添加比例为1-5U/mg生淀粉。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH03103177A (ja) * | 1988-07-01 | 1991-04-30 | Showa Denko Kk | 耐熱性アミラーゼ並びにその製造及び使用方法 |
JPH0799979A (ja) * | 1993-09-30 | 1995-04-18 | Tax Adm Agency | 新規遺伝子、それを用いた形質転換体及び その利用 |
CN101955887A (zh) * | 2010-09-02 | 2011-01-26 | 广西大学 | 产生生淀粉酶的青霉菌及其制备的生淀粉酶制剂 |
CN103710325A (zh) * | 2014-01-06 | 2014-04-09 | 安徽大学 | 一种α-淀粉酶AmyASS及其在生淀粉降解中的应用 |
CN108823186A (zh) * | 2018-07-03 | 2018-11-16 | 江西省科学院微生物研究所 | 一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体及其制备方法和应用 |
-
2019
- 2019-01-07 CN CN201910010979.3A patent/CN109679937B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH03103177A (ja) * | 1988-07-01 | 1991-04-30 | Showa Denko Kk | 耐熱性アミラーゼ並びにその製造及び使用方法 |
JPH0799979A (ja) * | 1993-09-30 | 1995-04-18 | Tax Adm Agency | 新規遺伝子、それを用いた形質転換体及び その利用 |
CN101955887A (zh) * | 2010-09-02 | 2011-01-26 | 广西大学 | 产生生淀粉酶的青霉菌及其制备的生淀粉酶制剂 |
CN103710325A (zh) * | 2014-01-06 | 2014-04-09 | 安徽大学 | 一种α-淀粉酶AmyASS及其在生淀粉降解中的应用 |
CN108823186A (zh) * | 2018-07-03 | 2018-11-16 | 江西省科学院微生物研究所 | 一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体及其制备方法和应用 |
Non-Patent Citations (4)
Title |
---|
WEI FANG ET AL.: "AmyZ1: a novel α-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches", 《BIOTECHNOLOGY FOR BIOFUELS》, vol. 12, no. 95, 23 April 2019 (2019-04-23), pages 1 - 15 * |
XUE, S.: "Pontibacillus sp. strain ZY alpha-amylase gene, partial cds, MH325467.1", 《GENBANK》, 12 September 2018 (2018-09-12), pages 1 - 2 * |
YANG LIU ET AL.: "Identification and Phylogenetic Characterization of a New Subfamily of α-Amylase Enzymes from Marine Microorganisms", 《MAR BIOTECHNOL》, vol. 14, 11 November 2011 (2011-11-11), pages 253 - 260, XP035039506, DOI: 10.1007/s10126-011-9414-3 * |
景洁等: "罗耳阿太菌源生淀粉糖化酶发酵条件的优化及鉴定", 《西北农林科技大学学报(自然科学版)》, vol. 45, no. 11, 9 October 2017 (2017-10-09), pages 122 - 131 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113444754A (zh) * | 2020-07-30 | 2021-09-28 | 吉林中粮生化有限公司 | 一种水解生淀粉的方法及其应用 |
CN113444754B (zh) * | 2020-07-30 | 2023-10-20 | 吉林中粮生化有限公司 | 一种水解生淀粉的方法及其应用 |
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