CN114250213A - 一种耐高温酸性α-淀粉酶及其基因和应用 - Google Patents
一种耐高温酸性α-淀粉酶及其基因和应用 Download PDFInfo
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- CN114250213A CN114250213A CN202111611313.7A CN202111611313A CN114250213A CN 114250213 A CN114250213 A CN 114250213A CN 202111611313 A CN202111611313 A CN 202111611313A CN 114250213 A CN114250213 A CN 114250213A
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Abstract
本发明提供了一种α‑淀粉酶,其氨基酸序列包括SEQ ID NO.1或与SEQ ID NO.1具有63%以上同一性的序列。本发明提供的α‑淀粉酶是一种耐高温酸性α‑淀粉酶,具有较好的pH稳定性和热稳定性。除可作用于可溶性淀粉外,对于土豆淀粉、红薯淀粉有一定的降解作用。该酶在玉米淀粉深加工、酒精生产、氨基酸和有机酸发酵和饲料行业具有非常广阔的应用前景。
Description
技术领域
本发明属于基因工程领域,具体涉及一种耐高温酸性α-淀粉酶AMY-L18及其基因、包含该基因的重组载体和应用。
背景技术
淀粉是一种碳水化合物,几乎完全是以α-D-葡萄糖为单位聚合组成的,链的平均长度为500-1000个葡萄糖残基。淀粉中能溶于热水的一部分叫直链淀粉,不能溶解的叫支链淀粉,这两种成分是淀粉颗粒的主要成分,含量可以达到75-80%。淀粉几乎存在于所有绿色植物多数组织中,是植物营养物质的一种储存形式,在显微镜下以颗粒形状存在于植物有机体中。当前,能够作为淀粉原料的主要作物是玉米,其次是薯类、稻谷和小麦。淀粉酶是可以水解淀粉的一类酶的总称,广泛存在于植物和微生物中。根据淀粉降解酶的作用方式主要分为三类:外切淀粉酶、内切淀粉酶和分支淀粉酶。在实际生产中最常使用的主要是α-淀粉酶、β-淀粉酶、葡萄糖淀粉酶、支链淀粉酶。其中,α-淀粉酶是从淀粉内部任意水解α-1,4-D葡萄糖苷键,导致淀粉部分解聚,是内切酶。根据α-淀粉酶的热稳定性和最适反应温度,通常把α-淀粉酶分成高温α-淀粉酶、中温耐热型α-淀粉酶、非耐热性α-淀粉酶和低温α-淀粉酶。高温α-淀粉酶作用最适温度90-100℃,95-105℃耐热性好,中温耐热型α-淀粉酶作用最适温度70-80℃,90℃以上处理一般会失活,非耐热性α-淀粉酶作用最适温度50℃左右,低温α-淀粉酶则一般在10-20℃还能表现出活性。根据α-淀粉酶作用的最适pH和对酸碱的稳定性,把α-淀粉酶分成酸性α-淀粉酶、中性α-淀粉酶和碱性α-淀粉酶。大多数α-淀粉酶都属于中性α-淀粉酶,一般最适pH 6.0-7.0,酸性α-淀粉酶最适pH<5.5,碱性α-淀粉酶最适pH>8.0。
对淀粉酶的研究已有半个世纪,主要研究集中在适合于麦芽糖浆、食品烘培、纺织退浆工业等方面的淀粉酶,已经从不同来源的植物与微生物中分离到大量的不同类型不同功能的淀粉酶,并分离出多种淀粉酶基因,现已工业化生产多种淀粉酶产品。近年来,耐高温淀酸性粉酶已经引起研究人员的广泛关注,在饲料、玉米淀粉深加工、氨基酸和有机酸发酵中添加淀粉酶已经很普遍了,缺乏耐高温性能优异,并且具有在酸性条件酶活较高的淀粉酶。目前许多工业用淀粉酶最适温度要105℃左右甚至更高,要求在酸性条件下酶活稳定,并且具有很好的降解效果。因此,急需开发在高温和酸性条件下,对淀粉也能发挥显著水解作用的高效淀粉酶。此外,在动物饲料行业添加耐高温酸性的淀粉酶更有利于在动物胃肠的酸性环境中发挥水解作用,有助于营养物质的吸收。
发明内容
为了解决上述问题,本发明提供了一种耐高温酸性α-淀粉酶。
一方面,本发明提供了一种α-淀粉酶。
所述的α-淀粉酶的氨基酸序列包括SEQ ID NO.1。
在GenBank中进行BLAST比对,该序列与来源于Bacillus thuringiensis的α-淀粉酶氨基酸序列一致性为63%,本发明同时保护与SEQ ID NO.1具有63%以上同一性的序列。
优选地,所述的α-淀粉酶N端还包含信号肽,所述的信号肽的序列为SEQ ID NO.2。
优选地,所述的α-淀粉酶的氨基酸序列为SEQ ID NO.3或与SEQ ID NO.3有63%以上同一性的序列。
另一方面,本发明提供了一种核苷酸序列。
所述的核苷酸序列编码前述的α-淀粉酶。
优选地,所述的核苷酸序列包括SEQ ID NO.4或与SEQ ID NO.4编码相同氨基酸序列的核苷酸序列。
所述的与SEQ ID NO.4编码相同氨基酸序列的核苷酸序列是指在密码子简并性的基础上对SEQ ID NO.4进行碱基替换的序列。
优选地,所述的核苷酸序列还包括编码信号肽的序列。
所述的编码信号肽的序列为SEQ ID NO.5或与SEQ ID NO.5编码相同信号肽的序列。
所述的与SEQ ID NO.5编码相同信号肽的序列是指在密码子简并性的基础上对SEQ ID NO.5进行碱基替换后的序列。
优选地,所述的核苷酸序列为SEQ ID NO.6。
再一方面,本发明提供了一种重组载体。
所述的重组载体包含前述的核苷酸序列。
优选地,所述的重组载体为pPIC-amy-L18,即前述核苷酸序列插入pPIC载体构建的重组载体。
所述的重组载体的构建中包括:将本发明的α-淀粉酶基因去掉信号肽后插入到表达载体合适的限制性酶切位点之间,使其核苷酸序列可操作的与表达调控序列相连接。
作为本发明的一个最优选的实施方案,将本发明的α-淀粉酶基因插入到质粒pPIC9上的EcoR I和Not I限制性酶切位点之间,使该核苷酸序列位于AOX1启动子的下游并受其调控,得到重组毕赤酵母表达质粒pPIC9-amy-L18。
又一方面,本发明提供了一种细胞。
所述的细胞中包括前述的重组载体。
所述的细胞用于表达前述的α-淀粉酶。
所述的细胞可以为工程菌或具有蛋白表达能力的任何细胞。
优选地,所述的细胞为重组菌株;优选菌株为大肠杆菌、酵母菌、芽孢杆菌或乳酸杆菌;更优选为重组菌株GS115/amy-L27,即表达本发明所述的α-淀粉酶基因的毕赤酵母细胞(Pichia pastoris)GS115。
又一方面,本发明提供了一种α-淀粉酶的制备方法。
所述的制备方法中包括使用重组细胞表达前述的α-淀粉酶。
具体地,所述的的制备方法中通过将表达前述α-淀粉酶的载体转化菌株进行表达。
具体地,所述的制备方法中包括以下步骤:
S1、表达载体构建;
S2、转化菌株;
S3、菌株培养,诱导α-淀粉酶表达;
S4、α-淀粉酶回收纯化。
优选地,所述的表达载体为包括前述核苷酸序列的表达载体。
优选地,所述的表达载体为pPIC-amy-L18,即前述核苷酸序列插入pPIC载体构建的重组载体。
优选地,所述的表达载体的构建中包括:将本发明的α-淀粉酶基因插入到质粒pPIC9上的EcoR I和Not I限制性酶切位点之间,使该核苷酸序列位于AOX1启动子的下游并受其调控,得到重组毕赤酵母表达质粒pPIC9-amy-L18。
又一方面,本发明提供了前述的α-淀粉酶和/或核苷酸序列和/或重组载体和/或细胞在淀粉深加工、酒精生产、氨基酸和有机酸发酵、饲料、洗涤、食品、轻工或能源行业中的应用。
所述的应用中包括使用前述的α-淀粉酶对淀粉进行分解。
又一方面,本发明提供了一种活性酶制剂。
所述的酶活性制剂包括前述的α-淀粉酶和/或核苷酸序列和/或重组载体和/或细胞。
所述的酶活性制剂可以用于淀粉深加工、酒精生产、氨基酸和有机酸发酵、饲料、洗涤、食品、轻工或能源行业。
所述的酶活性制剂通过分解淀粉进行应用。
所述的淀粉可以是可溶性淀粉或不可溶性淀粉。
所述的淀粉可以是直链淀粉或支链淀粉。
又一方面,本发明提供了一种淀粉加工产品。
所述的淀粉加工产品以淀粉为原料,通过前述的α-淀粉酶和/或核苷酸序列和/或重组载体和/或细胞进行制备。
又一方面,本发明提供了一种饲料。
所述的饲料通过前述的α-淀粉酶和/或核苷酸序列和/或重组载体和/或细胞进行制备。
本发明的有益效果:
本发明提供的α-淀粉酶是一种耐高温酸性α-淀粉酶,具有较好的pH稳定性和热稳定性,最适pH5.5,在pH4.5-7.5条件下仍具有80%以上的活性,并且在pH 4.0-8.0具有良好的稳定性;最适温度105℃,在115℃条件下仍具有75%以上的活性。比活为12098.6U/mg。除可作用于可溶性淀粉外,对于土豆淀粉、红薯淀粉有一定的降解作用。该酶在玉米淀粉深加工、酒精生产、氨基酸和有机酸发酵和饲料行业具有非常广阔的应用前景。
附图说明
图1为重组耐高温酸性α-淀粉酶的最适pH。
图2为重组耐高温酸性α-淀粉酶的pH稳定性。
图3为重组耐高温酸性α-淀粉酶的最适温度。
图4为重组耐高温酸性α-淀粉酶的热稳定性。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中:
毕赤酵母表达载体pPIC9及菌株GS115购自于Invitrogen公司;
限制性内切酶和T4 DNA连接酶购自Fermentas公司;
可溶性淀粉购自Sigma公司;
其它均为国产试剂,可从普通生化试剂公司购买得到。
大肠杆菌培养基LB:1%蛋白胨、0.5%酵母提取物、1%NaCl,pH自然;
BMGY培养基:1%酵母提取物、2%蛋白胨、1.34%YNB、0.00004%Biotin、1%甘油(V/V);
BMMY培养基:除以0.5%甲醇代替甘油,其余成份均与BMGY相同,pH自然。
说明:以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
实施例1枯草芽孢杆菌α-淀粉酶基因amy-L18的克隆
利用康为世纪细菌基因组DNA提取试剂盒(CW0552S)提取枯草芽孢杆菌CGMCCNO.14442基因组DNA(下述步骤中提到的溶菌酶,蛋白酶K,Buffer GW1,Buffer GW2,吸附柱DM,洗脱液GE等均来自该试剂盒):
(1)取0.5-2mL培养菌液,10000rpm,离心30s,尽可能的吸取上清,收集菌体;
(2)对于革兰氏阳性菌:加入180μL溶菌酶,颠倒混匀,37℃水浴30-60min;
(3)加入20μL蛋白酶K震荡混匀,加入200μL Buffer GL,震荡混匀;
(4)56℃水浴30min;
(5)加入200μL无水乙醇,震荡混匀;
(6)将上一步混合物(包括可能有的沉淀)加入一个吸附柱DM中,吸附柱放入收集管中,13000rpm离心30-60s,弃掉废液;
(7)向吸附柱DM中加入500μL Buffer GW1,13000rpm离心1min,弃废液;
(8)吸附柱DM中加入500μL Buffer GW2,13000rpm离心1min,弃废液;
(9)重复步骤8;
(10)将吸附柱DM放回空收集管中,13000rpm离心2min,尽量除去漂洗液GW2,室温数分钟彻底晾干;
(11)取出吸附柱DM,放入一个干净的离心管中,在吸附膜的中间部位加100μL洗脱缓冲液GE,室温放置3-5min,12000rpm离心1min。将得到的溶液重新加入离心吸附柱中,室温放置2min,12000rpm离心1min;
(12)得到的DNA于-20℃保存。
从NCBI基因数据库中获取芽孢杆菌来源α-淀粉酶基因序列进行序列比对分析,设计合成简并引物P1,P2:
P1:SEQ ID NO.7;
P2:SEQ ID NO.8。
以枯草芽孢杆菌总DNA为模板进行PCR扩增。PCR反应参数为:94℃变性5min;然后94℃变性30sec,45℃退火30sec,72℃延伸2min,30个循环后72℃保温10min,获得一条约1700bp大小的片段,将该片段回收后与pEASY-T3载体进行连接转化后送北京睿博新科生物技术有限公司测序。通过基因测序得到1698bp的基因片段(SEQ ID NO.6),编码565个氨基酸和一个终止密码子,预测该基因所编码的成熟蛋白的理论分子量为62.4kDa(SEQ IDNO.3),该基因上包括编码信号肽的序列SEQ ID NO.5,编码的信号肽为SEQ ID NO.2。
实施例2重组α-淀粉酶AMY-L18的制备
根据获得的α-淀粉酶AMY-L18的基因序列设计合成表达引物:
P3:SEQ ID NO.9;
P4:SEQ ID NO.10。
重新以枯草芽孢杆菌总DNA为模板进行PCR扩增,获得带有重组酶切位点的α-淀粉酶AMY-L18基因序列。将表达载体pPIC9进行双酶切(EcoR I+Not I),同时将编码α-淀粉酶的基因amy-L18双酶切(EcoR I+Not I),酶切出编码成熟α-淀粉酶的基因片段与表达载体pPIC9连接,获得含有枯草芽孢杆菌α-淀粉酶基因amy-L18的重组质粒pPIC-amy-L18并电击转化毕赤酵母GS115,获得重组毕赤酵母菌株GS115/amy-L18。
将含有重组质粒的GS115/amy-L18菌株,接种于400mL BMGY培养液中,30℃250rpm振荡培养48h后,离心收集菌体。然后利用200mL BMMY培养基重悬,30℃250rpm振荡培养。诱导72h后,离心收集上清。测定α-淀粉酶的活力,重组α-淀粉酶的表达量为15100.3U/mL。
实施例3重组α-淀粉酶的活性分析
采用DNS法对重组α-淀粉酶进行活性分析:在pH 5.5,45℃条件下,1mL的反应体系包括100μL适当的稀释酶液,900μL底物,反应10min,加入1.5mL DNS终止反应,沸水煮5min。冷却后540nm测定OD值。1个酶活单位(U)定义为在给定的条件下每分钟释放出1μmol还原糖的酶量。
测得比活为12098.6U/mg。
实施例4重组α-淀粉酶AMY-L18的性质测定
1、重组α-淀粉酶AMY-L18的最适pH和pH稳定性的测定
将实施例4的重组淀粉酶在不同的pH条件下进行酶促反应以测定其最适pH。底物用不同pH的0.1mol/L柠檬酸-磷酸氢二钠缓冲液中80℃下进行α-淀粉酶活力测定。结果(图1)表明,重组酶AMY-L18的最适pH为5.5,在pH 3.0-6.5条件下有60%以上的相对酶活性。α-淀粉酶于上述各种不同pH的缓冲液中37℃处理60min,再在pH5.5缓冲液体系中80℃条件下测定酶活性,以研究酶的pH耐性。结果(图2)表明淀粉酶在pH 2.0-7.0之间均很稳定,在此pH范围内处理60min后剩余酶活性在75%以上,这说明此酶具有较好的pH稳定性。
2、α-淀粉酶的最适温度及热稳定性测定
α-淀粉酶的最适温度的测定为在柠檬酸-磷酸氢二钠缓冲液(pH5.5)缓冲液体系及不同温度下进行酶促反应。耐温性测定为淀粉酶在不同温度下处理不同时间,再在80℃下进行酶活性测定。α-淀粉酶反应最适温度测定结果(图3)表明其最适温度为80℃。酶的热稳定性试验表明(图4),AMY-L18具有良好的热稳定性,在85℃下温育10min,能保持60%以上的酶活。
3、α-淀粉酶的Km值测定
用不同浓度的可溶性淀粉为底物,在柠檬酸-磷酸氢二钠缓冲液(pH5.5)缓冲液体系中,80℃条件下测定酶活性,计算出其在80℃下的Km值。经测定,以可溶性淀粉为底物时的Km、Vmax分别为6.8和930.3mg/mL。
4、不同金属离子化学试剂对AMY-L18酶活的影响
在酶促反应体系中加入不同浓度的不同的金属离子及化学试剂,研究其对酶活性的影响,各种物质终浓度为1和5mmol/L。在80℃、pH5.5条件下测定酶活性。结果表明,大多数离子和化学试剂在浓度为1mmol时重组α-淀粉酶的活力没有明显变化,只有SDS微弱抑制其活力。当Cu2+和β-巯基乙醇浓度为5mmol时可以部分抑制AMY-L18酶活力,5mmol SDS使其活力全部丧失。
5、重组α-淀粉酶的底物特异性
该酶除可作用于可溶性淀粉外,对于土豆淀粉、红薯淀粉有一定的降解作用(表1)。
表1.淀粉酶AMY-L18底物特异性分析
序列表
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cactccgaga aaaaaccacg ttctttctgg gacgctaaag aaaacatgta ctatccgccg 840
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tacggcaccg aaatcgcgct ggacggtggt agcgttccgg acaaccgtcg tctgatggac 960
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tactatccgc cgtctcgcct gaaactggcg ctgacttacc tgctgacttc cccgggtatc 960
ccgaacttct attacggcac cgaaatcgcg ctggacggtg gtagcgttcc ggacaaccgt 1020
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aatgcgcaga aagatatgca ggtaatcaaa aaagactttc gcatcatgac caaagaggac 1140
agcgaataca atgaaaaaat tgtggaatcc ttctccaaag ctgatgtatc cgtgaaatct 1200
ctgtacgacg tctccaaaaa agaaggtgaa ttcgttacct ttctggatga ccaggaaacc 1260
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aaaagcctgt atgacgtgtc caaaaaggaa ggtgaatttg ttaccttcct ggatgatcaa 1440
gaaaccaaac gctacgcacg tatcgctaaa gagaacatgt actatccgcc gagccgcctg 1500
aaactggccc tgacctacct gctgacctct ccgggtatcc cgaacttcga taatcgtcgt 1560
ctgatggatt ttaaatctga tgaaaaattc atgcagcaca tcaccaaacc tcgtggtctg 1620
aaaaccggca aatactacgg cacggaaatc gcactggacg gcggcagcgt accgaaccgc 1680
ctgaccaacg caggtccg 1698
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtngtnyaag rgtggcayga 20
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atrttawryt ctartcttrt g 21
<210> 9
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cggaattctt ttctaaagat gtaattt 27
<210> 10
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctgcggccgc tggtcctgca tttgtt 26
Claims (14)
1.一种α-淀粉酶,其特征在于,其氨基酸序列包括SEQ ID NO.1或与SEQ ID NO.1具有63%以上同一性的序列。
2.根据权利要求1所述的α-淀粉酶,其特征在于,还包括N端的信号肽,所述的信号肽的序列为SEQ ID NO.2。
3.根据权利要求2所述的α-淀粉酶,其特征在于,氨基酸序列为SEQ ID NO.3或与SEQID NO.3具有63%以上同一性的序列。
4.一种核苷酸序列,其特征在于,编码权利要求1-3任一项所述的α-淀粉酶。
5.根据权利要求4所述的核苷酸序列,其特征在于,包括SEQ ID NO.4或与SEQ ID NO.4编码相同氨基酸序列的核苷酸序列。
6.根据权利要求5所述的核苷酸序列,其特征在于,还包括编码信号肽的序列,所述编码信号肽的序列为SEQ ID NO.5或与SEQ ID NO.5编码相同氨基酸序列的核苷酸序列。
7.一种重组载体,其特征在于,包含权利要求4-6任一项所述的核苷酸序列。
8.一种细胞,其特征在于,所述的细胞中包括权利要求7所述的重组载体。
9.一种α-淀粉酶的制备方法,其特征在于,包括使用重组细胞表达权利要求1-3任一项所述的α-淀粉酶。
10.根据权利要求9所述的制备方法,其特征在于,包括以下步骤:
S1、表达载体构建;
S2、转化菌株;
S3、菌株培养,诱导α-淀粉酶表达;
S4、α-淀粉酶回收纯化。
11.权利要求1-3任一项所述的α-淀粉酶和/或权利要求4-6任一项所述的核苷酸序列和/或权利要求7所述的重组载体和/或权利要求8所述的细胞在淀粉深加工、酒精生产、氨基酸和有机酸发酵、饲料、洗涤、食品、轻工或能源行业中的应用。
12.一种活性酶制剂,其特征在于,包括权利要求权利要求1-3任一项所述的α-淀粉酶和/或权利要求4-6任一项所述的核苷酸序列和/或权利要求7所述的重组载体和/或权利要求8所述的细胞。
13.一种淀粉深加工产品,其特征在于,通过权利要求1-3任一项所述的α-淀粉酶和/或权利要求4-6任一项所述的核苷酸序列和/或权利要求7所述的重组载体和/或权利要求8所述的细胞进行制备。
14.一种饲料,其特征在于,通过权利要求1-3任一项所述的α-淀粉酶和/或权利要求4-6任一项所述的核苷酸序列和/或权利要求7所述的重组载体和/或权利要求8所述的细胞进行制备。
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CN101298604A (zh) * | 2008-06-25 | 2008-11-05 | 天津科技大学 | 高温耐酸性α-淀粉酶的突变株及其构建方法 |
CN103740673A (zh) * | 2014-01-17 | 2014-04-23 | 北京科为博生物科技有限公司 | 一种低温酸性α-淀粉酶AMY-L27及其基因和应用 |
CN110423737A (zh) * | 2019-09-10 | 2019-11-08 | 白银赛诺生物科技有限公司 | 来源于嗜热脂肪土芽孢杆菌的耐热型α-淀粉酶及其应用 |
CN112626053A (zh) * | 2020-12-01 | 2021-04-09 | 自然资源部第三海洋研究所 | 一种酸性α淀粉酶及其制备方法与应用 |
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CN101298604A (zh) * | 2008-06-25 | 2008-11-05 | 天津科技大学 | 高温耐酸性α-淀粉酶的突变株及其构建方法 |
CN103740673A (zh) * | 2014-01-17 | 2014-04-23 | 北京科为博生物科技有限公司 | 一种低温酸性α-淀粉酶AMY-L27及其基因和应用 |
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CN112626053A (zh) * | 2020-12-01 | 2021-04-09 | 自然资源部第三海洋研究所 | 一种酸性α淀粉酶及其制备方法与应用 |
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