CN102703482A - 一种耐有机溶剂碱性蛋白酶 - Google Patents
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Abstract
本发明属于蛋白酶基因工程技术领域,具体涉及一种耐有机溶剂蛋白酶,具体涉及其突变基因及其编码的蛋白质。从序列表的SEQ ID NO:1或SEQ ID NO:3可见,本发明所述耐有机溶剂碱性蛋白酶在原核苷酸序列的第385或第436发生了定点突变,即其成熟肽段的第24位突变氨基酸为谷氨酸或者谷氨酰胺;第41位氨基酸为丙氨酸或者赖氨酸。突变后的四种编码后的蛋白质的有机溶剂,特别是亲水性有机溶剂的耐受性明显大大加强。
Description
技术领域
本发明属于蛋白酶基因工程技术领域,具体涉及一种耐有机溶剂蛋白酶,具体涉及其突变基因及其编码的蛋白质。
技术背景
蛋白酶是指能催化肽键水解的一类酶,自1914年被试验用于洗涤剂的添加成分以来,由于具有重要的商业用途而被广泛关注。当今蛋白酶的产量占据酶市场的40%以上,广泛应用与洗涤剂、食品、制药、制革、诊断试剂、污水处理等领域。蛋白酶广泛存在于动物、植物、真菌及原核生物等所有生物中,其中微生物来源的蛋白酶占据目前蛋白酶生产总量的2/3以上。微生物来源的蛋白酶又可根据酶的反应pH、活性基团的特征等分为金属蛋白酶、天冬氨酸蛋白酶和丝氨酸蛋白酶等。
蛋白酶催化的最普遍的反应是水解蛋白质中的肽键。通常情况下,蛋白酶催化水解肽键的反应是在特定的pH的缓冲液中进行的,而且蛋白酶对其所催化肽键的氨基酸残基具有特异性要求,即有着高度的区域选择性和立体选择性。随着酶工程和溶剂工程技术的发展,人们发现蛋白酶在有机溶剂中能够在有机溶剂中能催化一些水溶液中不能进行的反应。如,1984年,Klibanov等人发现蛋白酶在一些有机溶剂中保存较长时间仍然具有活性,而且可以催化合成肽键的反应。大量研究表明,有机溶剂中进行酶促反应有着许多的优点:1、增大各种有机底物在溶剂中,特别是在亲水性有机溶剂中的溶解度,提高反应效率;2、具有高度的立体选择性和区域选择性,有机介质可改变选择性;3、控制反应平衡向所需方向移动,如水解酶在有机介质中能催化脱水缩合反应;4、有效防止微生物污染,产物易于分离纯化等。这一发现促进了非水酶学的兴起。如本研究室利用自行筛选到的耐有机溶剂蛋白酶在亲水性有机溶剂二甲基亚砜(DMSO)中实现了甜味剂Aspartame的前体和内吗啡呔1的合成,产率达到90%以上,而且在实现了底物与产物的反应分离耦合,大大提高了产率,简化了产物的分离纯化。说明在亲水性有机溶剂体系中进行酶催化反应的市场前景是非常巨大的。
有机溶剂体系中酶催化的诱人前景与酶易失活的矛盾推动了酶的改造与非水酶学的发展。过去20多年,研究者致力于提高酶在有机溶剂中的稳定性,常用方法:1、使用疏水性有机溶剂作为反应介质并控制介质中的含水量,以保证酶的“必需水”;2、在反应介质中加入保护剂甘油、乙二醇、多元醇聚合物等,或加入环糊精等冷冻干燥保护剂,以提高酶分子在有机介质中的稳定性。3、利用固定化、包埋法、大分子修饰等理化修饰方法提高酶在有机介质中的活性和稳定性;4、利用基因改造提高酶的有机溶剂稳定性。例如,Arnold小组利用易错PCR定向改造枯草杆菌蛋白酶(Subtilisin),提高其在有机相中的活力,对编码该蛋白酶成熟肽的DNA片段进行突变,并筛选得到在高浓度二甲基甲酰胺(DMF)中酶活性明显提高的突变株,其中突变体PC3在60%和85%的DMF中的酶活力分别是天然酶的256和131倍。此后再在PC3的基础上进行突变,得到的突变体13M酶活力比PC3还要高3倍。
虽然近年来在利用基因改造提高酶的有机溶剂稳定性方面取得了一定的进展,但往往出发酶的有机溶剂耐受性相对较低,有机溶剂耐性,特别是在亲水性的有机溶剂中提高的幅度不大,对工业应用还有一定的差距。
发明内容
本发明的技术目的在于提供一种高耐有机溶剂碱性蛋白酶的突变基因,使得本发明所述的这种蛋白酶相对于现有技术的同类蛋白酶而言,其有机溶剂,特别是亲水性有机溶剂的耐受性进一步大大提高。
为了实现本发明的技术目的,本发明的技术方案在于:
一、一种耐有机溶剂碱性蛋白酶,其特征在于其具有SEQ ID NO:1所示的核苷酸序列,其氨基酸序列示于SEQ ID NO:2;或者其具有SEQ ID NO:3所示的核苷酸序列,其氨基酸序列示于SEQ ID NO:4。
本发明所述的耐有机溶剂碱性蛋白酶基因的出发原始蛋白酶基因来自于中国专利申请(专利申请公开号CN101215534A)所述的耐有机溶剂碱性蛋白酶基因,其核苷酸序列如本发明序列表SEQ ID NO:5所示。从序列表的SEQ ID NO:1或SEQ ID NO:3可见,本发明所述耐有机溶剂碱性蛋白酶在原核苷酸序列的第385或第436发生了定点突变,即其成熟肽段的第24位突变氨基酸为谷氨酸或者谷氨酰胺;第41位氨基酸为丙氨酸或者赖氨酸。突变后的四种编码后的蛋白质的有机溶剂,特别是亲水性有机溶剂的耐受性明显大大加强。
二、本发明所述耐有机溶剂碱性蛋白酶基因的克隆及制备方法,包括如下步骤。
(1)选用专利申请公开号CN101215534A中所述Bacillus licheniformi YP1A(CCTCC NO: M207021)耐有机溶剂碱性蛋白酶编码基因的克隆方法克隆编码耐有机溶剂碱性蛋白酶基因,同时根据Bacillus subtilis 168中p43启动子的基因序列(GenBank号:K02714)设计引物扩增p43启动子,用重叠PCR的方法把启动子p43和上述耐有机溶剂碱性蛋白酶基因连接,最后与表达载体pHY300(由南京农业大学李顺鹏教授惠赠)连接,转化枯草芽孢杆菌宿主WB800(由南京农业大学李顺鹏教授惠赠),构建好重组菌WB800-pHY300-p43-YP1A进行表达。
(2)选用构建好的基因工程菌WB800-pHY300-p43-YP1A于37℃摇床培养过夜,提取质粒作为定向进化的模板。
(3)根据易错滚环复制的原理,设计3’末端硫代修饰的六聚体引物:5’-NpNpNpNpsNpsN-3’。
(4)以获得的质粒DNA为模板,用Φ 29 DNA 聚合酶(Fermentas #EP0097)进行易错滚环复制扩增,程序如下:95℃变性3min;迅速置冰上冷却至室温;30℃反应24 h。
(5)反应产物用DpnⅠ酶(Fermentas #ER1701)消化1 h,进行胶回收浓缩。
(6)胶回收产物转化枯草杆菌WB800,涂布牛奶平板进行初筛。
(7)选择有透明圈的突变株进行摇瓶培养7天,离心取上清液即为粗酶液,进行有机溶剂耐受性复筛,发现命名为K8和P8的突变株在50%(v/v)的DMF中的酶活力分别是原始酶的2.29和1.88倍;而命名为Z8和Z25的突变株在50%(v/v)的DMF中的酶活力分别是原始酶的1.76和1.8倍,如表二。
(8)对步骤(7)所述的突变株的主要性质进行了研究,发现四种突变体最适反应pH值,pH值稳定性都没有发生明显变化;三种突变体P8、Z8、Z25的最适反应温度和温度稳定性也没显著改变;而K8的最适反应温度和温度稳定性都有所提高了;突变株在各种有机溶剂中的稳定性都有显著的提高。核苷酸及氨基酸分析发现,突变基因与YP1A基因相比,分别从起始密码子后385 bp(K8/P8)和436 bp(Z8/Z25)处发生了突变,在第385位分别从GCG突变为CAG和GAG;氨基酸分别从Ala突变为Gln和Glu;从GAC突变为GCA和AAG;氨基酸从Asp突变为Ala和Lys。
三、本发明所述耐有机溶剂碱性蛋白酶在有机溶剂中催化合成小肽中的应用。
本发明的有益效果在于:
(1)本发明从天然耐有机溶剂碱性蛋白酶出发,通过定向进化的手段对其基因改造,获得了四个各种有机溶剂,特别是亲水性有机溶剂耐受性更强的突变基因,突变株在50%(v/v)亲水性有机溶剂中的酶活比原始酶活高1.5倍以上,在丙酮和乙腈溶剂中的酶活是原始酶活的30倍以上。目前未见有相关报道。
(2)本发明的YP1A基因的开放阅读框含有1140bp,编码379个氨基酸。核苷酸及氨基酸分析发现,突变基因与YP1A基因相比,分别从起始密码子后385 bp(K8/P8)和436 bp(Z8/Z25)处发生了突变;从序列表的SEQ ID NO:1或SEQ ID NO:3可见,本发明所述耐有机溶剂碱性蛋白酶在原核苷酸序列的第385或第436发生了定点突变,即其成熟肽段的第24位突变氨基酸为谷氨酸或者谷氨酰胺;第41位氨基酸为丙氨酸或者赖氨酸。突变后的四种编码后的蛋白质的有机溶剂,特别是亲水性有机溶剂耐受性明显大大加强。
(3)本发明所述的耐有机溶剂碱性蛋白酶能应用于在有机溶剂中催化合成小肽中,其超高的有机溶剂,特别是亲水性有机溶剂耐受性相对于现有技术的耐有机溶剂蛋白酶而言更加适用于小肽的合成领域。
附图说明
图1显示易错滚环复制电泳图和突变株做牛奶平板上的初步筛选。
图2显示突变株最适反应pH值。
图3显示突变株pH值稳定性。
图4显示突变株最适反应温度。
图5显示突变株温度稳定性。
图6和7显示突变株在DMF/DMSO中的稳定性。
具体实施方式
下面结合具体实例对本发明作进一步详细的描述。
实施例1
本实施例说明本发明所述耐有机溶剂碱性蛋白酶基因的定向进化。
选用专利申请公开号CN101215534A中所述Bacillus licheniformi YP1A(CCTCC NO: M207021)耐有机溶剂碱性蛋白酶编码基因的克隆方法克隆编码耐有机溶剂碱性蛋白酶基因,同时根据Bacillus subtilis 168中p43启动子的基因序列(GenBank号:K02714)设计引物扩增p43启动子,用重叠PCR的方法把启动子p43和耐有机溶剂碱性蛋白酶基因连接,最后与表达载体pHY300(由南京农业大学李顺鹏教授惠赠)连接,转化枯草芽孢杆菌宿主WB800(由南京农业大学李顺鹏教授惠赠),构建好重组菌WB800-pHY300-p43-YP1A进行表达,具体实施方法如下:
(1)设计扩增YP1A基因和启动子p43引物。
YP1A引物:
YP1AF:5-GAGAGGAATGTACACATGATGAGGAAAAAG-3;
YP1AR:5-CGGATCCTTATTGAGCGGCAGCTTC-3。
启动子引物:
p43F:5-GCAGATCTTGATAGGTGGTATGTTTTCGCT-3;
p43R:5-CTCTTTTTCCTCATCATGTGTACATTCCTC-3。
(2)分别按以下程序扩增YP1A基因和启动子p43。
①YP1A基因的扩增:94℃预变性5min;94℃变性30 sec;55℃退火30 sec;72℃
延伸1 min;30循环后,72℃保温10 min,根据该反应条件,扩增到了约1.1 kb的PCR片段。
②启动子p43的扩增:94℃预变性5 min;94℃变性30 sec;50℃退火30 sec;72℃
延伸30 sec;30循环后,72℃保温10 min,根据该反应条件,扩增到了约300 bp的PCR片段。
③YP1A-p43扩增:通过重叠PCR的方法连接YP1A基因与启动子p43,94℃预变性5 min;94℃变性30 sec;65℃退火30 sec(退火温度设计梯度下降1℃);72℃延伸2 min;19循环后;94℃变性30 sec;45℃退火30 sec;72℃延伸2 min;16循环后,72℃保温10 min。
④将重叠PCR产物进行纯化后用限制性内切酶BglⅡ(大连宝生物公司)与BamHⅠ(大连宝生物公司) 进行酶切,同时用同样的酶对载体pHY300进行酶切,纯化酶切产物用T4(大连宝生物公司)连接酶在16℃下连接过夜,转化枯草芽孢杆菌WB800进行表达。
选用构建好的基因工程菌WB800-pHY300-p43-YP1A于37℃摇床培养过夜,提取质粒作为突变模板;根据易错滚环复制的原理,设计3’末端硫代修饰的六聚体引物:5’-NpNpNpNpsNpsN-3’;按照以下程序进行突变:
(1)50 μL反应体系:Tris-HCl(pH7.5) 终浓度50 mM;硫酸铵终浓度10mM,MgCl2终浓度10 mM,DTT终浓度4 mM,BSA终浓度200 ng/μL,dNTP0.2 mM,引物六聚体4 μM,质粒DNA40 pM,MnCl2 0.8 mM, Φ 29 DNA 聚合酶5U。
(2)反应程序:把上述反应组分除聚合酶和MnCl2外,加到500 μL反应管中混匀,于95℃下变性3 min;把反应管迅速置于冰上冷却至室温;加入聚合酶和MnCl2;于30℃反应24 h。
(3)反应产物按照一定的体系加入DpnⅠ酶消化1 h,进行胶回收。
(4)回收产物直接转化到枯草杆菌WB800表达系统,于牛奶平板上选择有透明圈突变株进一步筛选(如图1)。
(5)选择有透明圈的突变株进行摇瓶培养7天,离心取上清液即为粗酶液,按照下列程序进行有机溶剂耐受性筛选。
把突变株接种于发酵培养基中培养7天,离心回收上清液即为突变株的粗酶液,在冰浴上取45%(V/V)体积的粗酶液,加入55%(V/V)体积亲水性有机溶剂(DMF)于3 mL密封小瓶子中,于37℃,200 rpm振荡1.5 h后测量残留蛋白酶活力。对照组为加入55%体积(V/V)缓冲液。进行有机溶剂耐受性筛选,选择耐受性变化明显的突变株提取质粒测序,确定突变位点。发现突变株K8和P8在55%的DMF中的耐受性分别提高了55%和30%;而Z8和Z25分别提高了29%和51%,如表一:
表一 突变筛选结果
本发明还对突变株的主要性质进行了研究,发现四种突变体最适反应pH值,pH值稳定性都没有发生明显变化(如图2,3);三种突变体P8、Z8、Z25的最适反应温度和温度稳定性也没显著改变(如图4,5);而K8的最适反应温度和温度稳定性都有所提高了(如图4,5);突变株在各种有机溶剂,特别是亲水性有机溶剂中的稳定性都有显著的提高(如表二,图6,7)。本发明的YP1A基因的开放阅读框含有1140bp,编码379个氨基酸。核苷酸及氨基酸分析发现,突变基因与YP1A基因相比,分别从起始密码子后385 bp(K8/P8)和436 bp(Z8/Z25)处发生了突变,在第385位分别从GCG突变为CAG和GAG;氨基酸分别从Ala突变为Gln和Glu;从GAC突变为GCA和AAG;氨基酸从Asp突变为Ala和Lys。
表二 突变株溶剂稳定性
注:DMSO浓度为65%(v/v),其它溶剂浓度为50%(v/v)。
序列表
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-40 -35 -30
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
-25 -20 -15 -10
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
-5 -1 1 5
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
10 15 20
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
25 30 35
Pro Xaa Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
40 45 50 55
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
60 65 70
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
75 80 85
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
90 95 100
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
105 110 115
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
120 125 130 135
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
140 145 150
Ala Gly Asn Ser Gly Pro Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
155 160 165
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
170 175 180
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
185 190 195
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
200 205 210 215
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
220 225 230
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
235 240 245
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
250 255 260
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
265 270
<210> 5
<211> 1140
<212> DNA
<213> Bacillus licheniformis YP1A蛋白酶基因
<400> 5
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttaatgct cgtgttcacg 60
atggcattca gcgattccgc ttctgctgct caactggcga aaaatgttga aaaggattat 120
atcgtcggat ttaagtcagg agtgaaaacc gcatccgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcaca catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtatcct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggaccttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggcgcgg tagactctaa cagcaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
Claims (1)
1.一种耐有机溶剂碱性蛋白酶,其特征在于其具有SEQ ID NO:1所示的核苷酸序列,其氨基酸序列示于SEQ ID NO:2;或者其具有SEQ ID NO:3所示的核苷酸序列,其氨基酸序列示于SEQ ID NO:4。
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WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101215534A (zh) * | 2007-12-28 | 2008-07-09 | 南京工业大学 | 一种耐有机溶剂碱性蛋白酶产生菌以及该耐有机溶剂碱性蛋白酶的基因和应用 |
WO2010126156A2 (en) * | 2009-04-30 | 2010-11-04 | Kao Corporation | Alkaline protease variants |
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CN101215534A (zh) * | 2007-12-28 | 2008-07-09 | 南京工业大学 | 一种耐有机溶剂碱性蛋白酶产生菌以及该耐有机溶剂碱性蛋白酶的基因和应用 |
WO2010126156A2 (en) * | 2009-04-30 | 2010-11-04 | Kao Corporation | Alkaline protease variants |
Non-Patent Citations (1)
Title |
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何小丹等: "地衣芽孢杆菌YP1A耐有机溶剂蛋白酶基因的克隆与功能表达", 《生物加工过程》 * |
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WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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