CN111607575B - 一种转氨酶phta、制备方法和应用 - Google Patents
一种转氨酶phta、制备方法和应用 Download PDFInfo
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- CN111607575B CN111607575B CN202010263854.4A CN202010263854A CN111607575B CN 111607575 B CN111607575 B CN 111607575B CN 202010263854 A CN202010263854 A CN 202010263854A CN 111607575 B CN111607575 B CN 111607575B
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- phta
- transaminase
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Abstract
本发明涉及一种转氨酶PHTA，所述转氨酶PHTA的氨基酸序列为SEQ ID NO.1。本发明转氨酶PHTA的最适温度35℃，最适pH为8.0，在pH 6‑8范围内仍保持50％以上的活性。在最适温度和最适pH下，本发明的转氨酶PHTA对HFB1的降解率为100％；本发明转氨酶PHTA性质优良，该酶可以应用于农业、饲料和食品等工业，减少伏马毒素FB1对动物及人类健康的危害。
Description
技术领域
本发明属于农业生物技术领域，尤其是一种转氨酶PHTA、制备方法和应用。
背景技术
伏马毒素是一类结构相关的代谢物，主要由镰刀菌和增殖镰刀菌产生。目前已鉴定出28种伏马毒素类似物，被分为A、B、C和P四个系列。其中伏马毒素B1(FB1)是最普遍、毒性最强的伏马毒素。它会引起马的白质脑软化和猪的肺水肿，也会影响家禽的肝脏和免疫系统。此外，FB1对大多数哺乳动物都有肝肾毒性。
消除食品和饲料中伏马毒素污染的缓解策略分为物理、化学和生物原理。物理方法，包括干法和湿磨法，浸泡、加热以及吸附剂的使用。然而，这些策略或因为使用仪器昂贵，或因为造成饲料中某些营养成分的损失，因此存在局限性。氨化和碱化是最常见的化学脱毒方法，但由于其潜在的毒性和对原料的口感与营养品质存在负面影响，其应用也受到限制。生物脱毒技术可以在不影响食品和饲料质量的前提下降低霉菌毒素毒性，被认为是一种很有前途的脱毒策略。酶制剂在存贮方面相比于微生物制剂而言具有更高的稳定性，因此，研发降解真菌毒素的酶制剂，可以很好地遏制被污染的粮食作物中的毒素的产生，挽回经济损失。
通过检索，尚未发现与本发明专利申请相关的专利公开文献。
发明内容
本发明目的在于克服现有技术的不足之处，提供一种转氨酶PHTA、制备方法和应用，该转氨酶PHTA能够降解伏马毒素中的氨基以解除其毒性。
本发明解决其技术问题所采用的技术方案是：
一种转氨酶PHTA，所述转氨酶PHTA的氨基酸序列为SEQ ID NO.1。
如上所述的转氨酶PHTA的制备方法，步骤如下：
⑴用包含编码所述转氨酶PHTA的基因的重组载体转化宿主细胞，得到重组菌株；
⑵培养重组菌株，诱导转氨酶PHTA表达；
⑶分离纯化获得的转氨酶PHTA。
而且，所述步骤⑴中宿主细胞为大肠细胞、啤酒酵母细胞或多型逊酵母细胞。
如上所述的转氨酶PHTA在降解伏马毒素方面中的应用。
一种编码如上所述的转氨酶PHTA的转氨酶PHTA基因。
而且，所述基因的核苷酸序列为SEQ ID NO.2。
包含如上所述的转氨酶PHTA基因的重组载体。
包含如上所述的转氨酶PHTA基因的重组载体pET28a(+)-PHTA。
包含如上所述的转氨酶PHTA基因的重组菌株。
而且，所述重组菌株为大肠杆菌。
本发明取得的优点和积极效果为：
1、本发明转氨酶PHTA的最适温度35℃，最适pH为8.0，在pH 6-8范围内仍保持50％
以上的活性。在最适温度和最适pH下，本发明的转氨酶PHTA对HFB1的降解率为100％；
本发明转氨酶PHTA性质优良，该酶可以应用于农业、饲料和食品等工业，减少伏马毒素FB1对动物及人类健康的危害。
2、本发明转氨酶PHTA可以降解催化水解伏马毒素FB1(HFB1)的氨基，消除伏马毒素的活性。由于氨基是使得伏马毒素具有毒性作用的关键官能团之一，因此去除氨基是解除伏马毒素毒性的关键点。本发明的转氨酶PHTA具有降解水解伏马毒素HFB1的活性，可以应用于农业、饲料和食品等工业，减少伏马毒素对动物及人类健康的危害。
附图说明
图1为本发明中转氨酶PHTA的SDS-PAGE纯化图；其中，M：蛋白marker；1:转氨酶PHTA；
图2为本发明中显示转氨酶PHTA的降解效果示意图；
图3为本发明中显示转氨酶PHTA的最适温度图；
图4为本发明中显示转氨酶PHTA的最适pH图。
具体实施方式
下面详细叙述本发明的实施例，需要说明的是，本实施例是叙述性的，不是限定性的，不能以此限定本发明的保护范围。
本发明中所使用的原料，如无特殊说明，均为常规的市售产品；本发明中所使用的方法，如无特殊说明，均为本领域的常规方法。
一种转氨酶PHTA，所述转氨酶PHTA的氨基酸序列为SEQ ID NO.1。
如上所述的转氨酶PHTA的制备方法，步骤如下：
⑴用包含编码所述转氨酶PHTA的基因的重组载体转化宿主细胞，得到重组菌株；
⑵培养重组菌株，诱导转氨酶PHTA表达；
⑶分离纯化获得的转氨酶PHTA。
较优地，所述步骤⑴中宿主细胞为大肠细胞、啤酒酵母细胞或多型逊酵母细胞，优选为大肠杆菌BL21(DE3)。
如上所述的转氨酶PHTA在降解伏马毒素方面中的应用。
一种编码如上所述的转氨酶PHTA的转氨酶PHTA基因。
较优地，所述基因的核苷酸序列为SEQ ID NO.2。
包含如上所述的转氨酶PHTA基因的重组载体。
包含如上所述的转氨酶PHTA基因的重组载体pET28a(+)-PHTA。
包含如上所述的转氨酶PHTA基因的重组菌株。
较优地，所述重组菌株为大肠杆菌。
具体地，所述转氨酶PHTA，其氨基酸序列如SEQ ID NO.1所示：
SEQ ID NO.1：
MTRQRAKDAELRERAYRVVPGGVYGHLSTALLPSGYPQFFRRGKGAHLWDVDDNMYIDYLCAYGPNLFGYGFEPIERAAVRQQNLGDTLTGPTEALVELAEAFVGMVTHADWAMFCKNGTDANTIALMISRAHTGRATVLVAEGAYHGAAPWSTPRPAGVTAGDRANIVTYRYNDPESLAAAYRAHRHDLAAIFATPFRHEVFADQEDLNVTYARLARELCDQAGALLVVDDVRAGFRIARDCSWSPSGVQPDLSAWGKCFANGYPISAVLGSDKARKAAAEIFVTGSFWMSATPMAAAIEGLKQIRETDYLERLVESGLALRHGLQRQAAAHGFTLRQTGPAQMPQILFEEDPDFRVGYAWAEACVERGVYFSPYHNMFLSTAHTDGVIRRTLEVTDVAFETVKRQRGSLRTPAQLVPYAQEMAARLAT
其中，该酶基因编码430个氨基酸，没有信号肽，因此，成熟的转氨酶PHTA的理论分子量为48.21kDa。
本发明提供了编码上述转氨酶PHTA。该基因的基因组序列如SEQ ID NO.2所示：
SEQ ID NO.2：
ATGACTAGACAGAGAGCTAAGGACGCTGAGTTGAGAGAGAGAGCCTACAGAGTTGTTCCAGGTGGTGTTTACGGTCACTTGTCCACTGCTTTGTTGCCATCTGGTTACCCACAGTTCTTCAGACGTGGTAAGGGTGCTCATTTGTGGGATGTTGACGACAACATGTACATCGACTACTTGTGTGCCTACGGTCCAAACTTGTTCGGTTACGGTTTCGAGCCAATTGAGAGAGCTGCTGTCAGACAACAAAACTTGGGTGACACTTTGACTGGTCCAACCGAGGCTTTGGTTGAATTGGCTGAGGCTTTCGTTGGTATGGTTACTCATGCTGACTGGGCCATGTTCTGCAAGAACGGTACTGACGCTAACACTATCGCCTTGATGATTTCCAGAGCACACACTGGTAGAGCCACTGTTTTGGTTGCTGAAGGTGCTTATCATGGTGCTGCACCTTGGTCTACTCCAAGACCTGCTGGTGTTACTGCTGGTGATAGAGCTAACATCGTCACCTACAGATACAACGACCCAGAATCTTTGGCTGCTGCTTACAGAGCCCATAGACATGACTTGGCTGCTATCTTCGCTACCCCATTCAGACACGAAGTTTTCGCTGATCAAGAGGACCTGAACGTTACCTACGCTAGATTGGCCAGAGAATTGTGTGATCAGGCTGGTGCCTTGTTGGTTGTTGATGATGTTAGAGCCGGTTTCAGAATCGCCAGAGACTGTTCTTGGTCCCCATCTGGTGTTCAACCAGATTTGTCTGCTTGGGGTAAGTGTTTCGCTAACGGTTACCCAATCTCCGCTGTTTTGGGTTCTGACAAGGCTAGAAAAGCTGCCGCCGAGATTTTCGTTACTGGTTCTTTTTGGATGTCCGCCACTCCAATGGCTGCCGCTATTGAAGGTTTGAAGCAGATCAGAGAGACTGACTACTTGGAGAGATTGGTCGAATCCGGTTTGGCTTTGAGACACGGTCTGCAAAGACAAGCTGCTGCTCACGGTTTTACCTTGAGACAAACTGGTCCAGCTCAGATGCCACAGATTTTGTTCGAAGAGGACCCAGACTTCAGAGTTGGTTATGCTTGGGCTGAAGCCTGTGTTGAAAGAGGTGTTTACTTCTCCCCATACCACAACATGTTCTTGTCCACCGCTCACACTGACGGTGTTATCAGAAGAACTTTGGAGGTTACCGACGTCGCCTTCGAGACTGTTAAGAGACAAAGAGGTTCCCTGAGAACCCCAGCTCAATTGGTTCCATACGCTCAAGAAATGGCTGCTAGACTGGCTACT
本发明通过PCR的方法分离克隆了转氨酶PHTA，DNA全序列分析结果表明，转氨酶PHTA基因开放阅读框架序列(ORF)全长1290bp。
本发明还提供了包含上述转氨酶PHTA的重组载体，将优选重组载体命名为pET28a-PHTA。将本发明的转氨酶PHTA基因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，优选为将本发明的解毒酶基因插入到质粒pET28a上的EcoR I和Xhol I限制性酶切位点之间，使该核苷酸序列位于T7启动子的下游并受其调控，得到重组大肠表达质粒pET28a-PHTA。
更具体地，相关制备及检测如下：
试验材料和试剂：
1、菌株及载体：本发明得到一种新的转氨酶PHTA。大肠杆菌表达载体pET28a(+)及菌株BL21(DE3)为实验室保存。
2、酶类及其它生化试剂：内切酶购自TaKaRa公司，连接酶购自Invitrogen公司。购自Sigma公司，其它都为国产试剂(均可从普通生化试剂公司购买得到)。
3、培养基：
大肠杆菌培养基LB(1％蛋白胨、0.5％酵母提取物、1％NaCl，pH 7.0)。
说明：以下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。
一、转氨酶PHTA的克隆
利用人工化学合成的方法获得转氨酶PHTA的基因片段，并在5’端引入内切酶位点EcoR I、在3’端引入内切酶位点Xhol I。
二、重组转氨酶PHTA的制备
将表达载体pET28a进行双酶切(EcoR I+XholI)，同时将编码转氨酶PHTA的基因双酶切(EcoR I+XholI)，切出编码成熟转氨酶的基因片段与表达载体pET28a连接，获得含有转氨酶基因PHTA的重组质粒pET28a-PHTA并转化大肠杆菌BL21(DE3)，获得重组大肠杆菌菌株BL21/PHTA。
取含有质粒的BL21(DE3)菌株，接种于100mL LB培养液中，37℃、220rpm振荡培养约2h后，加入1mM IPTG，置于25℃220rpm进行诱导，约20h后测定胞内和胞外的转氨酶活力。在胞内检测到转氨酶酶的活性，经过镍柱纯化，SDS-PAGE结果表明，重组转氨酶得到了表达。如图1所示，泳道1为纯化后的结果。
三、重组转氨酶的性质测定
高效液相色谱检测转氨酶的酶活，具体方法如下：
(1)HFB1标准储备液：配制成浓度为100μg/mL的标准液，-20℃保存。
(2)样品的制备：取纯化好的转氨酶酶液850μL，加入100μL的HFB1标准储备液和50μL的丙酮酸溶液，使HFB1的终浓度为10μg/mL，37℃，220rpm，避光培养20min。
(3)样品衍生化：取待测样品100μL，加入400μL 50％乙腈水，500μL OPA衍生液，混匀30s，衍生2min内进样，过滤膜待测。通过与HFB1的标品的峰图对比，来确定转氨酶PHTA的酶活性。
1、测定伏马毒素降解酶的降解能力
在900μL的转氨酶PHTA的酶液与丙酮酸的混合液(柠檬酸-磷酸氢二钠缓冲液，pH＝7.0)中加入100μL的HFB1溶液，使得HFB1的终浓度为10μg/mL。将反应液置于37℃，pH＝7的条件下，反应12h，未添加纯化后的转氨酶PHTA的溶液作为对照。反应完成后沸水煮10min，让酶失活。冷却至室温后过膜，待高效液相色谱检测。
结果展示在图2中，其中图2a表示的是缓冲液与HFB1的混合溶液，图2b表示的是酶液与HFB1的反应液。可以看出HFB1在11.646min的时候会出现最高峰，而加入了纯化后的重组转氨酶PHTA的溶液中未检测到HFB1。因此，可以得出转氨酶PHTA具有将HFB1完全降解的能力，能够在12h内将10μg/mLHFB1降解完全。
2、测定伏马毒素降解酶的最适温度
以HFB1为底物，取100μL底物并加入850μL的酶液和50μL丙酮酸溶液，终浓度为10μg/mL，在柠檬酸-磷酸氢二钠缓冲液(pH 7.0)缓冲液体系及不同温度下，反应1h，然后沸水煮10min，让酶失活。冷却至室温后过膜，待高效液相色谱检测。
如图3所示，转氨酶PHTA的最适温度为35℃。温度高于35℃后，酶的活力呈现不断下降的现象，到70摄氏度时，仅具有不高于10％的相对活力。
转氨酶PHTA的最适温度35℃与哺乳类动物体温37℃非常接近，转氨酶PHTA在用于动物饲料时，在动物体内会发挥较大的效力。
3、测定转氨酶PHTA的最适pH
将纯化的重组转氨酶PHTA在不同的pH缓冲液下进行酶促反应，以测定其最适pH，所选用的缓冲溶液的缓冲梯度为100mM柠檬酸-磷酸氢二钠(pH3.0–8.0)，100mM的Tris-HCl(pH7.0-9.0)，100mM甘氨酸-NaOH(pH 9.0–12.0)。转氨酶PHTA在上述不同的缓冲液中与底物HFB1(终浓度为1μg/mL)在37℃反应1h，沸水煮10min，高效液相色谱检测。
结果如图4所示，转氨酶PHTA的最适pH为8.0。在pH＝3-8之间时，转氨酶PHTA的相对酶活力不断上升，由20％升至100％，但是在pH大于8之后，呈现梯度很大的下降，当pH＝10时，相对酶活力不到20％。转氨酶PHTA的酸碱能容度较高，无论是在添加进饲料存储还是进入动物肠胃，都会保持一定的活力，达到降解毒素的目的。
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例所公开的内容。
序列表
<110> 天津科技大学
<120> 一种转氨酶PHTA、制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 430
<212> PRT
<213> 转氨酶PHTA的氨基酸序列(Unknown)
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Pro Ser Gly Tyr Pro Gln Phe Phe Arg Arg Gly Lys Gly Ala His Leu
35 40 45
Trp Asp Val Asp Asp Asn Met Tyr Ile Asp Tyr Leu Cys Ala Tyr Gly
50 55 60
Pro Asn Leu Phe Gly Tyr Gly Phe Glu Pro Ile Glu Arg Ala Ala Val
65 70 75 80
Arg Gln Gln Asn Leu Gly Asp Thr Leu Thr Gly Pro Thr Glu Ala Leu
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Val Glu Leu Ala Glu Ala Phe Val Gly Met Val Thr His Ala Asp Trp
100 105 110
Ala Met Phe Cys Lys Asn Gly Thr Asp Ala Asn Thr Ile Ala Leu Met
115 120 125
Ile Ser Arg Ala His Thr Gly Arg Ala Thr Val Leu Val Ala Glu Gly
130 135 140
Ala Tyr His Gly Ala Ala Pro Trp Ser Thr Pro Arg Pro Ala Gly Val
145 150 155 160
Thr Ala Gly Asp Arg Ala Asn Ile Val Thr Tyr Arg Tyr Asn Asp Pro
165 170 175
Glu Ser Leu Ala Ala Ala Tyr Arg Ala His Arg His Asp Leu Ala Ala
180 185 190
Ile Phe Ala Thr Pro Phe Arg His Glu Val Phe Ala Asp Gln Glu Asp
195 200 205
Leu Asn Val Thr Tyr Ala Arg Leu Ala Arg Glu Leu Cys Asp Gln Ala
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Gly Ala Leu Leu Val Val Asp Asp Val Arg Ala Gly Phe Arg Ile Ala
225 230 235 240
Arg Asp Cys Ser Trp Ser Pro Ser Gly Val Gln Pro Asp Leu Ser Ala
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370 375 380
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<210> 2
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<212> DNA/RNA
<213> 转氨酶PHTA基因(Unknown)
<400> 2
atgactagac agagagctaa ggacgctgag ttgagagaga gagcctacag agttgttcca 60
ggtggtgttt acggtcactt gtccactgct ttgttgccat ctggttaccc acagttcttc 120
agacgtggta agggtgctca tttgtgggat gttgacgaca acatgtacat cgactacttg 180
tgtgcctacg gtccaaactt gttcggttac ggtttcgagc caattgagag agctgctgtc 240
agacaacaaa acttgggtga cactttgact ggtccaaccg aggctttggt tgaattggct 300
gaggctttcg ttggtatggt tactcatgct gactgggcca tgttctgcaa gaacggtact 360
gacgctaaca ctatcgcctt gatgatttcc agagcacaca ctggtagagc cactgttttg 420
gttgctgaag gtgcttatca tggtgctgca ccttggtcta ctccaagacc tgctggtgtt 480
actgctggtg atagagctaa catcgtcacc tacagataca acgacccaga atctttggct 540
gctgcttaca gagcccatag acatgacttg gctgctatct tcgctacccc attcagacac 600
gaagttttcg ctgatcaaga ggacctgaac gttacctacg ctagattggc cagagaattg 660
tgtgatcagg ctggtgcctt gttggttgtt gatgatgtta gagccggttt cagaatcgcc 720
agagactgtt cttggtcccc atctggtgtt caaccagatt tgtctgcttg gggtaagtgt 780
ttcgctaacg gttacccaat ctccgctgtt ttgggttctg acaaggctag aaaagctgcc 840
gccgagattt tcgttactgg ttctttttgg atgtccgcca ctccaatggc tgccgctatt 900
gaaggtttga agcagatcag agagactgac tacttggaga gattggtcga atccggtttg 960
gctttgagac acggtctgca aagacaagct gctgctcacg gttttacctt gagacaaact 1020
ggtccagctc agatgccaca gattttgttc gaagaggacc cagacttcag agttggttat 1080
gcttgggctg aagcctgtgt tgaaagaggt gtttacttct ccccatacca caacatgttc 1140
ttgtccaccg ctcacactga cggtgttatc agaagaactt tggaggttac cgacgtcgcc 1200
ttcgagactg ttaagagaca aagaggttcc ctgagaaccc cagctcaatt ggttccatac 1260
gctcaagaaa tggctgctag actggctact 1290
Claims (1)
1.转氨酶PHTA在降解伏马毒素B1方面中的应用，所述转氨酶PHTA的氨基酸序列为SEQID NO.1，所述应用为非疾病的治疗目的。
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