CN111004786A - 一种葡萄糖氧化酶及其载体与应用 - Google Patents
一种葡萄糖氧化酶及其载体与应用 Download PDFInfo
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- CN111004786A CN111004786A CN201911359714.0A CN201911359714A CN111004786A CN 111004786 A CN111004786 A CN 111004786A CN 201911359714 A CN201911359714 A CN 201911359714A CN 111004786 A CN111004786 A CN 111004786A
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- glucose oxidase
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Abstract
本发明涉及基因工程与发酵工程领域，本发明公开了一种葡萄糖葡萄糖氧化酶GOD及其基因、制备与应用，其具有如SEQ ID NO：1所示的氨基酸序列。本发明的技术方案利用基因工程等技术手段生产性质优良的葡萄糖氧化酶，通过3L发酵罐验证发酵酶活可达1600U/mL，比活力为296U/mg，在70度处理5min还有70％以上的酶活力，同时对沙门氏杆菌，猪巴氏杆菌，副猪嗜血杆菌，致病大肠杆菌，铜绿假单胞菌，金黄色葡萄球菌的生长可产生明显抑制作用，在养殖过程中适当添加本产品可降低料肉比，节约原料，可广泛应用于饲料，食品，医药等工业。
Description
技术领域
本发明涉及酶工程技术领域，更具体地，涉及一种葡萄糖氧化酶及其载体与应用。
背景技术
葡萄糖氧化酶(glucose oxidase，GOD)是一种需氧脱氢酶，能专一性的将葡萄糖与氧气催化生成葡萄糖酸和过氧化氢，因此，其系统命名又称为D-葡萄糖氧化还原酶，广泛分布于动物，植物，微生物体内，在饲料，食品，医药等领域应用广泛，并取得了可观的商业价值。在饲料中添加葡萄糖氧化酶能具有抗氧化功能，显著抑制霉变微生物的产生，对霉变饲料毒素有很好的降解作用，从而提高动物的免疫力，代替药物或抗生素发挥作用；葡萄糖氧化酶在面包，牛奶，果汁，啤酒等领域去氧保鲜效果良好；在医药领域中可以制成尿，糖，血糖试纸，在临床上广泛应用。
目前，GOD普遍采用微生物发酵法进行生产，如黑曲霉，青霉属等，但生产水平较低，生产成本高，抑菌效果不佳。且饲料中抗生素的滥用本身也具有很多危害，包括以下几个方面，细菌的耐药性、饲料中药物残留、微生态平衡问题、畜禽机体免疫力下降、抗生素残留对人类的直接影响和间接影响、细菌耐药性对人类的危害等等。
发明内容
本发明第一个方面的目的，在于提供一种葡萄糖氧化酶。
本发明第二个方面的目的，在于提供一种葡萄糖氧化酶基因。
本发明第三个方面的目的，在于提供一种载体。
本发明第四个方面的目的，在于提供一种重组菌株。
本发明第五个方面的目的，在于提供一种抑菌剂。
本发明第六个方面的目的，在于提供一种饲料。
本发明所采取的技术方案是：
本发明的第一个方面，提供一种葡萄糖氧化酶，其氨基酸序列如SEQ ID NO.1所示。
本发明的第二个方面，一种葡萄糖氧化酶基因，所述葡萄糖氧化酶基因编码本发明第一个方面所述的葡萄糖氧化酶。
根据本发明第二个方面所述的葡萄糖氧化酶基因，其核苷酸序列如SEQ ID NO.2所示。
本发明的第三个方面，提供一种载体，所述载体可表达本发明第一个方面所述的葡萄糖氧化酶或含有本发明第二个方面所述的葡萄糖氧化酶基因。
本发明的第四个方面，提供一种重组菌株，含有第三个方面所述的载体。
根据本发明第四个方面所述的重组菌株，所述重组菌株为细菌或真菌细胞。
根据本发明第四个方面所述的重组菌株，所述重组菌株为酵母。
本发明的第五个方面，提供一种抑菌剂，含有本发明第一个方面所述的葡萄糖氧化酶。
根据本发明第五个方面所述的抑菌剂，所述抑菌剂应用于饲料，食品，医药领域。
本发明的第六个方面，提供一种饲料，含有本发明第五个方面所述的抑菌剂。
本发明的有益效果是：
本发明针对现有技术中构建得到的葡萄糖氧化酶生产菌的发酵能力较低，导致该酶的生产成本居高不下，制约了葡萄糖氧化酶的广泛应用的技术不足，通过对葡萄糖氧化酶进行定向进化和自然诱变，提供一中酶活显著提高、耐热性更好的葡萄糖氧化酶。该葡萄糖氧化酶具有很好的抑菌效果，可以显著抑制沙门氏杆菌、猪巴氏杆菌、副猪嗜血杆菌、致病大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌，可以被广泛应用于饲料，食品，医药领域，特别是在饲料领域，可以改善饲料中抗生素滥用的情况。
附图说明
图1为本实施例2中工程菌上罐发酵酶活；
图2为本发明葡萄糖氧化酶的耐热性；
图3为葡萄糖氧化酶液对沙门氏杆菌的平板抑菌效果；
图4为葡萄糖氧化酶液对猪巴氏杆菌的平板抑菌效果；
图5为葡萄糖氧化酶液对副猪嗜血杆菌的平板抑菌效果；
图6为葡萄糖氧化酶液对致病大肠杆菌的平板抑菌效果；
图7为葡萄糖氧化酶液对铜绿假单胞菌的平板抑菌效果；
图8为葡萄糖氧化酶液对金黄色葡萄球菌的平板抑菌效果。
具体实施方式
以下实施例中未作具体说明的分子生物学试验方法，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；所述试剂盒生物材料，如无特殊说明，均可从商业途径获得。
葡萄糖氧化酶测定方法
葡萄糖氧化酶活性测定采用邻—联茴香胺分光光度法。在葡萄糖氧化酶的作用下，葡萄糖和氧反应，生成葡萄糖酸和过氧化氢，过氧化氢和无色的还原型邻联茴香胺在过氧化物酶的作用下，生成水和红色的氧化型邻联茴香胺。在540nm下测定反应液吸光值，依据标准曲线计算葡萄糖氧化酶的酶活。
培养基：
LB培养基：1％蛋白胨，0.5％酵母粉，1％氯化钠；
BMGY培养基：1％酵母粉，2％蛋白胨，1.34％YNB，4*10-5％生物素，1％甘油(V/V)；
BMMY培养基：除0.5％的甲醇替代甘油外，其余成分均与BMGY相同。
实施例1葡萄糖氧化酶基因的优化与载体构建
从NCBI文库中获取了一来源于黑曲霉的葡萄糖氧化酶基因，经过定向进化和自然诱变筛选获得一株比活力和耐热均显著提高的新葡萄糖氧化酶基因god。
新葡萄糖氧化酶基因god的氨基酸序列如SEQ ID NO.1所示，其核苷酸序列如SEQID NO.2所示。
SNGIEASLLKDPKEVAGRTYDYIIAGGGLTGLTVAAKLTENPNITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNRTALIRSGNGLGGSTLINGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGLNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWVEYIPENFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQLSSHVMTVFYAMALKIADAVLADYASMQAPQPVPEAYAVSDPEAHPDDFAGMDANQLQKRGFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY*(SEQ ID NO.1)。
TCTAATGGTA TTGAGGCTTC CTTGTTGAAA GACCCAAAAG AGGTCGCCGG TAGAACCTACGACTACATCA TTGCCGGTGG TGGTTTGACC GGTTTGACCG TTGCTGCTAA GTTGACCGAG AATCCTAACATCACTGTTTT GGTTATTGAG TCCGGTTCCT ACGAGTCTGA CCGTGGTCCA ATTATTGAGG ATTTGAATGCCTACGGTGAC ATCTTCGGAT CTTCTGTCGA CCACGCCTAT GAGACCGTTG AGTTGGCTAC TAACAATAGAACTGCTTTGA TCCGTTCCGG TAACGGTTTG GGAGGATCCA CTTTGATTAA CGGTGGAACC TGGACTAGACCACATAAAGC CCAAGTCGAC TCCTGGGAGACTGTCTTCGG AAACGAAGGT TGGAACTGGG ACTCTGTTGCTGCTTACTCC CTTCAGGCTG AAAGAGCTCG TGCCCCAAAT GCTAAGCAGA TCGCCGCTGG TCACTACTTTAACGCCTCTT GCCACGGTTT GAACGGTACT GTTCACGCTG GACCACGTGA TACTGGTGAT GACTACTCTCCAATCGTCAA GGCCTTGATG TCTGCTGTCG AAGATCGTGG AGTCCCTACC AAGAAGGACT TGGGTTGCGGAGACCCTCAT GGTGTCTCCA TGTTCCCAAA CACCTTGCAC GAGGACCAAG TTCGTTCCGA CGCTGCCAGAGAATGGTTGC TTCCTAACTA CCAGAGACCA AACTTGCAGG TCTTGACTGG TCAGTACGTC GGTAAGGTCTTGTTGTCTCA GAACGCTACC ACCCCAAGAG CTGTTGGTGT CGAGTTCGGT ACTCACAAGG GTAACACCCACAACGTCTAC GCTAAGCATG AGGTCCTTTT GGCCGCCGGT TCTGCCGTTT CCCCAACCAT CTTGGAGTATTCTGGAATTG GTATGAAATC TATTTTGGAG CCTTTGGGAA TCGACACCGT TGTTGACCTT CCAGTTGGTTTGAACTTGCA GGACCAGACC ACCTCCACTG TCCGTTCTCG TATTACTTCC GCTGGTGCTG GACAAGGTCAAGCTGCCTGG TTCGCTACCT TCAATGAGAC CTTTGGTGAT TACACCGAGA AGGCCCACGA GTTGTTGAACACCAAGTTGG AGCAATGGGC TGAAGAGGCT GTCGCTAGAG GTGGATTCCA TAATACCACC GCCTTGTTGATCCAATACGA AAATTATAGA GATTGGATTG TTAAGGACAA TGTTGCTTAC TCCGAGTTGT TTTTGGATACCGCCGGAGTC GCTTCCTTTG ACGTCTGGGA CTTGTTGCCT TTCACCCGTG GTTACGTTCA CATTTTGGACAAAGATCCTT ACTTGCGTCA CTTCGCCTAC GACCCACAGT ACTTCTTGAA CGAGTTGGAC TTGTTGGGTCAAGCTGCTGC TACTCAGTTG GCCCGTAACA TTTCTAACTC TGGTGCCATG CAAACCTACT TCGCTGGAGAGACCATTCCA GGAGACAACT TGGCCTACGA TGCCGACTTG TCTGCCTGGG TCGAGTACAT CCCTGAAAACTTCCGTCCAA ACTATCACGG TGTCGGAACC TGCTCCATGA TGCCAAAGGA AATGGGTGGA GTCGTCGACAATGCCGCTCG TGTTTACGGA GTCCAGGGTT TGAGAGTCAT CGACGGTTCT ATCCCACCAA CCCAATTGTCCTCCCACGTC ATGACTGTCT TCTACGCTAT GGCCTTGAAG ATCGCTGACG CTGTTCTTGC TGACTACGCTTCTATGCAGG CACCCCAGCC TGTTCCCGAG GCTTACGCTG TTTCTGATCC CGAGGCTCAT CCTGACGATTTTGCTGGTAT GGATGCGAAC CAACTTCAGA AACGTGGATT TGGATGCAAT GGTCCTTGGG ATGAGGATGATATGCAGTGC CACAATCACT GCAAGTCTAT TAAGGGTTAC AAGGGAGGTT ATTGTGCTAA GGGGGGCTTTGTTTGCAAGT GTTACTAG(SEQ ID NO.2)。
将该葡萄糖氧化酶基因整合至毕赤酵母中进行高效表达。设计引物扩增葡萄糖氧化酶基因god，将PCR扩增，纯化产物经EcoRⅠ和NotⅠ双酶切，然后与经同样酶切的表达载体pPICZαA相连，连接产物转化大肠杆菌TOP10感受态细胞，经抗性平板筛选后，获得相应的阳性转化子。提取阳性克隆转化子质粒进行测序，测序结果与葡萄糖氧化酶基因god一致，表明重组表达载体pPICZαA-god构建成功。
所用扩增引物如下：
god-F：5’CTGAATTCTCTAATGGTATTGAGGCTTCC 3’(SEQ ID NO.3)
god-R：5’CTGGCGGCCG CCTAGTAACA CTTGCAAACA AAG 3’(SEQ ID NO.4)
将上述重组表达载体pPICZαA-god经PmeⅠ进行线性化后，电击转化毕赤酵母X33，涂布含Zeo的抗性平板进行筛选，获得葡萄糖氧化酶表达水平最高的重组工程菌X33/GOD。
实施例2葡萄糖氧化酶工程菌的发酵培养
将筛选出产酶活较高转化子接种于30mL YPD种子培养基中，培养48h后，转接到200mL BMGY液体培养基中，30℃，200rpm摇床震荡培养48h，进行菌体富集，4000rpm离心5min，弃上清，将菌体转接到100mL含有1％甲醇的BMMY液体培养基中，30℃，200rpm诱导培养72h，诱导培养期间，每隔24h补充一次终浓度为1％的甲醇溶液，诱导培养结束，离心，检测上清中葡萄糖氧化酶酶活力，摇瓶诱导发酵72h，上清中葡萄糖氧化酶活力可达116U/mL。经3L发酵罐进一步放大培养，测定发酵过程中的酶活力，结果如图1所示，发酵185h，上清中葡萄糖氧化酶活力可达1600U/mL，其比活力为296U/mg，文献报道来源于黑曲霉野生型的葡萄糖氧化酶比活为228U/mg，本发明所提供的葡萄糖氧化酶比活力相较野生型有一定的提高。
实施例3葡萄糖氧化酶的热稳定性试验
分别取适量实施例2中发酵所获得的葡萄糖氧化酶样品，分别在不同温度下处理5min后，按照葡萄糖氧化酶检测方法进行测定样品酶活力，以未处理样品酶活力为100％，计算其它温度处理后的剩余相对酶活力，结果如图2所示。
文献报道野生型葡萄糖氧化酶经70℃处理2min后，剩余酶活仅为50％，从图2可以看出，本发明所提供的葡萄糖氧化酶在70℃处理5min后，还具有70％以上酶活力，表明本发明中的葡萄糖氧化酶样品具有较好的耐温性。
实施例4葡萄糖氧化酶的抑菌实验
待测菌液的制备：从甘油管中转接少量以下6种待测菌：沙门氏杆菌、猪巴氏杆菌、副猪嗜血杆菌、致病大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌，接种至LB液体培养基中，37℃，200rpm过夜培养活化，稀释到一定的OD600，制成待测菌液。
待测抑菌剂的制备：本实施例中的待测抑菌剂为本实施例2中葡萄糖氧化酶工程菌的发酵上清液，用灭过菌的蒸馏水稀释一定的倍数制成待测抑菌剂。
抑菌方法：采用牛津杯做抑菌实验。先配置若干LB琼脂培养基(灭菌后放置在50℃烘箱中)待用。吸取1ml指示菌菌液(10-8)到空白平板培养基上，倒入LB琼脂培养基，并轻轻晃匀，在每个平板上均匀放置实验设计的灭菌牛津杯，取待测菌(或待测抑菌剂)菌液200ul加在牛津杯中，放置在4℃冰箱中静置24hr(扩散)。37℃培养静置24hr，观察抑菌情况，并测量抑菌圈直径大小。
表1葡萄糖氧化酶体外抑菌实验
结果如附图3至附图8所示。所形成抑菌圈直径大小如下表1所示。说明本实验中所构建的工程菌发酵液对沙门氏杆菌，猪巴氏杆菌，副猪嗜血杆菌，致病大肠杆菌，铜绿假单胞菌，金黄色葡萄球菌等均能形成抑菌圈，说明其具有明显的抑菌作用，为其在饲料领域应用推广奠定基础。
实施例5葡萄糖氧化酶添加在饲料中的实践
本试验日粮为粉料，由金钱公司生产，选用健壮、活泼、均匀的1日龄罗斯308白羽肉鸡42羽，随机分为3个试验组，每个试验组14个重复，1组为负对照组，日粮中不再额外添加其他物质；2组为正对照，日粮中添加相应抗生素；3组为加酶组，添加本发明的葡萄糖氧化酶，分组具体情况见下表2。试验期为小鸡阶段(1-21日龄)、中大鸡阶段(22-42日龄)，分别测定各组白羽肉鸡不同生长阶段的生长性能，结果如表3和表4所示。
表2实验设计分组
表3各试验组白羽肉鸡前期生长性能
表4各试验组白羽肉鸡后期生长性能
上述结果表明在试验过程中，在养鸡的日粮中加入本发明中的葡萄糖氧化酶，可以显著降低料肉比，节约了原料成本，同时成活率与添加抗生素的试验组基本一致。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
SEQUENCE LISTING
<110> 广东溢多利生物科技股份有限公司
<120> 一种葡萄糖氧化酶及其载体与应用
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 655
<212> PRT
<213> 人工序列
<400> 1
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Glu Val Ala
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Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu
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Thr Val Ala Ala Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
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Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
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Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr
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Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser
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Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
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Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
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Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Ala Gly Pro Arg
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Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala
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225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala
275 280 285
Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
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Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
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Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
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Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
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<210> 2
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tctaatggta ttgaggcttc cttgttgaaa gacccaaaag aggtcgccgg tagaacctac 60
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gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
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gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900
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ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
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gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
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tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
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ctggcggccg cctagtaaca cttgcaaaca aag 33
Claims (10)
1.一种葡萄糖氧化酶，其特征在于，其氨基酸序列如SEQ ID NO.1所示。
2.一种葡萄糖氧化酶基因，其特征在于，所述葡萄糖氧化酶基因编码权利要求1所述的葡萄糖氧化酶。
3.根据权利要求2所述的葡萄糖氧化酶基因，其特征在于，其核苷酸序列如SEQ IDNO.2所示。
4.一种载体，其特征在于，所述载体可表达权利要求1所述的葡萄糖氧化酶或含有权利要求2或3所述的葡萄糖氧化酶基因。
5.一种重组菌株，其特征在于，含有权利要求4所述的载体。
6.根据权利要求5所述的重组菌株，其特征在于，所述重组菌株为细菌或真菌细胞。
7.根据权利要求6所述的重组菌株，其特征在于，所述重组菌株为酵母。
8.一种抑菌剂，含有权利要求1所述的葡萄糖氧化酶。
9.根据权利要求8所述的抑菌剂，所述抑菌剂应用于饲料，食品，医药领域。
10.一种饲料，含有权利要求8所述的抑菌剂。
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| CN114395541A (zh) * | 2022-02-22 | 2022-04-26 | 广东溢多利生物科技股份有限公司 | 一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用 |
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| CN114395541A (zh) * | 2022-02-22 | 2022-04-26 | 广东溢多利生物科技股份有限公司 | 一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用 |
| CN114395541B (zh) * | 2022-02-22 | 2024-02-06 | 广东溢多利生物科技股份有限公司 | 一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用 |
| CN115029328A (zh) * | 2022-04-24 | 2022-09-09 | 广东溢多利生物科技股份有限公司 | 葡萄糖氧化酶突变体GOx-MUT1~6及其编码基因和应用 |
| CN115029327A (zh) * | 2022-04-24 | 2022-09-09 | 广东溢多利生物科技股份有限公司 | 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用 |
| CN115029328B (zh) * | 2022-04-24 | 2024-03-15 | 广东溢多利生物科技股份有限公司 | 葡萄糖氧化酶突变体GOx-MUT1~6及其编码基因和应用 |
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