WO2020125700A1 - 一种葡萄糖氧化酶突变体及其在工业化生产中的应用 - Google Patents

一种葡萄糖氧化酶突变体及其在工业化生产中的应用 Download PDF

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WO2020125700A1
WO2020125700A1 PCT/CN2019/126445 CN2019126445W WO2020125700A1 WO 2020125700 A1 WO2020125700 A1 WO 2020125700A1 CN 2019126445 W CN2019126445 W CN 2019126445W WO 2020125700 A1 WO2020125700 A1 WO 2020125700A1
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glucose oxidase
seq
oxidase mutant
mutant according
wild
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郑斐
严婷
朱继东
徐红
孙艳
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南京百斯杰生物工程有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12R2001/84—Pichia

Definitions

  • the invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a glucose oxidase mutant and its application in industrial production.
  • Glucose oxidase (GOD, E.C. 1.1.3.4) is an aerobic dehydrogenase.
  • the enzyme molecule is a dimer and contains two subunits with a molecular weight of about 160 kDa. Each subunit is combined with a FAD molecule. It can use molecular oxygen as an electron acceptor to specifically catalyze ⁇ -D-glucose to produce gluconic acid and hydrogen peroxide.
  • GOD is widely distributed in animals, plants and microorganisms. The extraction of GOD from animal and plant tissues has certain limitations, the amount of enzyme is not rich, and the amount of enzyme produced by bacteria GOD is small. Microbial fermentation is the main source of GOD production. At present, most commercial products on the market are produced by fermentation of Pichia pastoris and filamentous fungi, such as Aspergillus niger and Aspergillus oryzae.
  • glucose oxidase Because of its catalytic specificity and high efficiency, glucose oxidase has been widely used in food, chemical, pharmaceutical, agricultural, feed and other fields. It has attracted much attention in recent years and the market demand is also increasing. Due to the deoxidation and anti-oxidation effects of glucose oxidase, it is widely used in food, medicine, feed, etc. In the food industry, glucose oxidase as a food preservative has a significant effect in preventing beer aging, maintaining the original flavor of the product, and extending the shelf life. It can also be used as a flour improver and bread quality improver to improve food quality. In the field of medicine, glucose oxidase electrode method, glucose oxidase-peroxidase coupling method, etc.
  • glucose oxidase can improve the animal intestinal environment, improve feed utilization, and promote animal growth.
  • glucose oxidase is increasingly used in various fields, the industry, especially the feed industry, has increasingly higher requirements for its existing performance. For example, at normal temperature, the enzyme activity will not decrease for a long time. Heat and extreme pH conditions are tolerant and resistant to digestive enzymes. Among them, the thermal stability of the enzyme is very critical for the application of glucose oxidase. In the preparation process of the enzyme and under extreme reaction conditions (high temperature), the enzyme with strong heat resistance has a relatively large advantage. Therefore, improving the thermal stability of glucose oxidase is of great practical significance for the widespread promotion and application of glucose oxidase
  • the invention provides a glucose oxidase mutant. Compared with the wild-type glucose oxidase, its thermal stability has been significantly improved, which is beneficial to the application of the enzyme in industrial production.
  • the present invention provides a glucose oxidase mutant at a position corresponding to SEQ ID NO: 1, compared with the wild-type glucose oxidase shown in SEQ ID NO: 1, 19th and 25th. , 67, 92, 96, 121, 141, 142, 278, 305, 362, 449, 453, 477, 506, 521, 526, 528, 536, 560 or 572 amino acids were substituted.
  • the substitutions are T19Y, A25V, A67Y, A92Q, S96F, S121A, L141K, Q142K, N278Y, M305P, S362F, A449M, Q453N, S477Y, Y506W, C521A, K526R, M528L, A536L, V560L or S572A.
  • the substitution is A25V, A67Y, A92Q, S96F, S121A, L141K, Q142K, A449M, Q453N, S477Y, Y506W, C521A, K526R, M528L, A536L, V560L or S572A.
  • the glucose oxidase mutant is at a position corresponding to SEQ ID NO: 1, compared with the wild-type glucose oxidase shown in SEQ ID NO: 1, section 67, 92. , 96, 121, 142, 453, 477, 506, 528, 560 or 572 amino acids were substituted.
  • substitution is A67Y, A92Q, S96F, S121A, Q142K, Q453N, S477Y, Y506W, M528L, V560L, or S572A.
  • the glucose oxidase mutant is at the position corresponding to SEQ ID NO: 1, compared with the wild-type glucose oxidase shown in SEQ ID NO: 1, position 96 Or the 572th amino acid has been substituted.
  • substitution is S96F or S572A.
  • the glucose oxidase mutant at the position corresponding to SEQ ID NO: 1, is compared with the wild-type glucose oxidase shown in SEQ ID NO: 1. The amino acid was substituted.
  • the substitution is S96F.
  • the glucose oxidase mutant at a position corresponding to SEQ ID NO: 1, is compared with the wild-type glucose oxidase shown in SEQ ID NO: 1. The amino acid was substituted.
  • the substitution is S572A.
  • substitution means that the amino acid at the position is replaced with another amino acid.
  • amino acid at position 572 is “substituted”, which is represented as S572A.
  • the present invention also provides a polynucleotide encoding the glucose oxidase mutant.
  • the present invention also provides a recombinant expression vector comprising the above polynucleotide encoding the glucose oxidase mutant.
  • the host cell described above is a fungal cell, preferably a yeast cell or a filamentous fungal cell, more preferably a Pichia pastoris cell or an Aspergillus niger cell.
  • the present invention provides the application of the glucose oxidase mutant in the fields of food, chemical industry, medicine, agriculture or feed.
  • the glucose oxidase mutant of the present invention has a significantly improved thermal stability. Specifically, after treatment at 70°C for 3 minutes, it is preferably compared with the wild-type glucose oxidase.
  • the enzyme activity of the glucose oxidase mutant is increased by more than 30%, the enzyme activity of the more preferred glucose oxidase mutant is increased by more than 40%, and the enzyme activity of the particularly preferred glucose oxidase mutant is increased by more than 50%, especially preferred
  • the enzyme activity of the glucose oxidase mutant has increased by more than 80%, and it is particularly suitable for industrial production, for example, in the fields of food, chemicals, medicine, agriculture and feed.
  • Example 1 Construction of a single point thermostable mutant library of glucose oxidase
  • the underlined sites are EcoR I and Not I sites.
  • the reaction conditions were: pre-denaturation at 94 °C for 10 min, denaturation at 94 °C for 60 s, annealing at 58 °C for 60 s and extension at 72 °C for 2 min, a total of 30 cycles to recover the target gene fragment.
  • each well contains 150 ⁇ L LB medium (containing 1mM IPTG, 50ng/mL ampicillin), 30 °C, 220rpm shaking culture for 12h, the well plate was placed at -20 °C, repeated freezing and thawing to break the wall, to obtain crude enzyme solution containing glucose oxidase. Take 5 ⁇ l of crude enzyme solution to two new 96-well plates, one of which was treated at 70°C for 3 min, and the other was placed on ice as a control.
  • LB medium containing 1mM IPTG, 50ng/mL ampicillin
  • Both 96-well plates were added with methanol buffer and glucose buffer containing o-anisidine
  • the coloring solution of the liquid and horseradish peroxidase solution was added to 100 ⁇ L of 2M sulfuric acid to stop the reaction after reaction at 37°C for 3 min.
  • the residual enzyme activity was determined according to the coloring reaction. Take the strain with higher residual activity than wild-type glucose oxidase into a new 96-well culture plate and repeat the screening. Through two rounds of screening comparison.
  • the applicant screened T19Y, A25V, A67Y, A92Q, S96F, S121A, L141K, Q142K, N278Y, M305P, S362F, A449M, Q453N, S477Y, Y506W, which can significantly improve the heat resistance mutation of glucose oxidase GOD.
  • the above expression plasmids were linearized with pmeI, and the linearized fragments were purified and collected with a fragment purification kit (TaKaRa MiniBEST DNA Fragment Purifibation Kit), and then transformed into Pichia pastoris GS115 by electro transformation, respectively, and coated on MD plates.
  • the colonies grown on the MD plate were spread on a YPD plate of geneticin with a concentration of 1 mg/mL to screen multiple copies of positive transformants to obtain a recombinant strain of Pichia pastoris. Pick the transformants of each gene and transfer them to BMGY medium. After shaking culture at 220°C and 220 rpm for 18h, centrifuge to obtain bacterial cells.
  • the enzyme activity measurement system contains 2.5mL of o-anisidine solution, 0.3mL of 18% glucose, 0.1mL of 90U/mL horseradish peroxidase, after incubation at 35°C for 2min, add the diluted enzyme solution sample to the test tube 0.1mL, after 3min of reaction, add 2mol/L sulfuric acid to stop the reaction, remove the test tube, measure the absorbance of OD540, and use the heat-inactivated enzyme solution as a blank control. Based on the results of the standard curve, calculate the glucose oxidase activity units.
  • Solution of o-dianisidine accurately weigh 0.1 g of o-anisidine and dissolve it in 10 ml of methanol. This is a storage solution, which can be effectively stored for 3 days at 4°C. Before the experiment, take 0.1ml of the storage solution and dissolve it in 12ml of 0.1mol/L, pH 5.5 of the above phosphate buffer solution.
  • Glucose Accurately weigh 9.000g of glucose dried to constant weight (AR), dissolve in a small amount of distilled water, and dilute to 50ml with distilled water, and store at 4°C.
  • GOD standard buy sigma glucose oxidase standard with enzyme activity of 10,000 units, add 5mL of distilled water accurately, mix well, and store at -20°C until use.
  • horseradish peroxidase purchase horseradish peroxidase standard (enzyme activity>250units/mg, 100mg), accurately add 1mL of distilled water, fully dissolve horseradish peroxidase, and store at -20°C for future use. When using, take appropriate amount of standard product and dilute it to 90U/ml for enzyme activity.
  • the fermentation supernatant described in Example 2 was diluted to about 100 U/mL with a phosphate buffer of pH 5.5 and treated at 70°C for 3 min, respectively, and the residual enzyme activity was determined.
  • the enzyme activity of the untreated sample was 100% , Calculate the relative enzyme activity. The results are shown in the table below,
  • the thermal stability of the glucose oxidase mutant of the present invention has been significantly improved compared to the wild-type glucose oxidase, and the enzyme activity of the preferred glucose oxidase mutant has been increased by 30%
  • the enzyme activity of the more preferred glucose oxidase mutant is increased by more than 40%
  • the enzyme activity of the particularly preferred glucose oxidase mutant is increased by more than 50%
  • the enzyme activity of the particularly preferred glucose oxidase mutant is increased by more than 80% , Especially suitable for industrial production, for example, in the fields of food, chemical, pharmaceutical, agriculture and feed.

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Abstract

提供一种葡萄糖氧化酶突变体及其在工业化生产中的应用。所述葡萄糖氧化酶突变体，与野生型葡萄糖氧化酶相比，其热稳定性有了显著的提升，适合应用于工业化生产中，例如，应用于食品、化工、医药、农业和饲料领域中。

Description

一种葡萄糖氧化酶突变体及其在工业化生产中的应用
本申请要求于2018年12月20日提交中国专利局、申请号为201811566096.2、发明名称为“一种葡萄糖氧化酶突变体及其在工业化生产中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于基因工程和酶工程领域，具体涉及一种葡萄糖氧化酶突变体及其在工业化生产中的应用。
背景技术
葡萄糖氧化酶(Glucose oxidase,GOD,E.C 1.1.3.4)是一种需氧脱氢酶，该酶分子为二聚体，含有两个亚基，分子量约160kDa，每个亚基结合一个FAD分子。它能利用分子氧作为电子受体，专一催化β-D-葡萄糖生成葡萄糖酸和过氧化氢。GOD广泛分布于动植物和微生物体内，从动植物组织中提取GOD有一定的局限，酶量亦不丰富，细菌GOD产酶量少。微生物发酵法是生产GOD的主要来源。目前市场上大部分商业产品都是由毕赤酵母和丝状真菌，如黑曲霉，米曲霉等发酵生产。
葡萄糖氧化酶由于具有催化专一性和高效性的优点，在食品、化工、医药、农业、饲料等领域有比较广泛的应用，近年来倍受关注，市场需求量也越来越大。由于葡萄糖氧化酶的除氧和抗氧化作用，使其在食品、医药、饲料等方面应用非常广泛。在食品工业中，葡萄糖氧化酶作为一种食品保鲜剂，在防止啤酒老化、保持产品原有风味、延长保质期方面有显著效果，还可作为面粉改良剂和面包品质改良剂提高食品质量。在医药领域中普遍采用葡萄糖氧化酶电极法、葡萄糖氧化酶-过氧化物酶偶联法等检测血液、血清中葡萄糖含量。作为一种新型的饲料添加剂，葡萄糖氧化酶能够改善动物肠道环境，提高饲料利用率，促进动物生长。随着葡萄糖氧化酶越来越多的被应用于 各个领域，工业上尤其是饲料工业对其现有性能有了越来越高的要求，如在常温下较长时间保持酶活力不下降，对热和极端pH条件具有耐受性，对消化酶具有耐受性。其中酶的热稳定性对于葡萄糖氧化酶的应用非常关键，在酶的制备过程中和极端反应条件下(高温)，耐热性强的酶有着比较大的优势。因此，提高葡萄糖氧化酶热稳定性对葡萄糖氧化酶的广泛推广和应用具有重要的现实意义
发明内容
本发明提供葡萄糖氧化酶突变体，与野生型葡萄糖氧化酶相比，其热稳定性有了显著的提升，有利于该酶在工业化生产中应用。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供一种葡萄糖氧化酶突变体，所述葡萄糖氧化酶突变体在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第19、25、67、92、96、121、141、142、278、305、362、449、453、477、506、521、526、528、536、560或572位氨基酸发生了取代。
在本发明的一些具体实施方案中，所述取代为T19Y、A25V、A67Y、A92Q、S96F、S121A、L141K、Q142K、N278Y、M305P、S362F、A449M、Q453N、S477Y、Y506W、C521A、K526R、M528L、A536L、V560L或S572A。
在本发明的另一些实施方案中，所述的葡萄糖氧化酶突变体在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第25、67、92、96、121、141、142、449、453、477、506、521、526、528、536、560或572位氨基酸发生了取代。
在本发明的另一些具体实施方案中，所述取代为A25V、A67Y、A92Q、S96F、S121A、L141K、Q142K、A449M、Q453N、S477Y、Y506W、C521A、K526R、M528L、A536L、V560L或S572A。
在本发明的又一些实施方案中，所述的葡萄糖氧化酶突变体在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相 比，第67、92、96、121、142、453、477、506、528、560或572位氨基酸发生了取代。
在本发明的又一些具体实施方案中，所述取代为A67Y、A92Q、S96F、S121A、Q142K、Q453N、S477Y、Y506W、M528L、V560L或S572A。
在本发明的一些优选实施方案中，所述的葡萄糖氧化酶突变体在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第96、121、506、560或572位氨基酸发生了取代。
在本发明的一些优选具体实施方案中，所述取代为S96F、S121A、Y506W、V560L或S572。
在本发明的一些更优选实施方案中，所述的葡萄糖氧化酶突变体在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第96位或第572位氨基酸发生了取代。
在本发明的一些更优选具体实施方案中，所述取代为S96F或S572A。
在本发明的一些特别优选实施方案中，所述的葡萄糖氧化酶突变体，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第96位氨基酸发生了取代。
在本发明的一些特别优选具体实施方案中，所述取代为S96F。
在本发明的一些特别优选实施方案中，所述的葡萄糖氧化酶突变体，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第572位氨基酸发生了取代。
在本发明的一些特别优选具体实施方案中，所述取代为S572A。
本发明中A，R，C，Q，N，L，K，M，F，P，S，T，W，Y，V分别是丙氨酸Ala，精氨酸Arg，半胱氨酸Cys，谷氨酰胺Gln，天冬酰胺Asn，亮氨酸Leu，赖氨酸Lys，甲硫氨酸Met，苯丙氨酸Phe，脯氨酸Pro，丝氨酸Ser，苏氨酸Thr，色氨酸Trp，酪氨酸Tyr，缬氨酸Val的缩写。
术语“取代”指所在位置的氨基酸被替换成另一个氨基酸，例如，572位的氨基酸发生“取代”，表示为S572A。
本发明还提供编码所述葡萄糖氧化酶突变体的多核苷酸。
本发明还提供了一种重组表达载体，其包含上述编码所述葡萄糖氧化酶突变体的多核苷酸。
本发明还提供了一种宿主细胞，其包含上述重组表达载体。
在本发明的一些实施方案中，上述所述的宿主细胞是真菌细胞，优选酵母细胞或丝状真菌细胞，更优选毕赤酵母细胞或黑曲霉细胞。
本发明再一方面提供所述葡萄糖氧化酶突变体在食品、化工、医药、农业或饲料领域中的应用。
本发明的葡萄糖氧化酶突变体，与野生型葡萄糖氧化酶相比，其热稳定性有了显著的提升，具体的，在70℃条件下处理3min后，与野生型葡萄糖氧化酶相比，优选的葡萄糖氧化酶突变体的酶活提高了30％以上，更优选的葡萄糖氧化酶突变体的酶活提高了40％以上，特别优选的葡萄糖氧化酶突变体酶活提高了50％以上，尤其优选的葡萄糖氧化酶突变体酶活提高了80％以上，特别适合应用于工业化生产中，例如，应用于食品、化工、医药、农业和饲料领域中。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步详细的说明，需要指出的是，本实施例仅用于解释本发明，而非对本发明范围的限制。
实施例1：葡萄糖氧化酶单点耐热突变体库的构建
来源于黑曲霉(Aspergillus niger)的葡萄糖氧化酶基因GOD-wt由583个氨基酸构成(如SEQ ID NO：1所示)，采用全基因合成的方法合成了该葡萄糖氧化酶基因GOD-wt(如SEQ ID NO：2所示)，所述基因合成由南京金斯瑞生物科技有限公司完成。合成的基因两端带有EcoR I和Not I酶切位点。以合成的该基因为模板扩增葡萄糖氧化酶GOD-wt基因，使GeneMorph II随机突变PCR试剂盒(Stratagene)随机引入突变。所用引物为：
5’-GCGC GAATTCCGCTGCGGCCCTGCCACACTAC-3’，
5’-TAAA GCGGCCGCTCACTGCATGGAAGCATAATCTTC-3’。
下划线处分别为EcoR I和Not I酶切位点。
反应条件为：94℃预变性10min，94℃变性60s，58℃退火60s和72℃延伸2min，共30个循环，回收目的基因片段。将目的片段用EcoR I和Not I双酶切消化后，与经过相同酶切的pET21a(+)载体(氨苄抗性基因)用Ligase进行连接反应。将连接好的片段转化至大肠杆菌BL21(DE3)，涂布含有氨苄青霉素的LB平板，37℃倒置培养，待平板上出现转化子后，挑取单克隆至96孔板，每孔中含有150μL LB培养基(含有1mM IPTG，50ng/mL氨苄青霉素)，30℃，220rpm震荡培养12h，将孔板置于-20℃，反复冻融破壁，获得含有葡萄糖氧化酶的粗酶液。分别取出5μl粗酶液至两块新的96孔板，其中一块于70℃处理3min，另一块置于冰上作为对照，两块96孔板都加入含有邻联茴香胺甲醇缓冲液、葡萄糖缓冲液、辣根过氧化物酶溶液的显色液，37℃反应3min后加入100μL 2M硫酸终止反应，根据显色反应测定残余酶活。取残余活性比野生型葡萄糖氧化酶高的菌株到新的96孔培养板中，进行重复筛选。通过两轮的筛选比较。最终，申请人筛选到能显著提高葡萄糖氧化酶GOD的耐热性突变位点T19Y,A25V，A67Y，A92Q，S96F，S121A，L141K，Q142K，N278Y，M305P，S362F，A449M，Q453N，S477Y，Y506W，C521A，K526R，M528L，A536L，V560L，S572A。
实施例2：毕赤酵母工程菌株的构建
使用实施例1中所述引物，以实施例1中的葡萄糖氧化酶野生型序列和突变体序列为模板进行PCR扩增，PCR反应条件与实施例1相同。将扩增得到的实施例1所述葡萄糖氧化酶突变体基因片段，以及野生型基因片段，通过EcoR I和Not I位点与表达载体pPIC9K相连接，将表达载体转入大肠杆菌DH5α感受态，挑取转化子后大量提取质粒。将以上表达质粒用pmeI进行线性化，线性化片段用片段纯化试剂盒(TaKaRa MiniBEST DNA Fragment Purifibation Kit)纯化收集后，通过电转化分别转化毕赤酵母GS115，涂布MD平板。将在MD平板上生长出的菌落涂布到浓度为1mg/mL的遗传霉素的YPD平板上筛选多拷贝的阳性转化子，得到毕赤酵母重组菌株。分别挑取每个基因的转化子转接于BMGY培养基中，30℃，220rpm振荡培养18h后，离心获得菌体，将适量菌体转入 BMMY培养基中，使菌体浓度达到OD600＝1，30℃，250rpm继续振荡培养，每24h添加培养体积1％的甲醇。诱导表达4d后，将培养液离心获得上清，将上清液进行葡萄糖氧化酶活力测定和热稳定性测定。
实施例3 葡萄糖氧化酶的酶活测定
在有氧条件下，GOD催化葡萄糖脱氢产生H 2O 2，在辣根过氧化物酶(POD)的作用下，氧供体邻-联(二)茴香胺(DH2)被氧化成棕色产物。根据其在540nm处吸光度的变化及标准曲线，可换算GOD的活性。酶活测定体系中包含2.5mL邻联茴香胺溶液，0.3mL的18％葡萄糖，0.1mL的90U/mL辣根过氧化物酶，35℃保温2min后，向试管中加入稀释好的酶液样品0.1mL，反应3min后，加入2mol/L硫酸终止反应，取出试管，测OD540的吸光值，以热失活的酶液做空白对照。根据标准曲线的结果，计算葡萄糖氧化酶活力单位。
试剂和溶液
0.1mol/L pH5.5磷酸氢二钠-柠檬酸钠缓冲液：准确称取14.32g磷酸氢二钠，8.4056g一水合柠檬酸，溶解于400ml蒸馏水中，用磷酸氢二钠调节pH至5.5，备用。
邻联茴香胺溶液：准确称取0.1g邻联茴香胺，溶于10ml甲醇中，此为储存液，4℃有效保存3天。实验前取0.1ml储存液，溶于12ml0.1mol/L，pH5.5的上述磷酸缓冲液，即得。
18％葡萄糖：准确称取9.0000g烘干至恒重的葡萄糖(AR)，溶解于少量蒸馏水中，并用蒸馏水定容至50ml，4℃保存。
2mol/L H 2SO 4：准确称取40.00g H 2SO 4，缓慢加入160mL蒸馏水中，定容至200mL，备用。
GOD标准品：购买酶活为10000单位的sigma葡萄糖氧化酶标准品，准确加入5mL蒸馏水，混匀，-20℃保存备用。
90U/mL辣根过氧化物酶：购买辣根过氧化物酶标准品(酶活力＞250units/mg，100mg)，准确加入1mL蒸馏水，充分溶解辣根过氧化物酶，-20℃保存备用。使用时取适量标准品稀释至酶活为90U/ml后使用，需现稀释现用。
酶活力的测定
(1)标准曲线的制作
将GOD标准品分别稀释成0.4，0.8，1.2，1.6，2.0，2.4U/mL，在试管中加入2.5mL邻联茴香胺溶液.和0.3mL的18％葡萄糖溶液，再加入0.1mL 90U/mL辣根过氧化物酶溶液，35℃预热2min后，以15s的时间间隔加入0.1mL稀释好的GOD标准品，准确反应3min后立即加入2ml2mol/L H 2SO 4终止反应，取出混匀，在540nm处测定吸光值，以吸光值为横坐标，标准酶活力为纵坐标，绘制标准曲线y＝Kx+b。
(2)样品的测定
在试管中加入2.5mL邻联茴香胺溶液和0.3mL的18％葡萄糖溶液，加入0.1mL90U/mL辣根过氧化物酶溶液，35℃预热2min后，以15s的时间间隔加入0.1mL稀释好的待检样品(稀释标准为该样品的检测吸光值在线性范围内)，准确反应3min后立即加入2ml 2mol/L H 2SO 4终止反应，取出混匀，在540nm处测定吸光值A，计算酶活。
(3)酶活力的计算
X＝(K*A+b)*n
式中：
X---样品的酶活力U/ml             A---样品检测吸光值
n---酶液的稀释倍数               K----标准曲线斜率
b----标准曲线截距
实施例4 葡萄糖氧化酶及其突变体的耐热性测定
将实施例2所述发酵上清液用pH5.5的磷酸缓冲液稀释至约100U/mL，在70℃条件下分别处理3min后，测定残余酶活，以未处理样品的酶活为100％，计算相对酶活。结果如下表所示，
Figure PCTCN2019126445-appb-000001
Figure PCTCN2019126445-appb-000002
从表中的数据可以看出，与野生型葡萄糖氧化酶相比，本发明的葡萄糖氧化酶突变体的热稳定性有了显著的提升，优选的葡萄糖氧化酶突变体的酶活提高了30％以上，更优选的葡萄糖氧化酶突变体的酶活提高了40％以上，特别优选的葡萄糖氧化酶突变体酶活提高了50％以上，尤其优选的葡萄糖氧化酶突变体酶活提高了80％以上，特别适合应用于工业化生产中，例如，应用于食品、化工、医药、农业和饲料领域中。

Claims (17)

  1. 一种葡萄糖氧化酶突变体，其特征在于，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第25、67、92、96、121、141、142、449、453、477、506、521、526、528、536、560或572位氨基酸发生了取代。
  2. 根据权利要求1所述的葡萄糖氧化酶突变体，其特征在于，所述取代为A25V、A67Y、A92Q、S96F、S121A、L141K、Q142K、A449M、Q453N、S477Y、Y506W、C521A、K526R、M528L、A536L、V560L或S572A。
  3. 根据权利要求1所述的葡萄糖氧化酶突变体，其特征在于，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第67、92、96、121、142、453、477、506、528、560或572位氨基酸中发生了取代。
  4. 根据权利要求3所述的葡萄糖氧化酶突变体，其特征在于，所述取代为A67Y、A92Q、S96F、S121A、Q142K、Q453N、S477Y、Y506W、M528L、V560L或S572A。
  5. 根据权利要求3所述的葡萄糖氧化酶突变体，其特征在于，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第96、121、506、560或572位氨基酸发生了取代。
  6. 根据权利要求5所述的葡萄糖氧化酶突变体，其特征在于，所述取代为S96F、S121A、Y506W、V560L或S572A。
  7. 根据权利要求5所述的葡萄糖氧化酶突变体，其特征在于，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第96位或第572位氨基酸发生了取代。
  8. 根据权利要求7所述的葡萄糖氧化酶突变体，其特征在于，所述取代为S96F或S572A。
  9. 根据权利要求7所述的葡萄糖氧化酶突变体，其特征在于，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第96位氨基酸发生了取代。
  10. 根据权利要求9所述的耐热的葡萄糖氧化酶突变体，其特征在于，所述的取代为S96F。
  11. 根据权利要求7所述的葡萄糖氧化酶突变体，其特征在于，在对应于SEQ ID NO:1的位置，与SEQ ID NO:1所示的野生型葡萄糖氧化酶相比，第572位氨基酸发生了取代。
  12. 根据权利要求11所述的葡萄糖氧化酶突变体，其特征在于，所述的取代为S572A。
  13. 一种多核苷酸，其特征在于，其编码权利要求1-12中任一项所述的葡萄糖氧化酶突变体。
  14. 一种重组表达载体，其特征在于，所述重组表达载体包含权利要求13所述的编码葡萄糖氧化酶突变体的多核苷酸。
  15. 一种宿主细胞，其特征在于，所述的宿主细胞包含权利要求14所述的重组表达载体。
  16. 根据权利要求15所述的宿主细胞，其特征在于，所述的宿主细胞是真菌细胞。
  17. 权利要求1-12中任一项所述的葡萄糖氧化酶突变体在食品、化工、医药、农业或饲料领域中的应用。
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