CN105950577A - Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof - Google Patents

Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof Download PDF

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CN105950577A
CN105950577A CN201610528932.2A CN201610528932A CN105950577A CN 105950577 A CN105950577 A CN 105950577A CN 201610528932 A CN201610528932 A CN 201610528932A CN 105950577 A CN105950577 A CN 105950577A
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g0d
glucose oxidase
mutant
amino acid
thermal stability
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CN201610528932.2A
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肖志壮
张稳
李俊安
赵志强
武传菊
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青岛红樱桃生物技术有限公司
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/0006Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y101/00Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004Glucose oxidase (1.1.3.4)

Abstract

The invention discloses a glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof. According to the invention, an error-prone PCR (Polymerase Chain Reaction) method is used to mutate wild type glucose oxidase genes, and positive mutations are detected by means of a high-throughput screening method. By means of the mutant library constructing and screening method, three glucose oxidase mutants with obviously improved thermal stability are obtained, the thermal stability is improved by 1-3 times compared with the wild type glucose oxidase genes, and therefore the glucose oxidase mutant with improved thermal stability has a good market application prospect and industrial value.

Description

热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用 Improved thermal stability of glucose oxidase mutant genes and their applications

技术领域 FIELD

[0001] 本发明属于基因工程和酶工程领域,具体内容涉及热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用。 [0001] The present invention belongs to the field of genetic engineering and enzyme engineering, particularly relates to improving the thermal stability of glucose oxidase mutant genes and their applications.

背景技术 Background technique

[0002] 葡萄糖氧化酶(glucose oxidase,EC 1.1.3.4,G0D)有氧条件下能专一性地催化β-D-葡糖生成葡萄糖酸和过氧化氢。 [0002] Glucose oxidase (glucose oxidase, EC 1.1.3.4, G0D) under aerobic conditions can catalyze specific β-D- gluconic generate gluconic acid and hydrogen peroxide. 葡萄糖氧化酶是目前主要的工具酶之一,被广泛应用于在食品工业、畜牧养殖和医疗检测等领域。 Glucose oxidase is one of the main tools enzyme, it is widely used in the food industry, animal husbandry and medical testing and other areas. 葡萄糖氧化酶作为公认的安全抗氧剂应用于食品加工工艺中,在医疗产业中,葡萄糖氧化酶可以用于血糖测定等方向,作为一种饲料添加剂,葡萄糖氧化酶可以改善动物肠道环境,促进动物生长。 Glucose oxidase as an antioxidant generally recognized as safe processes used in food processing, in the medical industry, the glucose oxidase and the like may be used for blood glucose direction, as a feed additive, glucose oxidase can improve animal intestinal environment to promote animal growth. 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。 With glucose oxidase increasingly being used in various fields, especially in the industry prior to its feed industry has increasingly higher performance requirements, such a long time at normal temperature does not drop to maintain enzyme activity, for pH conditions and extreme heat resistant, resistant to digestive enzymes. 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。 Wherein the thermostable enzyme glucose oxidase for the application is critical, during the preparation of the enzyme and under extreme reaction conditions (high temperature), high heat resistance of the enzyme has a relatively large advantage.

[0003] 易错PCR(error-prone PCR)技术是一种在DNA序列中制造随机突变的方法,其基本原理是在PCR扩增目的基因时,通过改变传统PCR过程的反应条件(如提高酶离子浓度,改变体系中4种dNTP的浓度等),使用较低保真度的Taq酶,从而以一定频率随机引入错误碱基,从而形成序列不同的突变体库。 [0003] Error-prone PCR (error-prone PCR) technique is a method for producing random mutations in the DNA sequence, which is the basic principle in the target gene PCR amplification, by varying the reaction conditions of the conventional PCR process (such as improved enzyme ion concentration, change four kinds of dNTP concentration in the system, etc.) using a lower fidelity of Taq DNA polymerase, whereby a certain frequency error introduced randomly base, thereby forming a sequence of different mutant library.

发明内容 SUMMARY

[0004] 本发明提供了热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用,本发明提供的葡萄糖氧化酶突变体热稳定性有了显著的提升。 [0004] The present invention provides improved thermal stability of glucose oxidase mutant and coding gene and application, the present invention provides a mutant glucose oxidase thermal stability has been significantly improved.

[0005] 为实现上述发明目的,本发明采用以下技术方案予以实现: [0005] In order to achieve the above object, the present invention employs the following technical solutions to be achieved:

[0006] 本发明提供了一种热稳定性提高的葡萄糖氧化酶突变体G0D-H-2,所述的葡萄糖氧化酶突变体G0D-H-2的氨基酸序列如SEQ ID N0:4所示,所述的突变体G0D-H-2序列由氨基酸序列为SEQ ID NO: 1的葡萄糖氧化酶的第143位氨基酸由丙氨酸变为脯氨酸、第461位氨基酸由苯丙氨酸变为异亮氨酸获得。 [0006] The present invention provides an improved thermal stability of glucose oxidase mutant G0D-H-2, the glucose oxidase mutant amino acid sequence G0D-H-2 as SEQ ID N0: 4, mutant according G0D-H-2 sequence as the amino acid sequence of SEQ ID NO: 143 amino acid at position 1 of glucose oxidase is changed from alanine proline at amino acid 461 changed from phenylalanine isoleucine obtained.

[0007] 本发明提供了所述的葡萄糖氧化酶突变体G0D-H-2的葡萄糖氧化酶编码基因。 Glucose oxidase encoding gene thereof in G0D-H-2 [0007] The present invention provides a mutant of the glucose oxidase.

[0008] 本发明提供了含有所述的葡萄糖氧化酶编码基因的重组菌株。 [0008] The present invention provides a recombinant strain containing glucose oxidase encoding gene according to the.

[0009] 本发明提供了一种热稳定性提高的葡萄糖氧化酶突变体G0D-H-5,所述的葡萄糖氧化酶突变体G0D-H-5的氨基酸序列如SEQ ID勵:7所示,所述的突变体600-!1-5序列由氨基酸序列为SEQ ID NO: 1的葡萄糖氧化酶的第29位氨基酸由异亮氨酸变为亮氨酸、第143位氨基酸由丙氨酸变为脯氨酸、第390位氨基酸由甘氨酸变为精氨酸、第461位氨基酸由苯丙氨酸变为异亮氨酸获得。 [0009] The present invention provides an improved thermal stability of glucose oxidase mutant G0D-H-5, the amino acid sequence shown in SEQ ID excitation G0D-H-5 of the glucose oxidase mutant: 7, the mutant 600-1-5 sequence as the amino acid sequence of SEQ ID NO:! amino acid position 29 glucose oxidase 1 from isoleucine becomes leucine, amino acid 143 is changed from alanine is a proline at amino acid 390 glycine to arginine by the first 461 amino acids of isoleucine phenylalanine becomes available.

[0010] 本发明提供了所述的葡萄糖氧化酶突变体G0D-H-5的葡萄糖氧化酶编码基因。 [0010] The present invention provides the glucose oxidase mutant G0D-H-5 gene encoding glucose oxidase.

[0011] 本发明提供了含有所述的葡萄糖氧化酶编码基因的重组菌株。 [0011] The present invention provides a recombinant strain contains the gene encoding the enzyme glucose oxidase.

[0012] 本发明提供了一种热稳定性提高的葡萄糖氧化酶突变体G0D-H-6,所述的葡萄糖氧化酶突变体G0D-H-6的氨基酸序列如SEQ ID N0:8所示,所述的突变体G0D-H-6序列由氨基酸序列为SEQ ID NO: 1的葡萄糖氧化酶的第143位氨基酸由丙氨酸变为脯氨酸、第241位氨基酸由脯氨酸变为天门冬氨酸、第359位氨基酸由苏氨酸变为丝氨酸、第461位氨基酸由苯丙氨酸变为异亮氨酸获得。 [0012] The present invention provides an improved thermal stability of glucose oxidase mutant G0D-H-6, the amino acid sequence G0D-H-6 as SEQ ID N0 said mutant glucose oxidase: 8, the mutant G0D-H-6 sequence as the amino acid sequence of SEQ ID NO: 143 amino acid at position 1 of glucose oxidase becomes proline by alanine, proline at amino acid position 241 changed tianmen aspartic acid, amino acid 359 by a threonine to serine, amino acid 461 by a phenylalanine isoleucine becomes available.

[0013] 本发明提供了所述的葡萄糖氧化酶突变体G0D-H-6的葡萄糖氧化酶编码基因。 [0013] The present invention provides a glucose oxidase said glucose oxidase-encoding gene G0D-H-6 mutant.

[0014] 本发明提供了含有所述的葡萄糖氧化酶编码基因的重组菌株。 [0014] The present invention provides a recombinant strain containing glucose oxidase encoding gene according to the.

[0015] 本发明提供了葡萄糖氧化酶突变体G0D-H-2、G0D-H-5和G0D-H-6在用于制备动物饲料添加剂中的应用。 [0015] The present invention provides a glucose oxidase G0D-H-2, G0D-H-5 and G0D-H-6 in the preparation of an animal feed additive for mutants.

[0016] 本发明的优点和技术效果是:本发明的目的是采用定向进化技术对来源于黑曲霉(Aspergillus niger)的葡萄糖氧化酶进行蛋白质工程改造,为达到上述发明目的,本发明使用易错PCR方法对葡萄糖氧化酶基因进行突变,再通过高通量筛选方法将正突变检出,得到热稳定性提高的突变体,相比野生型葡萄糖氧化酶GOD-1,本发明的6个突变体G0D-H-1、 600-!1-2、600-!1-3、600-!1-4、600-!1-5、600-!1-6热稳定性明显提高,在80°(:处理3111丨11后,残余酶活分别提高了1.3、1.6、1.9、2.1、2.5、2.3倍。本发明提供的突变体具有良好的市场应用前景和工业价值 [0016] The advantages and technical effects of the invention are: object of the present invention is the use of directed evolution technology derived from Aspergillus niger (Aspergillus niger) glucose oxidase protein engineering, to achieve the above object, the present invention is the use of error-prone PCR was performed for the glucose oxidase gene mutation, high throughput screening methods and then by the positive detection of mutation, the mutant obtained improved thermal stability, compared with the wild-type glucose oxidase GOD-1, 6 mutants according to the present invention G0D-H-1, 600-! 1-2,600-! 1-3,600-! 1-4,600-! 1-5,600-! 1-6 significantly improved thermal stability at 80 ° ( : 3111 Shu 11 after treatment, were increased residual enzyme mutant 1.3,1.6,1.9,2.1,2.5,2.3 times provided by the invention has a good market prospect and industrial value.

附图说明 BRIEF DESCRIPTION

[0017]图1是葡萄糖氧化酶突变体G0D-H-1与野生型氨基酸序列比对图; [0017] FIG. 1 is glucose oxidase mutant G0D-H-1 than the wild-type amino acid sequence of FIG;

[0018] 图2是葡萄糖氧化酶突变体G0D-H-2与野生型氨基酸序列比对图; [0018] FIG. 2 is glucose oxidase mutant G0D-H-2 and the wild type amino acid sequence alignment of FIG;

[0019] 图3是葡萄糖氧化酶突变体G0D-H-3与野生型氨基酸序列比对图; [0019] FIG. 3 is glucose oxidase mutant G0D-H-3 and the wild type amino acid sequence alignment of FIG;

[0020] 图4是葡萄糖氧化酶突变体G0D-H-4与野生型氨基酸序列比对图; [0020] FIG. 4 is glucose oxidase mutant G0D-H-4 and wild type amino acid sequence alignment of FIG;

[0021 ]图5是葡萄糖氧化酶突变体G0D-H-5与野生型氨基酸序列比对图; [0021] FIG. 5 is glucose oxidase mutant G0D-H-5 and the wild type amino acid sequence alignment of FIG;

[0022] 图6是葡萄糖氧化酶突变体G0D-H-6与野生型氨基酸序列比对图; [0022] FIG. 6 is glucose oxidase mutant G0D-H-6 compared with wild-type amino acid sequence of FIG;

[0023] 图7为葡萄糖氧化酶突变体G0D-H-1、G0D-H-2、G0D-H-3、G0D-H-4、G0D-H-5、G0D-H-6在不同温度下的残余酶活。 [0023] FIG. 7 is glucose oxidase mutant G0D-H-1, G0D-H-2, G0D-H-3, G0D-H-4, G0D-H-5, G0D-H-6 at different temperatures the residual enzyme activity.

具体实施方式 Detailed ways

[0024]下面结合附图和具体实施例对本发明的技术方案做进一步详细的说明。 [0024] DRAWINGS specific embodiments and further detailed description of the technical solution of the present invention.

[0025] 本发明用到了分子生物学领域使用的常规技术和方法。 [0025] The present invention uses conventional techniques and methods used in the art of molecular biology. 实施例仅是用于解释本发明,不限制本发明的保护范围。 Example embodiments are merely to illustrate the invention without limiting the scope of the present invention.

[0026] 实施例1:易错PCR(err〇r-pr〇ne PCR)方法构建葡萄糖氧化酶G0D-1突变文库 [0026] Example 1: Construction of glucose oxidase G0d Yi-1 mutant libraries wrong PCR (err〇r pr〇ne-PCR) method

[0027] 来源于黑曲霉(Aspergillus niger)的葡萄糖氧化酶基因G0D-1由589个氨基酸构成(如SEQ ID N0:1所示),采用全基因合成的方法合成了该葡萄糖氧化酶基因G0D-1(如SEQ ID N0:2所示),合成的基因两端还带有EcoR I和Not I酶切位点。 [0027] derived from Aspergillus niger (Aspergillus niger) glucose oxidase gene G0D-1 of 589 amino acids (e.g., SEQ ID N0: 1 as shown), using the whole gene synthesis method of the synthesis of the glucose oxidase gene G0D- 1 (e.g., SEQ ID N0: 2 as shown), both ends of the synthetic gene also with Not I and EcoR I restriction sites. 以合成的该基因为模板扩增葡萄糖氧化酶G0D-1基因,使用GeneMorph II随机突变PCR试剂盒(Stratagene)随机引入突变。 In this synthetic gene was amplified G0D-1 glucose oxidase gene using PCR random mutagenesis GeneMorph II kit (Stratagene) introducing mutations randomly.

[0028] 所用引物为:5' -GCGCGAATTCCGCTGCGGCCCTGCCACACTAC-3' (SEQ ID No:9), [0028] As used primers: 5 '-GCGCGAATTCCGCTGCGGCCCTGCCACACTAC-3' (SEQ ID No: 9),

[0029] 57-TAAAGCGGCCGCTCACTGCATGGAAGCATAATCTTCCAAGATAGCATCC-3 7(SEQ ID No: 10)〇 [0029] 57-TAAAGCGGCCGCTCACTGCATGGAAGCATAATCTTCCAAGATAGCATCC-3 7 (SEQ ID No: 10) square

[0030]下划线处分别为EcoR I和Not頂每切位点。 [0030] EcoR I are underlined and Not top of each cleavage site.

[0031] 反应条件为:94°C预变性10min,94°C变性60s,58°C退火60s和72°C延伸2min,共30 个循环,回收目的基因片段。 [0031] The reaction conditions were: 94 ° C denaturation 10min, 94 ° C denaturation 60s, 58 ° C and 72 ° C annealing 60s extending 2min, 30 cycles, target gene fragments were recovered.

[0032] 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pET 21a( + )载体(氨苄抗性基因)用Ligase进行连接反应。 [0032] The fragment, and digested pET 21a through the same (+) vector (ampicillin resistance gene) are connected with the reaction Ligase After EcoR I and Not I double digestion. 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°C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150uL LB培养基(含有ImM IPTG,50ng/mL氨苄青霉素),30°C220rpm震荡培养12h,将孔板置于-20°C,反复冻融破壁,获得含有葡萄糖氧化酶的粗酶液。 After the ligated fragment transformed into E. coli BL21-DE3, LB plates containing ampicillin coated, 37 ° C inverted culture, transformants appeared to be on the plate, the monoclonal picked to 96-well plates each well containing 150uL LB medium (containing ImM IPTG, 50ng / mL ampicillin), 30 ° C220rpm shaking culture 12h, the plate was placed -20 ° C, repeated freezing and thawing broken, to obtain a crude enzyme solution containing glucose oxidase. 分别取出5ul裂解液至两块新的96孔板,其中一块于80°C处理3min,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 °C反应3min后加入100uL2M硫酸终止反应,根据显色反应测定残余酶活。 Lysates were taken 5ul to two new 96-well plate, wherein a process at 80 ° C 3min, 96 are two o-dianisidine was added methanol containing buffer, dextrose buffer, horse radish peroxidase solution the color developing solution, 37 ° C 3min reaction 100uL2M sulfuric acid was added to terminate the reaction, the residual enzyme activity was measured in accordance with the color reaction.

[0033]取残余活性比野生型G0D-1高的菌株到新的96孔培养板中,进行重复筛选。 [0033] The residue was taken activity than the wild type strain G0D-1 high to fresh 96-well culture plate, repeated screening. 筛选到2个突变体,分别为G0D-H-1和G0D-H-2,残余活性是野生型对照的1-3倍,对这两个突变体进行DNA测序。 Screening of the two mutants were 1-3 times G0D-H-1 and G0D-H-2, the residual activity of wild-type control, the two mutant DNA sequencing.

[0034] 测序结果显示,如图1和图2所示,本轮易错PCR获得了一个含K122N和G492E的两点突变的突变体G0D-H-1,其氨基酸序列为SEQ ID N0:3,一个含A143P和F461I的两点突变的突变体G0D-H-2,其氨基酸序列为SEQ ID N0:4。 [0034] Sequence analysis showed that 1 and 2, FIG. 2 PCR round of error prone obtained a mutant containing two o'clock and G492E mutation K122N the G0D-H-1, amino acid sequence of SEQ ID N0: 3 , a mutant containing two mutations A143P and F461I of G0D-H-2, the amino acid sequence of SEQ ID N0: 4.

[0035] G0D-H-1:该酶的第122位的赖氨酸K变为天冬酰胺N(AAG变为AAC),第492位的甘氨酸G变为谷氨酸E(GGG变为GAG)。 [0035] G0D-H-1: enzyme K lysine at position 122 changed to an asparagine N (AAG changed to AAC), glycine at position 492 to glutamic acid G E (GGG GAG becomes ).

[0036] G0D-H-2:该酶的第143位的丙氨酸A变为脯氨酸P(GCC变为CCC),第461位的苯丙氨酸F变为异亮氨酸I(TTC变为ATC)。 [0036] G0D-H-2: 143 enzyme alanine-proline A to P (GCC changed to CCC), phenylalanine at position 461 being altered to isoleucine F I ( TTC becomes ATC).

[0037]实施例2:第二轮易错PCR突变文库的构建和筛选突变文库的构建[0038]将第一轮易错PCR方法筛选到的耐热性提高的两个突变体G0D-H01和G0D-H02分别提取质粒作第二轮易错PCR的模板,突变文库的构建过程,使用的引物,PCR反应条件,同实施例1。 [0037] Example 2: The second round of error-prone PCR mutation library construction and screening of mutant library construction [0038] The improved heat resistance of the first round of error prone PCR and screened to two G0D-H01 and mutants G0D-H02 plasmids were extracted for a second round of error-prone PCR template mutant library construction process, the use of primers, PCR reaction conditions, same as in Example 1.

[0039]通过第二轮易错PCR,同样获得了大量的突变体基因片段。 [0039] by a second round of error-prone PCR, to obtain a large number of the same mutant gene fragment. 将构建得到的突变体转入大肠杆菌表达菌株BL21-DE3,筛选耐热性正突变时以G0D-H-1和G0D-H-2为对照,其余操作与实施例2相同,取残余活性比突变型G0D-H-1和G0D-H-2高的菌株到新的96孔培养板中, 进行重复筛选。 The mutant construct was transferred to E. coli expression strain BL21-DE3, heat resistance screened positive mutated to G0D-H-1 and is G0D-H-2 control, the same operation as in Example 2 to rest, taking the residual activity ratio of mutant G0D-H-1 and high G0D-H-2 strain to a fresh 96-well culture plate, repeated screening.

[0040] 本轮筛选共获得4个突变体,分别命名为G0D-H-3、G0D-H-4,G0D-H-5和G0D-H-6。 [0040] round of screening A total of four mutants, named G0D-H-3, G0D-H-4, G0D-H-5 and G0D-H-6. 其中G0D-H-3和G0D-H-4是以G0D-H01为模板获得的突变体,其热稳定性高于G0D-H01,G0D-H-5 和G0D-H-6是以G0D-H02为模板获得的突变体,其热稳定性高于G0D-H02,挑取突变体菌株送测序公司测序。 Wherein G0D-H-3 and G0D-H-4 is a mutant G0D-H01 as a template obtained, its thermal stability than G0D-H01, G0D-H-5 and G0D-H-6 is G0D-H02 mutants obtained as a template, its higher heat stability than G0D-H02, mutant strain were picked send sequenced sequenced.

[0041 ] 测序结果显示,如图3、图4、图5和图6所示,本轮易错PCR获得一个含D70Q、K122N和G492E的三点突变的突变体G0D-H-3,其氨基酸序列为SEQ ID N0:5。 [0041] Sequence analysis showed that 3, 4, 5, and 6, to obtain a round of error-prone PCR containing D70Q, three o'clock mutations K122N mutants G492E and the G0D-H-3, which is amino acids sequence SEQ ID N0: 5. 一个含K122N、Q263P、 R341S和G492E的四点突变的突变体G0D-H-4,其氨基酸序列为SEQ ID N0:6。 Containing a mutant K122N, Q263P, R341S, and the four point mutations G492E G0D-H-4, the amino acid sequence of SEQ ID N0: 6. 一个含I29L、 A143P、G390R和F461I的四点突变的突变体G0D-H-5,其氨基酸序列为SEQIDN0:7。 Containing a I29L, A143P, G390R and four point mutations F461I mutant G0D-H-5, which is the amino acid sequence SEQIDN0: 7. 一个含A143P、P241D、T359S和F4611的四点突变的突变体G0D-H-6,其氨基酸序列为SEQ ID NO: 8。 Containing a mutant A143P, P241D, T359S and four point mutations of F4611 G0D-H-6, the amino acid sequence of SEQ ID NO: 8.

[0042] G0D-H-3:该酶的第70位天冬氨酸D变为谷氨酸Q(DNA序列由GAC变为GAA),第122位的赖氨酸K变为天冬酰胺N(AAG变为AAC),第492位的甘氨酸G变为谷氨酸E(GGG变为GAG)。 [0042] G0D-H-3: 70 enzyme of aspartic acid to glutamic acid D Q (DNA sequence changes by a GAC ​​GAA), lysine at position 122 changed to an asparagine K N (AAG becomes AAC), glycine at position 492 to glutamic acid G E (GGG becomes GAG). [0043] G0D-H-4:该酶的第122位的赖氨酸K变为天冬酰胺N(AAG变为AAC),第263为的谷氨酰胺Q变为脯氨酸P(CAG变为CCG),第341位的精氨酸R变为丝氨酸S(CGC变为AGC),第492位的甘氨酸G变为谷氨酸E(GGG变为GAG)。 [0043] G0D-H-4: enzyme K lysine at position 122 changed to an asparagine N (AAG changed to AAC), 263 is changed to proline Q glutamine P (CAG Variable is CCG), arginine at position 341 changed to serine R S (CGC changed to AGC), glycine at position 492 to glutamic acid G E (GGG becomes GAG).

[0044] G0D-H-5:该酶的第29位的异亮氨酸I变为亮氨酸L(AUC变为CUC),第143位的丙氨酸A变为脯氨酸P(GCC变为CCC),第390位的甘氨酸G变为精氨酸R(GGC变为CGC),第461位的苯丙氨酸F变为异亮氨酸I(TTC变为ATC)。 [0044] G0D-H-5: at position 29 of the enzyme Isoleucine I Leucine becomes L (AUC becomes CUC), 143 alanine proline A to P (GCC becomes CCC), glycine at position 390 changed to arginine G R (GGC becomes CGC), the phenylalanine at position 461 being altered to isoleucine F I (TTC becomes ATC).

[0045] G0D-H-6:该酶的第143位的丙氨酸A变为脯氨酸P(GCC变为CCC),第241位的脯氨酸P变为天门冬氨酸D(CCC变为GAC),第359位的苏氨酸T变为丝氨酸S(ACC变为UCC),第461位的苯丙氨酸F变为异亮氨酸I(TTC变为ATC)。 [0045] G0D-H-6: enzyme alanine at position 143 changed to Proline P A (changed to the GCC CCC), proline at position 241 changed to aspartic acid P D (CCC becomes GAC), threonine at position 359 changed to serine T S (ACC becomes UCC), phenylalanine at position 461 being altered to isoleucine F I (TTC becomes ATC).

[0046] 实施例3:毕赤酵母工程菌株的构建 Construction of Pichia pastoris strains: [0046] Example 3

[0047] 使用实施例1中所述引物,以实施例1和实施例2所获得的突变体作为模板进行PCR 扩增,PCR反应条件与实施例1相同。 Example 1 The primer [0047] Using Example, Example 1 and the mutants obtained in Example 2 was amplified by PCR as a template, PCR reaction conditions were the same as in Example 1.

[0048] 将扩增得到的实施例1及实施例2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,构建表达载体?? [0048] The gene fragment amplified in Example 1, and the wild-type gene fragments and mutants of the glucose oxidase in Example 2, connected by EcoR I and Not I sites of the expression vector pPIC9K, construct an expression vector? ? 1091(-600-1、pPIC9K-G0D-H-1、pPIC9K-G0D-H-2、pPIC9K-G0D-H-3、pPIC9K-G0D-H-4、pPIC9K-G0D-H-5 和PPIC9K-G0D-H-6。将表达载体转入大肠杆菌DH5a感受态,挑取转化子后大量提取质粒。 1091 (-600-1, pPIC9K-G0D-H-1, pPIC9K-G0D-H-2, pPIC9K-G0D-H-3, pPIC9K-G0D-H-4, pPIC9K-G0D-H-5 and PPIC9K-G0D -H-6. the expression vectors transformed into E. coli DH5a competent, the plasmid was extracted after a large number of transformants were picked.

[0049] 将以上表达质粒用Sail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,通过电转化方法转化毕赤酵母GS115,涂布MD平板。 [0049] The above expression plasmids were linearized with Sail, linear fragment was collected and purified by fragment purification kit (TaKaRa MiniBEST DNA Fragment Purifibation Kit), transformed by electroporation transformation method pastoris GSl 15, coating MD plates. 将在MD平板上生长出的菌落涂布到浓度依次逐渐升高(lmg/mL,2mg/ mL,4mg/mL,8mg/mL)的遗传霉素的YH)平板上筛选多拷贝的阳性转化子,得到毕赤酵母重组菌株。 Colonies grown on MD plates are sequentially applied to a concentration gradually increased (lmg / mL, 2mg / mL, 4mg / mL, 8mg / mL) of Geneticin YH) positive transformants screened multiple copies on the plate to obtain a recombinant strain Pichia.

[0050] 将7个基因的转化子分别命名为毕赤酵母600-1(?丨吐丨3口38如^8 600-1)、600-H_l(Pichia pastoris G0D-H-1)、毕赤酵母G0D-H_2(Pichia pastoris G0D-H-2)、毕赤酵母G0D-H_3(Pichia pastoris G0D-H-3)、毕赤酵母G0D-H_4(Pichia pastoris G0D-H-4)、 毕赤酵母G0D-H_5(Pichia pastoris G0D-H-5)和毕赤酵母G0D-H_6(Pichia pastoris G0D-H-6),分别挑取每个基因的转化子转接于BMGY培养基中,30°C,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0D600 = 1,30°C,220rpm继续振荡培养,每24h添加培养体积1 %的甲醇。 [0050] Transformants seven genes were designated as 600-1 Pichia (? 3 Shu Shu jetting as 38 ^ 8 600-1), 600-H_l (Pichia pastoris G0D-H-1), Pichia yeast G0D-H_2 (Pichia pastoris G0D-H-2), Pichia G0D-H_3 (Pichia pastoris G0D-H-3), Pichia G0D-H_4 (Pichia pastoris G0D-H-4), Pichia G0d -H_5 (Pichia pastoris G0D-H-5), and Pichia G0D-H_6 (Pichia pastoris G0D-H-6), respectively, transformants were picked for each gene transfer in BMGY medium, 30 ° C, 220rpm after shaking the culture for 18 h, centrifuged to obtain bacterial cells, the amount of cells transferred to BMMY medium so that cell concentration 0D600 = 1,30 ° C, 220rpm shaking culture was continued, the culture was added every 24h 1% by volume of methanol. 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。 4D expression after induction, the culture was centrifuged to obtain a supernatant, the supernatant is measured glucose oxidase activity and thermostability assays.

[0051 ]实施例4:突变体和野生型表达产物酶活力及热稳定性的测定[0052]酶活力测定方法: [0051] Example 4: mutant and wild-type expression product of the enzyme activity and thermal stability assay [0052] Determination of enzyme activity:

[0053] 取邻联茴香胺缓冲液2.5mL(0 . lmLl %邻联茴香胺甲醇储备液加入到12mL 0.1M pH6.0磷酸缓冲液配成),18%葡萄糖溶液0.3mL,0.03 %过氧化物酶溶液0. lmL,加入到比色管中37°C保温5min,再加入0. lmL葡萄糖氧化酶酶液(空白管加入0. lmL蒸馏水),反应3min 后,加入2M硫酸2mL,混匀以终止反应。 [0053] o - dianisidine buffer 2.5mL (0. LmLl% o-dianisidine in methanol stock solution was added to 12mL 0.1M pH6.0 phosphate buffer dubbed), 18% glucose solution 0.3mL, 0.03% peroxide 0. lmL enzyme solution was added to the cuvette incubated 37 ° C 5min, then added 0. lmL glucose oxidase enzyme solution (blank tube of distilled water was added 0. lmL), 3min after the reaction, sulfuric acid was added 2M 2mL, mix to stop the reaction. 以标准空白样为空白对照,在540nm波长处测定空白(A〇)和试样溶液(心)的吸光值。 In the standard blank is blank, measuring a blank (A〇) and sample solution (core) is the absorbance at a wavelength of 540nm. 得出Δ A=Ai-Ao Stars Δ A = Ai-Ao

[0054]试样酶活力计算: [0054] Sample Calculation of enzyme activity:

[0055] X=( AAXnX3)/(11.3XtX0.1) [0055] X = (AAXnX3) / (11.3XtX0.1)

[0056] T一测定时间,min [0056] T a measurement time, min

[0057] 0.1-样品体积,mL [0057] 0.1 sample volume, mL

[0058] 11.3-消光系数 [0058] The extinction coefficient 11.3

[0059] N-稀释倍数[0060] 3-反应液体积,mL [0059] N- dilution [0060] 3- volume of the reaction solution, mL

[00611酶活单位与定义:在pH5.5、37°C的条件下,每分钟能把1. Ownol的β-D-葡萄糖氧化成葡萄糖酸和H202的酶量为一个单位。 [00611 unit of enzyme activity defined: under conditions of pH5.5,37 ° C, per minute can 1. Ownol the β-D- glucose is oxidized to gluconic acid and the amount of enzyme H202 is one unit.

[0062]将实施例3所述发酵上清液用pH 6.0的磷酸缓冲液稀释至约20U/mL,在80°C条件下处理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。 [0062] The embodiments of the Example 3 was diluted fermentation supernatant at pH 6.0 with phosphate buffer to about 20U / mL, after treatment at 80 ° C for 3min conditions, determine the residual enzyme activity, enzyme activity for the untreated sample 100%, the relative activity is calculated. 结果如图7所示,野生型葡萄糖氧化酶在80°C条件下处理3min,酶活仅剩23%,而突变体600-!1-1、6(®-!!- 2、600-!1-3、600-!1-4,600-!1-5和600-!1-6同样在80°(:条件下处理3111丨11仍能保持30-60%的酶活。其耐热性分别与野生型基因相比,分别提高了1.3、1.6、2.2、2.1、2.7、2.3倍。 Results As shown, treatment of wild-type glucose oxidase at 80 ° C for 7 conditions 3min, 23% remaining activity, while the mutant 600- 1-1,6 (® -! !! - 2,600-! 1-3,600- 1-4,600- 1-5 and 600-1-6 in the same (80 °:!!! 11 Shu process 3111 can maintain 30-60% of the enzyme under heat resistance, respectively. compared with the wild-type gene, it was increased 1.3,1.6,2.2,2.1,2.7,2.3 times.

[0063] 由此可见,突变后的葡萄糖氧化酶的耐热性与野生型相比有了较大提高,更加有利于其在工业中的应用。 [0063] Thus, the mutated glucose oxidase and heat resistance have been greatly improved compared to wild type, it is more advantageous application in industry.

[0064] 实施例5:葡萄糖氧化酶的养殖应用实验 [0064] Example 5: Application of breeding experiments glucose oxidase

[0065] 5.1实验设计 [0065] 5.1 Experimental Design

[0066]白羽肉鸡苗购自某种禽厂,共分为8栋鸡舍,每一鸡舍20000只鸡,其中1、2、3、4为对照组,5、6、7、8为实验组,实验组在基础日粮中添加0.2U/g本发明提供的葡萄糖氧化酶G0D-H-5,实验为期40天。 [0066] White Boilers plant seedlings purchased from a certain birds, divided into 8 sheds, each 20,000 chicken coop, wherein 1,2,3,4 control group, the experimental group 5,6,7,8 experimental group was added glucose oxidase G0D-H-5 0.2U / g of the present invention provides a basal diet, a 40-day experiment.

[0067] 5.2生产性能测定 [0067] 5.2 Performance of assay

[0068] 分别实验开始第2、40天对各组鸡空腹称重,记录初始重和末重。 [0068] start of the experiment were weighed 2,40 fasting day chickens in each group, and record the initial weight final weight. 饲养过程中,观察记录各组鸡的健康状况,并记录每周每笼采食量,统计实验期内的成活率、料重比及计算欧洲指数。 The feeding process, the recording state of health was observed in each group of chickens, and the survival rate recorded intake per cage, statistical experimental period weekly, feed conversion ratio and calculating European indices. 实验结果: The results:

[0069] 表1对照组生产性能数据 [0069] Table 1 Control group performance data

Figure CN105950577AD00071

[0071 ]表2实验组生产性能数据 [0071] Table 2 experimental group performance data

Figure CN105950577AD00081

[0073] 与对照组相比,实验组的均重有所增加,成活率也有所增加,而料重比有所下降, 经过计算欧洲指数上升了2.9 %,表明在日粮中添加了葡萄糖氧化酶G0D-H-5使养殖效益有所增加。 [0073] Compared with the control group, the experimental group the average weight increase, also increased the survival rate, and decreased feed conversion ratio, calculated to Europe rose 2.9%, indicating that the added glucose oxidation in the diet G0D-H-5 enzyme the breeding efficiency has increased.

[0074] 本实施例5为了便于体现本发明所述的葡萄糖氧化酶的应用,并不限于肉鸡的应用,因为所述的葡萄糖氧化酶可以添加到基础日粮中,可以用于其他畜禽类的饲喂。 [0074] Example 5 of the present embodiment in order to facilitate the application reflect the glucose oxidase of the present invention is not limited to application broiler, because the glucose oxidase may be added to the basal diet, it may be used for other livestock and poultry feeding. 通常可以在猪、兔和奶牛等养殖过程中在其配合饲料中添加。 Generally it may be added to feed in pigs, rabbits, and the like dairy cattle breeding process.

[0075] 以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。 [0075] The above embodiments are intended to illustrate the present invention, but not intended to be limiting;. Although the present invention has been described in detail embodiments, those of ordinary skill in the art, the foregoing can still the technical solutions described in the embodiments may be modified, or some technical features equivalents; as such modifications or replacements do not cause the essence of corresponding technical solutions to depart from the spirit and scope of the invention as claimed technical solution.

Claims (10)

1. 一种热稳定性提高的葡萄糖氧化酶突变体G0D-H-2,其特征在于:所述的葡萄糖氧化酶突变体G0D-H-2的氨基酸序列如SEQ ID N0:4所示,所述的突变体G0D-H-2序列由氨基酸序列为SEQ ID NO: 1的葡萄糖氧化酶的第143位氨基酸由丙氨酸变为脯氨酸、第461位氨基酸由苯丙氨酸变为异亮氨酸获得。 An improved thermal stability of glucose oxidase mutant G0D-H-2, wherein: the amino acid sequence of G0D-H-2 of the glucose oxidase mutations such as SEQ ID N0: 4 shown, the mutants described G0D-H-2 sequence as the amino acid sequence of SEQ ID NO: 143 amino acid at position 1 of glucose oxidase is changed from alanine proline at amino acid 461 by a phenylalanine isobutyl becomes leucine is obtained.
2. 权利要求1所述的葡萄糖氧化酶突变体G0D-H-2的葡萄糖氧化酶编码基因。 Glucose oxidase said glucose oxidase-encoding gene 1 G0D-H-2 mutant claim.
3. 含有权利要求2所述的葡萄糖氧化酶编码基因的重组菌株。 The recombinant strain of claim 2 gene encoding glucose-containing oxide according to claim 3.
4. 一种热稳定性提高的葡萄糖氧化酶突变体G0D-H-5,其特征在于:所述的葡萄糖氧化酶突变体G0D-H-5的氨基酸序列如SEQ ID N0:7所示,所述的突变体G0D-H-5序列由氨基酸序列为SEQ ID NO: 1的葡萄糖氧化酶的第29位氨基酸由异亮氨酸变为亮氨酸、第143位氨基酸由丙氨酸变为脯氨酸、第390位氨基酸由甘氨酸变为精氨酸、第461位氨基酸由苯丙氨酸变为异亮氨酸获得。 An improved thermal stability of glucose oxidase mutant G0D-H-5, wherein: the amino acid sequence of G0D-H-5 of the glucose oxidase mutations such as SEQ ID N0: 7 shown in the mutant G0D-H-5 amino acid sequence described by the sequence SEQ ID NO: 29, amino acids 1 glucose oxidase changed by isoleucine leucine, amino acid 143 alanine proline becomes acid, amino acid 390 by a glycine to arginine at amino acid 461 by a phenylalanine isoleucine becomes available.
5. 权利要求4所述的葡萄糖氧化酶突变体G0D-H-5的葡萄糖氧化酶编码基因。 Glucose oxidase claimed in claim 4 mutant G0D-H-5 gene encoding glucose oxidase.
6. 含有权利要求5所述的葡萄糖氧化酶编码基因的重组菌株。 6. The recombinant strain containing a gene encoding glucose oxidation as claimed in claim 5.
7. -种热稳定性提高的葡萄糖氧化酶突变体G0D-H-6,其特征在于:所述的葡萄糖氧化酶突变体G0D-H-6的氨基酸序列如SEQ ID N0:8所示,所述的突变体G0D-H-6序列由氨基酸序列为SEQ ID NO: 1的葡萄糖氧化酶的第143位氨基酸由丙氨酸变为脯氨酸、第241位氨基酸由脯氨酸变为天门冬氨酸、第359位氨基酸由苏氨酸变为丝氨酸、第461位氨基酸由苯丙氨酸变为异亮氨酸获得。 7. - species improved thermostability glucose oxidase mutant G0D-H-6, wherein: said glucose oxidase mutant G0D-H-6 amino acid sequence as SEQ ID N0: 8 shown, the said mutant G0D-H-6 sequence as the amino acid sequence of SEQ ID NO: 143 amino acids glucose oxidase becomes 1 by an alanine-proline, a proline at position 241 changes the amino acid asparagine acid, amino acid 359 by a threonine to serine, amino acid 461 by a phenylalanine isoleucine becomes available.
8. 权利要求7所述的葡萄糖氧化酶突变体G0D-H-6的葡萄糖氧化酶编码基因。 Glucose oxidase according to claim 7 glucose oxidase encoding gene G0D-H-6 mutant.
9. 含有权利要求8所述的葡萄糖氧化酶编码基因的重组菌株。 9. The recombinant strain containing a gene encoding glucose oxidation as claimed in claim 8.
10. 葡萄糖氧化酶突变体G0D-H-2、G0D-H-5和G0D-H-6在用于制备动物饲料添加剂中的应用。 10. Glucose oxidase mutant G0D-H-2, G0D-H-5 and G0D-H-6 in the preparation of additives for animal feed.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2415863A1 (en) * 2010-08-03 2012-02-08 B.R.A.I.N. Mutant glucose oxidase
CN103981159A (en) * 2014-06-05 2014-08-13 青岛蔚蓝生物集团有限公司 Glucose oxidase mutant and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2415863A1 (en) * 2010-08-03 2012-02-08 B.R.A.I.N. Mutant glucose oxidase
CN103981159A (en) * 2014-06-05 2014-08-13 青岛蔚蓝生物集团有限公司 Glucose oxidase mutant and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JULIA MARIN-NAVARRO等: "Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase", 《PLOS ONE》 *

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