CN118086257A - α-半乳糖苷酶突变体 - Google Patents
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Abstract
本发明涉及基因工程与蛋白质改造技术领域,具体涉及一种α‑半乳糖苷酶突变体。本发明以α‑半乳糖苷酶AG2为基础,提供了分别包含至少一个选自S41H、I108V、S146M、A174W、A176M、N201Y、V202P、K206Y、V211W、A230L、D233S、S239H、A268I、V328I、T347K、T347L、T347Q、H358E、T395K、T395R、V397M、N407A、A459C、A462D、A462Q、T478K、N487L、Q502C、Q539M、Q539Y、A552P、A577S、A581P、A581R、V587T、S601C、G613F、S654L、Q659I、Q659K、Q659M、Q681D、Q681S、S710R所述突变位点的α‑半乳糖苷酶突变体。与野生型相比,所述突变体的耐热性得到显著提升,有利于其在饲料中的广泛应用。
Description
技术领域
本发明属于基因工程与蛋白质改造技术领域,具体涉及一种α-半乳糖苷酶突变体。
背景技术
α-半乳糖苷酶,又叫蜜二糖酶(α-galactosidase,EC 3.2.1.22),可以催化不同半乳糖苷底物去除α-1,6-连接的半乳糖残基。α-半乳糖苷酶在自然界中广泛存在,尤其在细菌、真菌、酵母菌等微生物中分布最广。α-半乳糖苷酶的细菌来源有嗜热芽孢杆菌、嗜酸乳酸杆菌、发酵乳杆菌、青春双歧杆菌、短双歧杆菌等。另外从极端嗜热菌杜氏杆菌及海洋细菌假交替单胞菌也分离得到α-半乳糖苷酶。据报道丝状放线菌当中也含有α-半乳糖苷酶,例如从重红色链丝菌和红色糖多孢菌中可分离得到α-半乳糖苷酶。Brouns等从高酸度陆地火山地区发现的超嗜热古生菌硫磺矿硫化叶菌P2也可分离纯化得到α-半乳糖苷酶,这种α-半乳糖苷酶具有极高的热稳定性。此外,在真菌中,米曲霉、烟曲霉、藤仓赤霉、简青霉、嗜热棉毛菌及少孢根霉均可作为α-半乳糖苷酶的来源。Wang等从费希新萨托菌中分离得到一种α-半乳糖苷酶,研究结果表明,其对豆制品具有高度专一的水解活性。
大豆和其他豆类作为饲料当中丰富的蛋白质来源,其中含有高浓度的可溶性低聚糖如棉子糖和水苏糖等,其在动物胃肠道中不能被彻底消化,这些未消化完全的糖进入动物肠道后会促进有害菌群的增长,引起胀气和胃肠道紊乱,严重损害动物的健康,降低动物饲料的饲用效率。因此,在动物饲料中添加α-半乳糖苷酶制剂可以将这些多余的糖彻底清除,抑制其在肠道中发酵,减轻动物胃肠道胀气症状,避免有害致病菌对动物肠道的损害,提高动物的免疫力,增加动物肠道对营养物质的吸收,提高饲料利用率。
例如,Baucells等在生长猪和肥育猪的饲料中添加α-半乳糖苷酶制剂,研究结果表明,与对照组相比试验组生长猪和肥育猪的体重明显增加,各项指标明显提升。Ghazi等在肉鸡大豆饲料中添加α-半乳糖苷酶制剂,结果表明,肉鸡对大豆饲料的消化效率提高,增加了大豆饲料的营养价值。戴求仲等在饲料中添加α-半乳糖苷酶制剂研究其对黄羽肉鸡生产性能的影响,结果表明,添加酶制剂后可以降解饲料中的α-半乳糖苷,改变肠道底物组成,促进有益菌的生长,抑制有害菌的生长,改善肉鸡生长状况,能够明显降低肉鸡后期和全期料重比,改善试验鸡生产性能。缪志军等在饲料中添加α-半乳糖苷酶制剂研究其对番鸭增重和生产性能的影响,结果表明,其能够提高番鸭增重,改善生产性能。
目前在颗粒饲料生产过程中有一个短暂的高温阶段,将α-半乳糖苷酶直接添加到动物饲料中进行制粒,其残留酶活极低,而采用饲料制粒后将α-半乳糖苷酶喷涂到饲料上或拌入到饲料中不仅增加设备投入,而且对酶制剂的稳定性和在饲料中分布的均一性都无法得到很好的保证。因此,提高α-半乳糖苷酶的耐温性具有重要的现实意义。
发明内容
本发明为解决现有技术问题,提供了一种α-半乳糖苷酶突变体;是在α-半乳糖苷酶AG2的基础上通过大量的突变筛选,最终获得耐热性得到显著提高的突变体,为其在饲料领域的广泛使用奠定了基础。
本发明提供了一种α-半乳糖苷酶突变体,其包含与SEQ ID NO:2具有至少90%同一性的氨基酸序列,且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代:41、108、146、174、176、201、202、206、211、230、233、239、268、328、347、358、395、397、407、459、462、478、487、502、539、552、577、581、587、601、613、654、659、681、710。
在本发明的一些实施例中,所述突变体的氨基酸序列与SEQ ID NO:2相比具有至少91%,92%,93%,94%,95%,96%,97%,98%,或至少99%的同一性。
在一些更具体的实施例中,所述突变体的氨基酸序列与SEQ ID NO:2相比具有至少99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,或至少99.9%的同一性。
在本发明的一些实施例中,所述突变体包含下组中至少一个氨基酸的取代:S41H、I108V、S146M、A174W、A176M、N201Y、V202P、K206Y、V211W、A230L、D233S、S239H、A268I、V328I、T347K、T347L、T347Q、H358E、T395K、T395R、V397M、N407A、A459C、A462D、A462Q、T478K、N487L、Q502C、Q539M、Q539Y、A552P、A577S、A581P、A581R、V587T、S601C、G613F、S654L、Q659I、Q659K、Q659M、Q681D、Q681S、S710R。
在本发明的一些实施例中,所述突变体包含下组中至少一个氨基酸的取代组合:
S41H/I108V;
S41H/S146M;
S41H/A174W;
S41H/A176M;
S41H/N201Y;
S41H/V202P;
S41H/K206Y;
S41H/V211W;
S41H/A230L;
S41H/A268I;
S41H/T347K;
S41H/H358E;
S41H/T395K;
S41H/N407A;
S41H/N487L;
S41H/Q502C;
S41H/Q539Y;
S41H/A552P;
S41H/V587T;
S41H/G613F;
S41H/S654L;
S41H/Q659I;
S41H/Q659M;
S41H/Q681D;
S41H/S710R;
V211W/I108V;
V211W/S146M;
V211W/A174W;
V211W/A176M;
V211W/N201Y;
V211W/V202P;
V211W/K206Y;
V211W/A230L;
V211W/A268I;
V211W/T347K;
V211W/H358E;
V211W/T395K;
V211W/N407A;
V211W/N487L;
V211W/Q502C;
V211W/Q539Y;
V211W/A552P;
V211W/A581R;
V211W/V587T;
V211W/G613F;
V211W/S654L;
V211W/Q659I;
V211W/Q659M;
V211W/Q681D;
V211W/S710R;
A459C/I108V;
A459C/S146M;
A459C/A174W;
A459C/A176M;
A459C/N201Y;
A459C/V202P;
A459C/K206Y;
A459C/A230L;
A459C/A268I;
A459C/T347K;
A459C/H358E;
A459C/T395K;
A459C/N407A;
A459C/N487L;
A459C/Q502C;
A459C/Q539Y;
A459C/A552P;
A459C/A581R;
A459C/V587T;
A459C/G613F;
A459C/S654L;
A459C/Q659I;
A459C/Q659M;
A459C/Q681D;
A459C/S710R;
N487L/I108V;
N487L/S146M;
N487L/A174W;
N487L/A176M;
N487L/N201Y;
N487L/V202P;
N487L/K206Y;
N487L/A230L;
N487L/A268I;
N487L/T347K;
N487L/H358E;
N487L/T395K;
N487L/N407A;
N487L/Q502C;
N487L/Q539Y;
N487L/A552P;
N487L/A581R;
N487L/V587T;
N487L/G613F;
N487L/S654L;
N487L/Q659I;
N487L/Q659M;
N487L/Q681D;
N487L/S710R;
A581R/I108V;
A581R/S146M;
A581R/A174W;
A581R/A176M;
A581R/N201Y;
A581R/V202P;
A581R/K206Y;
A581R/A230L;
A581R/A268I;
A581R/T347K;
A581R/H358E;
A581R/T395K;
A581R/N407A;
A581R/A462Q;
A581R/Q502C;
A581R/Q539Y;
A581R/A552P;
A581R/V587T;
A581R/G613F;
A581R/S654L;
A581R/Q659I;
A581R/Q659M;
A581R/Q681D;
A581R/S710R;
S41H/I108V/S146M;
S41H/I108V/A174W;
S41H/S146M/A174W;
S41H/S146M/D233S;
S41H/S146M/S239H
I108V/S146M/N407A/V587T;
I108V/S146M/D233S/V587T;
I108V/S146M/V328I/H358E;
I108V/S146M/D233S/V397M;
I108V/S146M/A459C/Q502C;
A268I/H358E/N407A;
A268I/T347K/H358E/N407A;
A268I/T347K/H358E/T395K;
A268I/T347K/H358E/V587T;
A268I/T347K/H358E/N407A/A459C;
S41H/I108V/S146M/A174W;
S41H/I108V/S146M/A174W/D233S;
S41H/I108V/S146M/A174W/D233S/S239H;
S41H/I108V/S146M/D233S;
S41H/I108V/S146M/V328I;
S41H/I108V/S146M/H358E;
S41H/I108V/S146M/N407A;
S41H/I108V/S146M/V587T;
S41H/I108V/S146M/N407A/V587T;
S41H/I108V/S146M/D233S/V587T;
S41H/I108V/S146M/V328I/H358E;
S41H/I108V/S146M/D233S/V397M;
S41H/I108V/S146M/A459C/Q502C;
S41H/I108V/S146M/V328I/A459C/Q502C;
S41H/I108V/S146M/H358E/A459C/Q502C;
S41H/I108V/S146M/V328I/H358E/A459C/Q502C;
S41H/I108V/S146M/D233S/V397M/V587T;
S41H/I108V/S146M/N407A/A459C/Q502C;
S41H/I108V/S146M/D233S/V397M/A459C/Q502C;
S41H/I108V/S146M/H358E/N407A/V587T;
S41H/I108V/S146M/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/H358E/N407A/A459C/Q502C/V587T;
I108V/S146M/V328I/A459C/Q502C;
I108V/S146M/H358E/A459C/Q502C;
I108V/S146M/V328I/H358E/A459C/Q502C;
I108V/S146M/D233S/V397M/V587T;
S146M/H358E/N407A/A459C/Q502C/V587T;
S146M/A176M/H358E/N407A/A459C/Q502C/V587T;
S146M/N201Y/V202P/H358E/N407A/A459C/Q502C/V587T;
S146M/K206Y/H358E/N407A/A459C/Q502C/V587T;
S146M/V211W/H358E/N407A/A459C/Q502C/V587T;
S146M/A230L/H358E/N407A/A459C/Q502C/V587T;
S146M/T347K/H358E/N407A/A459C/Q502C/V587T;
A268I/H358E/N407A/A459C/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/Q502C/V587T;
A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/A462D/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/T478K/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/N487L/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T;
A268I/T347K/H358E/N407A/A459C/Q502C/A552P/V587T;
H358E/N407A/A459C/Q502C/V587T;
H358E/T395K/N407A/A459C/Q502C/V587T;
H358E/N407A/A459C/A462D/Q502C/V587T;
H358E/N407A/A459C/T478K/Q502C/V587T;
H358E/N407A/A459C/N487L/Q502C/V587T;
H358E/N407A/A459C/Q502C/Q539Y/V587T;
H358E/N407A/A459C/Q502C/A552P/V587T;
S41H/T95K/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/A462D/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/T478K/Q502C/V587T;
S41H/I108V/S146M/T347K/H358EN407A/A459C/N487L/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/A552P/V587T;
S41H/I108V/S146M/A176M/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/N201Y/V202P/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/K206Y/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/V211W/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A230L/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/A462D/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/T478K/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/N487L/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/A552P/V587T;S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/V587T;I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T;I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S710R;I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/G613F/S710R;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S654L/S710R;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q659I/S710R;
I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/V587TK/Q659M/S710R;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q681D/S710R;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/G613F;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S654L;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q659I;
S41H/I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/V587TK/Q659M;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q681D;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S710R;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I;
S41H/I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V587TK/Q659M;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q681D;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/A552P/V587T;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/A552P/V587T;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/G613F;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S654L;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I;
I108V/S146M/V211W/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V58
7TK/Q659M;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/Q681D;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/S654L/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/Q659I/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V58
7TK/Q659M/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/Q681D/S710R;
S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A5
52P/A581R/V587T/Q681D;
S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A5
52P/V587T/Q659I/S710R。
本发明还涉及编码上述α-半乳糖苷酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达载体。
本发明还涉及一种宿主细胞,包含上述重组表达载体。
所述宿主细胞为毕赤酵母(Pichia pastoris)。
所述宿主细胞为黑曲霉(Aspergillus niger)。
所述宿主细胞为里氏木霉(Trichoderma reesei)。
本发明以野生型α-半乳糖苷酶AG2为基础,提供了包含至少一个选自S41H、I108V、S146M、A174W、A176M、N201Y、V202P、K206Y、V211W、A230L、D233S、S239H、A268I、V328I、T347K、T347L、T347Q、H358E、T395K、T395R、V397M、N407A、A459C、A462D、A462Q、T478K、N487L、Q502C、Q539M、Q539Y、A552P、A577S、A581P、A581R、V587T、S601C、G613F、S654L、Q659I、Q659K、Q659M、Q681D、Q681S、S710R所述突变位点的α-半乳糖苷酶突变体。与α-半乳糖苷酶AG2相比,本发明提供的突变体耐热性得到显著提升。其中,本发明提供的包含上述单个突变位点的突变体,在67℃条件下处理3min后,其酶活残留率高达40.42%-72.06%,比野生型提高了10.47%-96.94%;本发明提供的包含上述至少两个突变位点的突变体,在68℃条件下处理3min后,酶活残留率达到24.92%-64.21%,比野生型提高了113.4%-449.7%。
综上,与α-半乳糖苷酶AG2相比,本发明提供的所述单点突变体及组合突变体的耐热性得到显著提升,从而有利于α-半乳糖苷酶在饲料中的广泛应用。
附图说明
图1:重组质粒pPIC9K-AG2图谱。
具体实施方式
下面结合实例对本发明的方法做进一步说明。下列实施例中未注明具体条件的实验方法,通常可按常规条件,如J.萨姆布鲁克(Sambrook)等编写的《分子克隆实验指南》中所述的条件,或按照制造厂商所建议的条件运行。本领域相关的技术人员可以借助实施例更好地理解和掌握本发明。但是,本发明的保护和权利要求范围不仅限于所提供的具体案例,而应包括本领域技术人员在本说明书基础上,不需经过创造性劳动就能扩展的保护范围。
实验材料和试剂:
菌株与载体:大肠杆菌DH5α、毕赤酵母GS115、载体pPIC9k购自Invitrogen公司。
酶与试剂盒:PCR酶及连接酶购买自Takara公司,限制性内切酶购自Fermentas公司,质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司,GeneMorph II随机突变试剂盒购自北京博迈斯生物科技有限公司。
培养基配方:
大肠杆菌培养基(LB培养基):0.5%酵母提取物,1%蛋白胨,1%NaCl,pH7.0;
LB+Amp培养基:LB培养基加100μg/mL氨苄青霉素;
LB+Kana培养基:LB培养基加50μg/mL卡那霉素;
酵母培养基(YPD培养基):1%酵母提取物、2%蛋白胨、2%葡萄糖;
酵母筛选培养基(MD培养基):2%葡萄糖、1.34% YNB、4×10-5%生物素、2%琼脂粉;
BMGY培养基:2%蛋白胨、1%酵母提取物、100mM磷酸钾缓冲液(pH6.0)、1.34%YNB、4×10-5%生物素、1%甘油;
BMMY培养基:2%蛋白胨、1%酵母提取物、100mM磷酸钾缓冲液(pH6.0)、1.34%YNB、4×10-5%生物素、0.5%甲醇;
下面结合实施例对本发明进行详细的描述。
实施例1α-半乳糖苷酶基因的合成与重组质粒的获得
将α-半乳糖苷酶基因命名为AG2,其核苷酸序列为SEQ ID NO:1,其编码氨基酸序列为SEQ ID NO:2。由上海捷瑞生物工程有限公司合成该基因。
根据基因5’端设计PCR引物含有EcoRI内切酶位点,3’端设计PCR引物含NotI内切酶位点,引物序列如下:
5’端引物AG2-F:GGCGAATTCCCCCTGCTATTGGTGCTTCTAA(下划线为限制性内切酶EcoRI识别位点);
3’端引物AG2-R:ATAGCGGCCGCTTATTGTCTTTCAAGAAAAACAAC(下划线为限制性内切酶NotI识别位点)。
以合成的α-半乳糖苷酶AG2基因SEQ ID NO:1为模板,用上述引物进行PCR扩增,PCR扩增体系为:模板1μL、上游引物AG2-F 1μL、下游引物AG2-R 1μL、5×PSBuffer 10μL、dNTPs(2.5mM)4μL、Primer-StarDNA聚合酶1μL、ddH2O 32μL,反应总体系为50μL。PCR循环程序为:95℃预变性5min,30cycles:94℃30sec、55℃30sec、72℃2min,72℃10min;胶回收PCR产物,EcoRI、NotI进行酶切处理后与经同样酶切后的pPIC-9k载体16℃过夜连接并转化大肠杆菌DH5a,涂布于LB+Amp平板,37℃倒置培养,待转化子出现后,菌落PCR验证阳性克隆,经测序验证后最后获得了正确的重组质粒pPIC9K-AG2(图1)。
实施例2α-半乳糖苷酶突变体筛选
为了进一步提高α-半乳糖苷酶AG2的热稳定性,对合成的α-半乳糖苷酶AG2基因进行蛋白结构分析,该蛋白有三个结构域:N端的285个氨基酸残基组成结构域1,中间349个氨基酸残基组成结构域2,C端的74个氨基酸残基组成结构域3,保守序列和活性中心均位于结构域2中,在不破坏蛋白二级结构与活性中心的前提下,进一步对该基因进行了大量突变位点的筛选,并通过实验进行效果验证:
设计PCR引物AG2-F1、AG2-R1:
AG2-F1:GGCCCATGGCCCCTGCTATTGGTGCTTCTAA(下划线为限制性内切酶NcoI识别位点);
AG2-R1:GGCCTCGAGTTATTGTCTTTCAAGAAAAACAAC(下划线为限制性内切酶XhoI识别位点)。
以α-半乳糖苷酶基因AG2为模板,以上述引物用GeneMorph II随机突变PCR试剂盒(Stratagene)进行PCR扩增,胶回收PCR产物,NcoI、XhoI进行酶切处理后与经同样酶切后的pET-28a载体连接,转化至大肠杆菌BL21(DE3)中,涂布于LB+Kana平板,37℃倒置培养,待转化子出现后,用牙签逐个挑至96孔板,每个孔中加入150ul含有0.1mM IPTG的LB+Kana培养基,37℃220rpm培养6h左右,离心弃上清,菌体用缓冲液重悬,反复冻融破壁,获得含有α-半乳糖苷酶的大肠杆菌细胞裂解液。
分别取出40ul裂解液至两块新的96孔板,其中一块板于65℃处理10min后,两块96孔板都加入40ul底物(3mg/ml的对硝基苯酚-α-D-吡喃半乳糖苷溶液),于37℃反应10min后加入160ul终止液(0.5mol/L的Na2CO3试剂),400nm处测定吸光值,不同的突变子高温处理后保持的活性不同。
实验结果表明,有些突变对α-半乳糖苷酶AG2蛋白耐热性没有影响,有些突变甚至使其耐热性或酶活变得更差了;另外还有些突变,虽然能提高α-半乳糖苷酶AG2蛋白对温度的耐受性,但突变后其酶学性质发生了显著的变化,这些均不符合要求。最终,得到既能显著提高α-半乳糖苷酶AG2的耐热性,又不会影响其酶活及原有酶学性质的突变位点:S41H、I108V、S146M、A174W、A176M、N201Y、V202P、K206Y、V211W、A230L、D233S、S239H、A268I、V328I、T347K、T347L、T347Q、H358E、T395K、T395R、V397M、N407A、A459C、A462D、A462Q、T478K、N487L、Q502C、Q539M、Q539Y、A552P、A577S、A581P、A581R、V587T、S601C、G613F、S654L、Q659I、Q659K、Q659M、Q681D、Q681S、S710R。
本发明在α-半乳糖苷酶AG2的基础上,提供了分别包含S41H、I108V、S146M、A174W、A176M、N201Y、V202P、K206Y、V211W、A230L、D233S、S239H、A268I、V328I、T347K、T347L、T347Q、H358E、T395K、T395R、V397M、N407A、A459C、A462D、A462Q、T478K、N487L、Q502C、Q539M、Q539Y、A552P、A577S、A581P、A581R、V587T、S601C、G613F、S654L、Q659I、Q659K、Q659M、Q681D、Q681S、S710R单个突变位点的突变体。
本发明还提供了包含选自上述耐热突变位点中的两个或多个突变位点组合的突变体,其耐热性比对应单点突变体得到进一步提高。
实施例3毕赤酵母工程菌株的构建
3.1表达载体的构建
依照毕赤酵母的密码偏爱性分别对α-半乳糖苷酶AG2及其突变体的基因序列进行优化,由上海捷瑞生物工程有限公司合成,并且在合成序列5’和3’两端分别加上EcoRI和NotI两个酶切位点。
分别用上述突变体为模板,用引物AG2-F、AG2-R扩增,胶回收PCR产物,EcoRI、NotI进行酶切处理后与经同样酶切后的pPIC-9k载体16℃过夜连接并转化大肠杆菌DH5a,涂布于LB+Amp平板,37℃倒置培养,待转化子出现后,菌落PCR验证阳性克隆,经测序验证后最后获得了正确的重组质粒。
3.2酵母感受态制备
将毕赤酵母GS115菌株进行YPD平板活化,30℃培养48h后接种活化的GS115单克隆于5mL YPD液体培养基中,30℃、220rpm,培养约18h后转接菌液于装有50ml YPD液体培养基的三角瓶中,30℃、220rpm培养约5h经紫外分光光度计检测其菌体密度,待其OD600值在1.1~1.3范围后,4℃6000rpm离心3min分别收集5ml菌体至灭菌EP管中,轻轻弃上清,用灭菌的滤纸吸干残留的上清后用预冷的2mL灭菌水重悬菌体,4℃、6000rpm离心3min,轻轻弃上清,用预冷的2mL山梨醇(1mol/L)重悬菌体;4℃6000rpm离心3min,轻轻弃上清,预冷的100-150μl山梨醇(1mol/L)轻柔重悬菌体。
3.3转化和筛选
分别将实施例2构建得到的重组表达质粒用Sac I进行线性化,线性化片段纯化回收后通过电穿孔法分别转化毕赤酵母GS115,在MD平板上筛选得到毕赤酵母重组菌株。挑取单个转化子转接于BMGY培养基中,30℃、250rpm振荡培养1d后,再转入BMMY培养基中30℃、250rpm振荡培养,每天添加0.5%的甲醇。诱导表达3d后,离心去除菌体,得到含α-半乳糖苷酶的上清液,测定α-半乳糖苷酶的活力。
结果显示,本发明构建得到的重组表达α-半乳糖苷酶AG2及其突变体的毕赤酵母重组菌株发酵上清液中α-半乳糖苷酶的活力为1140-2020U/ml。
α-半乳糖苷酶的酶活测定方法
(1)酶活单位的定义
在37℃、pH值为5.0的条件下,每分钟从浓度为1.5mg/ml的对硝基苯酚-α-D-吡喃半乳糖苷溶液中释放1μmol对硝基苯酚所需要的酶量即为一个酶活力单位U。
(2)测定方法
取0.5ml浓度为3mg/ml的对硝基苯酚-α-D-吡喃半乳糖苷溶液,加入到比色管中,37℃平衡5min,再加入0.5ml经pH5.0磷酸氢二钠-柠檬酸缓冲液适当稀释并经37℃平衡好的α-半乳糖苷酶酶液,混匀于37℃精确保温反应10min。反应结束后,加入4ml浓度为0.5mol/L的Na2CO3试剂,混匀以终止反应。然后冷却至室温,以标准空白样为空白对照,在400nm处测定吸光值AE,根据对硝基苯酚标准曲线用回归方程计算出相应的对硝基苯酚的ug数R=(AE-b)/K。
酶活计算公式:XD=(R×n)/(10×0.5×139.11)。
式中:XD为酶液中α-半乳糖苷酶的活力,U/ml;R为计算得到的对硝基苯酚ug数;10为反应时间10min;0.5为加入酶液体积0.5ml;n为酶液稀释倍数;139.11为对硝基苯酚的摩尔质量,139.11g/mol。
实施例4α-半乳糖苷酶突变体耐热性分析
用预热10min的pH5.0磷酸氢二钠-柠檬酸缓冲液分别将实施例3构建得到的表达α-半乳糖苷酶突变体的毕赤酵母重组菌株发酵上清液稀释至α-半乳糖苷酶酶活为200U/mL,混合均匀,67℃处理3min,结束时取样并冷却至室温,然后测定其α-半乳糖苷酶酶活,以未处理样品的酶活计100%,计算酶活残留率。具体结果见表1。
酶活残留率(%)=热处理后的酶活/热处理前的酶活×100%。
表1α-半乳糖苷酶单点突变体耐热性分析
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结果如表1所示,与野生型α-半乳糖苷酶AG2相比,本发明提供的分别含S41H、I108V、S146M、A174W、A176M、N201Y、V202P、K206Y、V211W、A230L、D233S、S239H、A268I、V328I、T347K、T347L、T347Q、H358E、T395K、T395R、V397M、N407A、A459C、A462D、A462Q、T478K、N487L、Q502C、Q539M、Q539Y、A552P、A577S、A581P、A581R、V587T、S601C、G613F、S654L、Q659I、Q659K、Q659M、Q681D、Q681S、S710R单个突变位点的α-半乳糖苷酶突变体的耐热性得到显著提高。67℃处理3min后,所述半乳糖苷酶单点突变体的酶活残留率达到40.42%-72.06%,比野生型提高了10.47%-96.94%,取得了意料不到的技术效果。
此外,在野生型α-半乳糖苷酶AG2基础上,本发明提供的S41H/I108V、S41H/S146M、A459C/Q502C、S41H/A174W两点突变体;S41H/I108V/S146M、S41H/I108V/A174W、S41H/S146M/A174W三点突变体;S41H/I108V/S146M/A174W、S41H/I108V/S146M/D233S、S41H/I108V/S146M/N407A、S41H/I108V/S146M/V328I、S41H/I108V/S146M/A174W四点突变体;S41H/I108V/S146M/A174W/D233S、S41H/I108V/S146M/D233S/V397M、S41H/I108V/S146M/A459C/Q502C五点突变体;S41H/I108V/S146M/A174W/D233S/S239H六点突变体;S41H/I108V/S146M/D233S/V397M/A459C/Q502C七点突变体,在68℃条件下处理3min后,酶活残留率达到24.92%-64.21%,比野生型提高了113.4%-449.7%,耐热性得到显著提高。
综上,与α-半乳糖苷酶AG2相比,本发明提供的所述单点突变体及组合突变体的耐热性得到显著提升,从而有利于α-半乳糖苷酶在饲料中的广泛应用。
Claims (10)
1.一种α-半乳糖苷酶突变体,其特征在于,所述突变体包含与SEQ ID NO:2具有至少90%同一性的氨基酸序列,且与SEQ ID NO:2相比在选自下组中的至少一个位置上包含氨基酸的取代:41、108、146、174、176、201、202、206、211、230、233、239、268、328、347、358、395、397、407、459、462、478、487、502、539、552、577、581、587、601、613、654、659、681、710。
2.如权利要求1所述的α-半乳糖苷酶突变体,其特征在于,所述突变体的氨基酸序列与SEQ ID NO:2相比具有至少91%,92%,93%,94%,95%,96%,97%,98%,或至少99%的同一性。
3.如权利要求2所述的α-半乳糖苷酶突变体,其特征在于,所述突变体的氨基酸序列与SEQ ID NO:2相比具有至少99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,或至少99.9%的同一性。
4.如权利要求1所述的α-半乳糖苷酶突变体,其特征在于,所述突变体包含下组中至少一个氨基酸的取代:S41H、I108V、S146M、A174W、A176M、N201Y、V202P、K206Y、V211W、A230L、D233S、S239H、A268I、V328I、T347K、T347L、T347Q、H358E、T395K、T395R、V397M、N407A、A459C、A462D、A462Q、T478K、N487L、Q502C、Q539M、Q539Y、A552P、A577S、A581P、A581R、V587T、S601C、G613F、S654L、Q659I、Q659K、Q659M、Q681D、Q681S、S710R。
5.如权利要求1所述的α-半乳糖苷酶突变体,其特征在于,所述突变体包含下组中至少一个氨基酸的取代组合:
S41H/I108V;
S41H/S146M;
S41H/A174W;
S41H/A176M;
S41H/N201Y;
S41H/V202P;
S41H/K206Y;
S41H/V211W;
S41H/A230L;
S41H/A268I;
S41H/T347K;
S41H/H358E;
S41H/T395K;
S41H/N407A;
S41H/N487L;
S41H/Q502C;
S41H/Q539Y;
S41H/A552P;
S41H/V587T;
S41H/G613F;
S41H/S654L;
S41H/Q659I;
S41H/Q659M;
S41H/Q681D;
S41H/S710R;
V211W/I108V;
V211W/S146M;
V211W/A174W;
V211W/A176M;
V211W/N201Y;
V211W/V202P;
V211W/K206Y;
V211W/A230L;
V211W/A268I;
V211W/T347K;
V211W/H358E;
V211W/T395K;
V211W/N407A;
V211W/N487L;
V211W/Q502C;
V211W/Q539Y;
V211W/A552P;
V211W/A581R;
V211W/V587T;
V211W/G613F;
V211W/S654L;
V211W/Q659I;
V211W/Q659M;
V211W/Q681D;
V211W/S710R;
A459C/I108V;
A459C/S146M;
A459C/A174W;
A459C/A176M;
A459C/N201Y;
A459C/V202P;
A459C/K206Y;
A459C/A230L;
A459C/A268I;
A459C/T347K;
A459C/H358E;
A459C/T395K;
A459C/N407A;
A459C/N487L;
A459C/Q502C;
A459C/Q539Y;
A459C/A552P;
A459C/A581R;
A459C/V587T;
A459C/G613F;
A459C/S654L;
A459C/Q659I;
A459C/Q659M;
A459C/Q681D;
A459C/S710R;
N487L/I108V;
N487L/S146M;
N487L/A174W;
N487L/A176M;
N487L/N201Y;
N487L/V202P;
N487L/K206Y;
N487L/A230L;
N487L/A268I;
N487L/T347K;
N487L/H358E;
N487L/T395K;
N487L/N407A;
N487L/Q502C;
N487L/Q539Y;
N487L/A552P;
N487L/A581R;
N487L/V587T;
N487L/G613F;
N487L/S654L;
N487L/Q659I;
N487L/Q659M;
N487L/Q681D;
N487L/S710R;
A581R/I108V;
A581R/S146M;
A581R/A174W;
A581R/A176M;
A581R/N201Y;
A581R/V202P;
A581R/K206Y;
A581R/A230L;
A581R/A268I;
A581R/T347K;
A581R/H358E;
A581R/T395K;
A581R/N407A;
A581R/A462Q;
A581R/Q502C;
A581R/Q539Y;
A581R/A552P;
A581R/V587T;
A581R/G613F;
A581R/S654L;
A581R/Q659I;
A581R/Q659M;
A581R/Q681D;
A581R/S710R;
S41H/I108V/S146M;
S41H/I108V/A174W;
S41H/S146M/A174W;
S41H/S146M/D233S;
S41H/S146M/S239H
I108V/S146M/N407A/V587T;
I108V/S146M/D233S/V587T;
I108V/S146M/V328I/H358E;
I108V/S146M/D233S/V397M;
I108V/S146M/A459C/Q502C;
A268I/H358E/N407A;
A268I/T347K/H358E/N407A;
A268I/T347K/H358E/T395K;
A268I/T347K/H358E/ V587T;
A268I/T347K/H358E/N407A/A459C;
S41H/I108V/S146M/A174W;
S41H/I108V/S146M/A174W/D233S;
S41H/I108V/S146M/A174W/D233S/S239H;
S41H/I108V/S146M/D233S;
S41H/I108V/S146M/V328I;
S41H/I108V/S146M/H358E;
S41H/I108V/S146M/N407A;
S41H/I108V/S146M/V587T;
S41H/I108V/S146M/N407A/V587T;
S41H/I108V/S146M/D233S/V587T;
S41H/I108V/S146M/V328I/H358E;
S41H/I108V/S146M/D233S/V397M;
S41H/I108V/S146M/A459C/Q502C;
S41H/I108V/S146M/V328I/A459C/Q502C;
S41H/I108V/S146M/H358E/A459C/Q502C;
S41H/I108V/S146M/V328I/H358E/A459C/Q502C;
S41H/I108V/S146M/D233S/V397M/V587T;
S41H/I108V/S146M/N407A/A459C/Q502C;
S41H/I108V/S146M/D233S/V397M/A459C/Q502C;
S41H/I108V/S146M/H358E/N407A/V587T;
S41H/I108V/S146M/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/H358E/N407A/A459C/Q502C/V587T ;
I108V/S146M/V328I/A459C/Q502C;
I108V/S146M/H358E/A459C/Q502C;
I108V/S146M/V328I/H358E/A459C/Q502C;
I108V/S146M/D233S/V397M/V587T;
S146M/H358E/N407A/A459C/Q502C/V587T ;
S146M/A176M/H358E/N407A/A459C/Q502C/V587T;
S146M/N201Y/V202P/H358E/N407A/A459C/Q502C/V587T;
S146M/K206Y/H358E/N407A/A459C/Q502C/V587T;
S146M/V211W/H358E/N407A/A459C/Q502C/V587T;
S146M/A230L/H358E/N407A/A459C/Q502C/V587T;
S146M/T347K/H358E/N407A/A459C/Q502C/V587T;
A268I/H358E/N407A/A459C/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/Q502C/V587T;
A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/A462D/Q502C/V587T ;
A268I/T347K/H358E/N407A/A459C/T478K/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/N487L/Q502C/V587T;
A268I/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T;
A268I/T347K/H358E/N407A/A459C/Q502C/A552P/V587T;
H358E/N407A/A459C/Q502C/V587T;
H358E/T395K/N407A/A459C/Q502C/V587T;
H358E/N407A/A459C/A462D/Q502C/V587T ;
H358E/N407A/A459C/T478K/Q502C/V587T;
H358E/N407A/A459C/N487L/Q502C/V587T;
H358E/N407A/A459C/Q502C/Q539Y/V587T;
H358E/N407A/A459C/Q502C/A552P/V587T;
S41H/T95K/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/A462D/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/T478K/Q502C/V587T;
S41H/I108V/S146M/T347K/H358EN407A/A459C/N487L/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/A552P/V587T;
S41H/I108V/S146M/A176M/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/N201Y/V202P/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/K206Y/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/V211W/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A230L/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/A462D/Q502C/V587T ;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/T478K/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/N487L/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/A552P/V587T;
S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/V587T;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T;I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S710R;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/G613F/S710R;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S654L/S710R;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q659I/S710R;
I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/V587TK/Q659M/S710R;
I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q681D/S710R;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/G613F;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S654L;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q659I;
S41H/I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/V587TK/Q659M;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q681D;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S710R;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I;
S41H/I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V587TK/Q659M;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q681D;
S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/A552P/V587T;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/A552P/V587T;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/G613F;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S654L;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I;
I108V/S146M/V211W/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V587TK/Q659M;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q681D;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S654L/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V587TK/Q659M/S710R;
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q681D/S710R;
S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A552P/A581R/V587T/Q681D;
S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A552P/V587T/Q659I/S710R。
6.编码权利要求1-5任一所述α-半乳糖苷酶突变体的DNA分子。
7.包含权利要求6所述DNA分子的重组表达质粒。
8.一种宿主细胞,其特征在于,所述的宿主细胞包含权利要求7所述的重组表达质粒;所述的宿主细胞为非植物细胞。
9.如权利要求8所述的宿主细胞,其特征在于,所述的宿主细胞为毕赤酵母(Pichia pastoris)、黑曲霉(Aspergillus niger)或里氏木霉(Trichoderma reesei)中的任意一种。
10.权利要求1-5任一所述α-半乳糖苷酶突变体在饲料生产中的应用。
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