该研究利用类弹性蛋白多肽(elastin-like polypeptides,ELP)标签和分子黏合剂“SpyTag/SpyCatcher”建立了快速高效的乳糖酶分离纯化方法,并研究了获得的重组乳糖酶(lactase-elastin-like polypeptides,Lac-ELP)的酶学性质及其在合成低聚半乳糖(galactooligosaccharides,GOS)中的应用。从产马乳酒乳杆菌(Lactobacillus kefiranofaciens)ZW3中克隆得到乳糖酶酶基因lac,并将lac连接到“Spytag”标签,在大肠杆菌DH5α中表达获得了含有Spytag的乳糖酶Lac-Spytag。通过基因合成获得了编码ELP的基因片段elp,并将elp接到“Spycatcher”编码基因的下游,在大肠杆菌DH5α中表达获得了含有SpyCatcher的ELP标签SpyCatcher-ELP。随后,通过SpyTag和SpyCatcher的自发连接,将Lac-SpyTag和SpyCatcher-ELP进行融合,获得重组乳糖酶Lac-SpyTag-SpyCatcher-ELP(Lac-ELP)。利用ELP标签的可逆相变循环技术,对Lac-ELP进行分离纯化,成功获得了分子质量大小为115 kDa的Lac-ELP,回收效率为73.8%。随后,比较ELP标签对Lac活性的影响。Lac-ELP和Lac的最适温度均为50 ℃,最适pH值均为7.0,但Lac-ELP比Lac表现出更好的热稳定性和pH稳定性。最后,以乳糖为底物研究了Lac-ELP在制备GOS中的应用。结果表明,Lac-ELP表现出较好的转糖苷活性,底物浓度400 mg/mL,Lac-ELP添加量10 U/g乳糖,35 ℃反应24 h后,GOS产率可达33.23%。综上,该研究建立的基于ELP的乳糖酶纯化技术操作简便,成本低,可以快速获得高活性的重组乳糖酶,为GOS的工业合成提供参考。
This study established a rapid and efficient method for separating and purifying lactase using elastin-like polypeptides (ELP) tags and molecular adhesive "SpyTag/SpyCatcher", and investigated the enzymatic properties of the obtained recombinant lactase (Lac-ELP) and its application in the synthesis of GOS.The lactase gene lac was cloned from Lactobacillus kefieranofaciens ZW3 and linked to the "Spytag".The lactase containing Spytag was expressed in E.coli DH5α.The gene fragment elp encoding ELP was obtained through gene synthesis and connected downstream of the Spycatcher encoding gene.The ELP tag containing SpyCatcher was expressed in E.coli DH5α.Subsequently, by spontaneously connecting of SpyTag and SpyCatcher, Lac-SpyTag and SpyCatcher-ELP were fused to obtain recombinant lactase Lac-SpyTag-SpyCatcher-ELP (Lac-ELP).The Lac-ELP was separated and purified by using the inverse transition cycling (ITC) technology, and a molecular weight of 115 KDa was successfully obtained, with a recovery efficiency of 73.8%.Subsequently, the influence of ELP tag on activity of Lac was compared.The optimal temperature for both Lac-ELP and Lac is 50 ℃, and the optimal pH is 7.0.However, Lac-ELP exhibits better thermal and pH stability than Lac.Finally, the application of Lac-ELP in the preparation of GOS was studied using lactose as a substrate.The results showed that Lac-ELP exhibited good transglycoside activity, with a substrate concentration of 400 mg/mL and a Lac-ELP addition of 10 U/g lactose.After 24 hours of reaction at 35 ℃, the yield of GOS reached 33.23%.In summary, the ELP based lactase purification technology established in this study is convenient and can quickly obtain highly active recombinant lactase, providing a reference for the industrial synthesis of GOS.
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