|Alternative Title||Heterologous Expression,Optimizing the Expression Condition of the Lysostaphin and Fowlicidin-1 in Kluyveromyces lactis and pichia pastoris|
|Place of Conferral||兰州|
|Keyword||乳酸克鲁维酵母 溶葡球菌酶 诱变 毕赤酵母 抗菌肽|
(1) 根据葡萄球菌的溶葡球菌酶基因序列以及乳酸克鲁维酵母密码子偏好性设计引物扩增溶葡球菌酶基因表达片段，构建溶葡球菌酶(lysostaphin，Lys)基因重组表达载体(pKLAC1-Lys)，转化乳酸克鲁维酵母，实现了Lys基因的分泌表达。(2) 对重组菌株(K. lactis GG799/pKLAC1- Lys)进行NTG随机化学诱变，获得高表达菌株mu4#，优化其表达条件，对蛋白进行纯化并研究其酶学性质。结果表明：诱变后Lys活性提高了约5.2倍（约8000U/L)。最适接种量为40g/L，诱导过程中每24h添加一次浓度为20g/L的半乳糖和NH4NO3可提高酶活，最适表达pH为7.0-7.5；最适反应pH为7.0-8.0，最适反应温度为37℃，实验表明，低于40℃，pH 3.0-6.0之间时，该酶较稳定。Sr2+对其活性具有促进作用，Ba2+、Ca2+、Zn2+、Cu2+、Mn2+、Mg2+ 对其具有抑制性。(3) 在扫描电镜下观察重组溶葡球菌酶对金黄色葡萄球菌细胞形态的影响，大幅度降低了金黄色葡萄球菌的浓度，细胞体积变小，细胞大量破裂聚集成块。通过对mu4#菌株做100L放大发酵实验，结果发酵上清液酶活性是原始菌株表达酶活的45倍，生物量达到210g/L。(4)通过毕赤酵母表达系统，成功筛选出重组高表达菌株(SMD1168/Ppic9k-F3)，质谱结果证明：鸡源抗菌肽Fowlicidin-1基因在毕赤酵母中成功分泌表达。该抗菌肽在中性及偏碱性条件下，对部分致病性病原菌有抑制、杀伤作用，但对乳酸杆菌无抑制作用。
Recently, the emergence of all kinds of drug-resistant strains leads to increase the difficulty of disease treatment, due to the overuse of antibiotics. These drug-resistant strains threaten our human beings health severely. It is important to hunt for antimicrobial substance of safety, effectivity and non-drug-resistant as for the replacement drugs of antibiotics. Lysostaphin can treat infection by the Staphylococcus aureus efficiently, and it can not cause the emergence of drug-resistant strains. Antimicrobial peptide has the characterization of small molecular and good stability. It does not generate drug resistance by the way of physical sterilization, so it has enormous application potential. In this paper, the Lysostaphin and Fowlicidin-1 were expressed by using recombinant gene technology. The main results were as follows.
(1) According to the sequence of lysostaphin gene from Staphylococcus simulans and codon bias of Kluyveromyces lactis , the PCR primer was designed to amplify the lysostaphin gene. The gene was inserted in pKLAC1, and transformed to K. lactis GG799. The K.lactis GG799/pKLAC1- Lys was cultivated to express Lys.
(2) We obtained a high expression strain (mu4#) by using powerful mutagen (N-methy1-N-nitro-N-nitrosoguanidine,NTG) on the recombinant and optimized the expression condition .The fermentation broth of mu4# was purified by Ni-NTA agarose and the enzyme characterization was studied. The result showed that the activity of Lys was approximately 5.2 times (8000U/L) higher in the mutation. The optimal inoculum dose of the mutant (mu4#) was 40g/L; Galactose and NH4NO3 (20g/L) were added in every 24 hours, Lys exhibited optimal expression at pH 7.0-7.5; Furthermore, the Lys enzyme optimal reaction performed at pH 7.0-8.0 and temperature at 37°C. The recombinant Lys was stable below 40 °C and pH between 3.0 and 6.0. Sr2+ stimulated its activity whereas Ba2+、Ca2+、Zn2+、Cu2+、Mn2+、Mg2+ inhibited the activities. This research accomplished Lys recombinant expression, yield improvement by chemical mutagenesis in K. lactis and characterization of lysostaphin. These research results provide profound guiding significance for the large-scale production and application of recombinant lysostaphin.
(3) Under the SEM, we observed the effect that lysostaphin sterilized the Staphylococcus aureus . It decreased the concentration of the Staphylococcus aureus. The volume of cells diminished and a number of cells bursted. We used mu4# strain do pilot scale test in a 100L fermentor. The result showed that the activity of Lys was approximately 45 times higher in the fermentor. The biomass added up to 210g/L.
(4) We constructed the high expression strain SMD1168/Ppic9k-F3 by the expression system of Pichia pastoris. MS experiment demonstrated that the strain SMD1168/Ppic9k-F3 successfully expressed Fowlicidin-1. The Fowlicidin-1 has good heat stability. The Fowlicidin-1 showed good antibacterial effect on Staphylococcus aureus, escherichia coli and clostridium perfringens,but without antibacterial effect to lactobacillus in the condition of approaching alkalinity.
|王曦. 溶葡球菌酶和鸡源抗菌肽Fowlicidin-1的酵母异源表达及优化[D]. 兰州. 兰州大学,2018.|
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