植酸酶基因在乳酪杆菌中的高效表达及生化特性研究
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摘要
本研究通过电击转化将来自无花果曲霉的植酸酶基因与原核表达载体pIAβ8构建的重组载体pIAβ8-phyA转入乳酪杆菌中,并对转基因乳酪杆菌的培养特性以及分泌的植酸酶进行酶学特性分析。选用不同包埋材料对转基因乳酪杆菌进行包埋,并运用不同的方法进行干燥处理。最后,通过测定乳酸菌对不同抗生素的耐受性及其抑菌作用,为转基因乳酪杆菌在生产中的应用提供依据。
利用MRS培养基培养乳酪杆菌并制成感受态细胞,之后通过电击转化将含有植酸酶基因的重组表达载体pIAβ8-phyA转入乳酪杆菌中。通过抗生素筛选、植酸酶活力测定以及SDS-PAGE蛋白电泳检测,证明植酸酶基因已成功地在乳酪杆菌中得到表达。转基因乳酪杆菌分泌到细胞内外的植酸酶活力分别达到4.57和21.31 U/ml(P<0.05)。SDS-PAGE蛋白电泳检测,转植酸酶基因乳酪杆菌可表达植酸酶蛋白,其分子量为39.2 kDa。
对转基因乳酪杆菌分泌植酸酶进行不同温度和pH处理并测定酶活力变化可知,所分泌的植酸酶最适pH为5.0(P<0.05),最适温度为70℃(P<0.05)。通过对转基因乳酪杆菌培养液的pH、干细胞重、光密度值以及活菌数的测定,可知培养基的pH在乳酪杆菌静止培养72 h后达到最低(3.35,P<0.05),干细胞重在培养96 h后达到最高(3.9 mg/ml,P<0.05),光密度值在培养144 h后达到最高(5.89,P<0.05),活菌数在培养48 h后达到最大(1.1×109 CFU/ml,P<0.05)。综合以上指标以及酶活力随时间的变化规律,可以确定转基因乳酪杆菌的最佳收获时间为厌氧培养96 h。
试验选用海藻酸钠、琼脂、聚乙烯醇和明胶对转基因乳酪杆菌进行包埋,之后运用不同的烘干方法进行干燥处理。结果表明,海藻酸钠在添加量为3%条件下包埋和冻干效果最好,活菌数可达7.55×105 CFU/g。采用琼脂包埋后则具有较好的耐热性,85℃烘干后活菌数保持在1.68×107 CFU/g。
最后,就乳酸菌对抗生素的耐受性及其抑菌作用进行测定。结果表明,当金霉素和硫酸粘杆菌素的添加量分别为大于0.10%和0.05%时,对乳酸菌生长有显著抑制作用(P<0.05)。另外,研究还证实,乳酸菌及其培养液都对致病性大肠杆菌的生长有显著抑制作用(P<0.05)。
In this experiment,recombinant expression vector pIAβ8-phyA which contains phytase gene from Aspergillus ficuum was transformed into Lactobacillus casei (L. casei) by electroporation , and then the expressed phytase characteristics and incubation condition were analysized.The transgenic L. casei was encapsulated by the different embedding materials to increase its availability and living ability.The ability of the transgenic L. casei to resist the antibiotics was also studied for its application in animal husbandry.
The competent cells of L. casei were prepared with MRS incubation medium,and then the recombinant expression vector pIAβ8-phyA was transformed into the competent L. casei by electroporation.Through the screening by antibiotics,the positive colonies with phytase activity were selected and incubated.The maximal phytase activities in the supernatant and cells were 21.31 and 4.57 U/ml(P<0.05),respectively.The phytase proteins from the supernatant and cells of the transgenic L. casei were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) for protein analyses,which indicated that the molecular weight of the expressed phytase protein was 39.2 kDa.
The phytase was used for thermostability and pH determination,the result demonstrated that the optimum pH and temperature of phytase were 5.0 and 70℃(P<0.05),respectively.The analysis of liquid fermentation showed that the pH value of the media got to the lowest value of 3.35 after 72 h incubation,cell dry weight reached the peak of 3.9 mg/ml after 96 h incubation,and cell optical density reached the peak of 5.89 after 144 h incubation,and the living cells reached the peak of 1.1×109 CFU/ml after 48 h incubation,respectively (P<0.05).Through the above analysis,the optimum harvest time was at the 4 th day of anaerobic incubation for the transgenic L. casei.
The transgenic L. casei was encapsulated by the different embedding materials,such as agar,sodium alginate,polyvinyl alcohol and gelatin,and then the thermostability and availability of the embedded transgenic L. casei were determined.The results indicated that the best embedding conditions were 3% sodium alginate and frozen drying,and the living cells were kept at 7.55×105 CFU/ml.After embedding with the agar,the embedded L.casei had good thermostability at 85℃,and the living cells got to 1.68×107 CFU/ml.
In order to know the relationship between probiotics and antibiotics ,chlortetracycline and colistin sulfate were used to determine their effects on L. casei growth. The results indicated that the 0.1% chlortetracycline and 0.05% colistin sulfate had significant resistant effect on L . casei growth,respectively(P<0.05).This study also showed that the L. casei and its supernatant had the effect to inhibit proliferation of the pathogenic Escherichia coli.
利用MRS培养基培养乳酪杆菌并制成感受态细胞,之后通过电击转化将含有植酸酶基因的重组表达载体pIAβ8-phyA转入乳酪杆菌中。通过抗生素筛选、植酸酶活力测定以及SDS-PAGE蛋白电泳检测,证明植酸酶基因已成功地在乳酪杆菌中得到表达。转基因乳酪杆菌分泌到细胞内外的植酸酶活力分别达到4.57和21.31 U/ml(P<0.05)。SDS-PAGE蛋白电泳检测,转植酸酶基因乳酪杆菌可表达植酸酶蛋白,其分子量为39.2 kDa。
对转基因乳酪杆菌分泌植酸酶进行不同温度和pH处理并测定酶活力变化可知,所分泌的植酸酶最适pH为5.0(P<0.05),最适温度为70℃(P<0.05)。通过对转基因乳酪杆菌培养液的pH、干细胞重、光密度值以及活菌数的测定,可知培养基的pH在乳酪杆菌静止培养72 h后达到最低(3.35,P<0.05),干细胞重在培养96 h后达到最高(3.9 mg/ml,P<0.05),光密度值在培养144 h后达到最高(5.89,P<0.05),活菌数在培养48 h后达到最大(1.1×109 CFU/ml,P<0.05)。综合以上指标以及酶活力随时间的变化规律,可以确定转基因乳酪杆菌的最佳收获时间为厌氧培养96 h。
试验选用海藻酸钠、琼脂、聚乙烯醇和明胶对转基因乳酪杆菌进行包埋,之后运用不同的烘干方法进行干燥处理。结果表明,海藻酸钠在添加量为3%条件下包埋和冻干效果最好,活菌数可达7.55×105 CFU/g。采用琼脂包埋后则具有较好的耐热性,85℃烘干后活菌数保持在1.68×107 CFU/g。
最后,就乳酸菌对抗生素的耐受性及其抑菌作用进行测定。结果表明,当金霉素和硫酸粘杆菌素的添加量分别为大于0.10%和0.05%时,对乳酸菌生长有显著抑制作用(P<0.05)。另外,研究还证实,乳酸菌及其培养液都对致病性大肠杆菌的生长有显著抑制作用(P<0.05)。
In this experiment,recombinant expression vector pIAβ8-phyA which contains phytase gene from Aspergillus ficuum was transformed into Lactobacillus casei (L. casei) by electroporation , and then the expressed phytase characteristics and incubation condition were analysized.The transgenic L. casei was encapsulated by the different embedding materials to increase its availability and living ability.The ability of the transgenic L. casei to resist the antibiotics was also studied for its application in animal husbandry.
The competent cells of L. casei were prepared with MRS incubation medium,and then the recombinant expression vector pIAβ8-phyA was transformed into the competent L. casei by electroporation.Through the screening by antibiotics,the positive colonies with phytase activity were selected and incubated.The maximal phytase activities in the supernatant and cells were 21.31 and 4.57 U/ml(P<0.05),respectively.The phytase proteins from the supernatant and cells of the transgenic L. casei were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) for protein analyses,which indicated that the molecular weight of the expressed phytase protein was 39.2 kDa.
The phytase was used for thermostability and pH determination,the result demonstrated that the optimum pH and temperature of phytase were 5.0 and 70℃(P<0.05),respectively.The analysis of liquid fermentation showed that the pH value of the media got to the lowest value of 3.35 after 72 h incubation,cell dry weight reached the peak of 3.9 mg/ml after 96 h incubation,and cell optical density reached the peak of 5.89 after 144 h incubation,and the living cells reached the peak of 1.1×109 CFU/ml after 48 h incubation,respectively (P<0.05).Through the above analysis,the optimum harvest time was at the 4 th day of anaerobic incubation for the transgenic L. casei.
The transgenic L. casei was encapsulated by the different embedding materials,such as agar,sodium alginate,polyvinyl alcohol and gelatin,and then the thermostability and availability of the embedded transgenic L. casei were determined.The results indicated that the best embedding conditions were 3% sodium alginate and frozen drying,and the living cells were kept at 7.55×105 CFU/ml.After embedding with the agar,the embedded L.casei had good thermostability at 85℃,and the living cells got to 1.68×107 CFU/ml.
In order to know the relationship between probiotics and antibiotics ,chlortetracycline and colistin sulfate were used to determine their effects on L. casei growth. The results indicated that the 0.1% chlortetracycline and 0.05% colistin sulfate had significant resistant effect on L . casei growth,respectively(P<0.05).This study also showed that the L. casei and its supernatant had the effect to inhibit proliferation of the pathogenic Escherichia coli.
引文
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