Yin Supeng, Zhu Hongbin, Shen Mengyu, Li Gang, Lu Shuguang, Zhao Yan, Le Shuai, Tan Yinling, Peng Yizhi, Hu Fuquan, Wang Jing
Department of Microbiology, Third Military Medical University (Army Medical University), Chongqing, China.
State Key Laboratory of Trauma, Burns, and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China.
Curr Microbiol. 2018 Oct;75(10):1362-1371. doi: 10.1007/s00284-018-1531-z. Epub 2018 Jun 19.
β-Galactosidase is an essential enzyme for the metabolism of lactose in human beings and has an important role in the treatment of lactose intolerance (LI). β-Galactosidase expressed by intestinal microflora, such as lactic acid bacteria (LAB), also alleviates LI. A promising approach to LI management is to exploit a food-grade LAB delivery system that can inhabit the human intestine and overproduce β-galactosidase. In this study, we constructed a food-grade β-galactosidase surface display delivery system and then integrated into the chromosome of Lactococcus lactis (L. lactis) NZ9000 using recombination. Western blot and immunofluorescence analyses confirmed that β-galactosidase was expressed on the cell surface of recombinant L. lactis stain NZ-SDL. The whole-cell biocatalyst exhibits V and K values of 121.38 ± 7.17 U/g and 65.36 ± 5.54 mM, based on ONPG hydrolysis. The optimum temperature for enzyme activity is 37 °C and the optimum pH is 5.0. Activity of the whole-cell biocatalyst is promoted by Mg, Ca, and K, but inhibited by Zn, Fe, and Fe. The system has a thermal stability similar to purified β-galactosidase but better pH stability, and is also more stable in artificial intestinal juice. Oral administration and intraperitoneal injections of NZ-SDL in mice cause no detectable health effects. In conclusion, we have successfully constructed a food-grade gene expression system in L. lactis that displays β-galactosidase on the cell surface. This system exhibits good enzyme activity and stability in vitro, and is safe in vivo. It is therefore a promising candidate for use in LI management.
β-半乳糖苷酶是人类乳糖代谢所必需的酶，在乳糖不耐受（LI）的治疗中具有重要作用。由肠道微生物群（如乳酸菌，LAB）表达的β-半乳糖苷酶也可缓解乳糖不耐受。一种有前景的乳糖不耐受管理方法是开发一种可定殖于人体肠道并过量产生β-半乳糖苷酶的食品级乳酸菌递送系统。在本研究中，我们构建了一种食品级β-半乳糖苷酶表面展示递送系统，然后通过重组将其整合到乳酸乳球菌（L. lactis）NZ9000的染色体中。蛋白质免疫印迹和免疫荧光分析证实β-半乳糖苷酶在重组乳酸乳球菌菌株NZ-SDL的细胞表面表达。基于对硝基苯-β-D-半乳糖苷（ONPG）水解，全细胞生物催化剂的V和K值分别为121.38±7.17 U/g和65.36±5.54 mM。酶活性的最适温度为37℃，最适pH为5.0。全细胞生物催化剂的活性受到Mg、Ca和K的促进，但受到Zn、Fe和Fe的抑制。该系统具有与纯化的β-半乳糖苷酶相似的热稳定性，但pH稳定性更好，并且在人工肠液中也更稳定。给小鼠口服和腹腔注射NZ-SDL未引起可检测到的健康影响。总之，我们成功地在乳酸乳球菌中构建了一种在细胞表面展示β-半乳糖苷酶的食品级基因表达系统。该系统在体外表现出良好的酶活性和稳定性，在体内是安全的。因此，它是用于乳糖不耐受管理的一个有前景的候选物。
