Barboza-Corona J Eleazar, Ortiz-Rodríguez Tomás, de la Fuente-Salcido Norma, Bideshi Dennis K, Ibarra Jorge E, Salcedo-Hernández Rubén
Campus Irapuato-Salamanca, División Ciencias de la Vida, Departamento de Ingeniería en Alimentos, Universidad de Guanajuato, Irapuato, Guanajuato, Mexico.
Antonie Van Leeuwenhoek. 2009 Jun;96(1):31-42. doi: 10.1007/s10482-009-9332-9. Epub 2009 Apr 1.
Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5' mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (+/-0.214) and 1.356 (+/-0.247) mum in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (+/-0.222) and 1.139 (+/-0.202) mum in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 x 10(9), 1.3 x 10(9)), compared to HD-73-pEHchiA74 (4.9 x 10(9), 5.3 x 10(9)) and HD-73 (6.8 x 10(9), 8.8 x 10(9)) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.
苏云金芽孢杆菌HD - 73用内切几丁质酶基因chiA74进行转化，该基因受强启动子（pcytA）和5' mRNA稳定序列（STAB - SD）调控（HD - 73 - pEBchiA74）。将其表达水平与野生型菌株（HD - 73）以及在其天然启动子调控下表达chiA74的重组HD - 73菌株（HD - 73 - pEHchiA74）的表达水平进行比较。HD - 73 - pEBchiA74的几丁质分解活性显著提高，分别比HD - 73 - pEHchiA74和亲本HD - 73高约58倍和362倍，代表了迄今为止报道的重组苏云金芽孢杆菌中几丁质酶表达的最高水平。透射电子显微镜下测量的伴孢晶体显示，当细菌分别在NBS和添加葡萄糖的NBS中生长时，HD - 73产生的晶体长度分别为1.235（±0.214）和1.356（±0.247）μm。否则，当HD - 73 - pEBchiA74分别在NBS和添加葡萄糖的NBS中培养时，合成的晶体长度分别为1.250（±0.222）和1.139（±0.202）μm，与在相同条件下生长的HD - 73 - pEHchiA74产生的晶体相比，长度值分别减少了约10%和20%。每毫升活孢子数的比较表明，当分别在NBS和添加葡萄糖的NBS中生长时，HD - 73 - pEBchiA74产生的活孢子最少（1.5×10⁹，1.3×10⁹），相比之下，HD - 73 - pEHchiA74（4.9×10⁹，5.3×10⁹）和HD - 73（6.8×10⁹，8.8×10⁹）。尽管几丁质酶过量产生，但未观察到细胞蛋白酶活性的变化。
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