Cloning And Expression Of Arginase From Bacillus Amyloliquefaciens And Its Application In The Synthesis Of L-ornithine

L-ornithine(L-Orn) due to a variety of physiological and biochemical function, is widely used in medicine, food, chemical and other industries. Arginase, which catalyzes the hydrolysis of L-arginine(L-Arg) to L-Orn, is a key enzyme in the urea cycle. In this study, we focused on the the production of L-Orn by bioconversion and fermentation based on the cloning and expression of argI.Arginase encoded gene argI was firstly cloned and expressed in Corynebacterium crenatum SYPA5-5 and Bacillus subtilis 168 from Bacillus amyloliquefaciens B10-127. The recombinant strain B. subtlis 168/pMA5-argI exhibited a high enzyme activity of arginase. The specific enzyme activity reached to 21.9 U?mg-1, which is 26.7 folds higher than the partental strain B. subtilis 168. Th e L-Arg-conversion experiment of recombinant strain showed that 200 g?L-1 L-Arg was almost converted to 148.7 g?L-1 L-Orn in 4 h, with a residue of 2.3 g?L-1 L-Arg, The mole conversion rate of L-Arg was 99.4%. The other recombinant strain C. crenatum SYPA 5-5/pXMJ19-argI exhibited an arginase activity of 24.3 U?mg-1. The whole cell of recombinant strain was used as biocatalyst for the bioconversion of L-Arg. The preliminary result showed that 200 g?L-1 L-Arg was complete transformed to 151.4 g?L-1 in 10 h. The mole conversion rate of L-Arg reached to 99.8%.The recombinant arginase was purified by Ni-TA and the characteristic of purified arginase was investigated. As shown that the opimum temperature and pH of arginase were 40 oC and 10.0. The enzyme stability experiment showed that arginas owns a high stability under the condition that temperature below 40 oC and pH between 7.0 and 10.0. In addition, the recombinant arginase exhibited a strong Mn2+ preference. The product L-Orn exhibited a inhibitive effect on the activity of arginase. The Km value of arginase is9.3 mmol?L-1, and its maximum reaction rate Vmax is 95.4 μmol?mg-1·min-1。The whole-cell of the recombinant strain B. subtilis 168/pMA5-argI was used for the bioconversion of L-Orn from L-Arg. Additionally, bioconversion condition was optimized. As the results showed, the optimum bioconversion temperature and pH were 40 oC and 10.0. The optimum Mn2+ concentration is 0.6 mmol?L-1. The optimum substrate concentrate is 140 g?L-1 and optimum conversion buffer is 0.2 mol?L-1 carbonate buffer. Fed-batch stragety was used for the conversion of L-Arg and 356.9 g?L-1 L-Orn was obtained within 12 h. The wholecell was recycle by cell immobilization for the next batch of conversion. The result showed that after 9 batchs, the efficient of whole-cell retained more than 90% of the highest activity.In order to reduced the cost of L-Orn producing. One-step fermentation for the production of L-Orn was based on the highly L-Arg productive strain C. crenatum SYPA 5-5. The recombinant strain C. crenatum SYPA 5-5/pXMJ19-argI was used for the production of L-Orn from glucose by fermentation without extra addition of L-Arg and the fermentation condition was optimized. The result showed that when the IPTG was added in the first 18 h of the fermentation in flsak, few L-Arg was accumulated, as the inductive time of arginase was postpone, the concentration of L-Arg was increased, inversely the production of L-Orn decreased. Subsequently, two-phase pH control strategy was used for the production of L-Orn in 5-L fermentator. In 114 h, 24.3 g?L-1 was obtained and the L-Arg concentration was 0.9 g?L-1. Additionally, co-fermentation with C. crenatum SYPA 5-5 and B. subtilis 168/pMA5-argI for the production of L-Orn was investigated. The ratio of these two strain was optimized. As shown, the optimum biosmass ration with C. crenatum SYPA 5-5 and B. subtilis 168/pMA5-argI was about 1:500-530. Finally, co-fermentation was performed in the 5-L fermentator. In 96 h, the production of L-Orn reached to 32.4 g?L-1, with only 0.3 g?L-1 L-Arg was detected in the fermentation broth.