| Natto is a traditional Japanese fermented soy food fermented by Bacillus subtilis natto. It has a high nutritional value and various healthy functions, however, B. subtilis natto produce a high content of ammonia during fermentation, which causes natto has a pungent odor. It is reported that the major metabolic pathways of ammonia production in B. subtilis natto are transdeamination and transamination catalyzed by glutamate dehydrogenase(GDH), and the hydrolysis of urea into carbon dioxide and ammonia, which is catalyzed by urease.urease is a kind of intracellular enzymes, which plays an important role in nitrogen metabolic pathway. It can catalyze the hydrolysis of urea to carbon dioxide and ammonia. While the research about the characterization of the B. subtilis natto urease and its effects on metabolism were limited.In this paper, we focused on the characterization of B. subtilis natto urease and expected to get a way to reduce urease production and produce natto with low ammonia odor through control fermentation conditions, and get an optimized fermentation condition for the activity study of urease. The concentration of urease under different culture conditions and the influence on its activity which were exerted by different temperatures, p H values and metal ions were studied.The results showed that the highest yield of urease was 57.2 μg/m L, which was occurred when gultamic acid was used as a nitrogen source, sucrose as a carbon source, and the strains cultured at 37℃, p H 7 for 12 h. The optimum reaction temperature and p H of urease were 35℃ and 7.0, respectively. Urease was inhibited by metal ions, the highest inhibition strength was 36.4%, which was caused by Cu2+, and the inhibition strength of a series of ions was as following: Cu2 +>Zn2+>Fe2+>Mg2+>Mn2+.The optimum urease producing conditions of B. subtilis natto were almost identical with its optimum growth condition, therefore, it is difficult to regulate the production of urease by control the fermentation conditions, without reduing natto’s quality. Consequently, a deletion mutant recombinant vector of ure was constructed, in accordance with the stratage of double-crossover homologous recombination, so as to construct a B.subtilis natto mutant of ure gene, which was expected to produce low ammonia natto. Primers were designed according to the bioinformatics analysis of ure upstream and downstream gene of B. subtilis natto. The upstream fragment ure12 and downstream fragment ure34 were amplified by PCR, using the DNA of B.subtilis natto as template. HindⅢ, Xhol and Bam HI restriction sites were added to ure12 gene sequence, and Sal I and Bam HI restriction sites were added into ure34. The ure12 and ure34 homology arms were ligated into p EASY-T1 vector to get p EASY-T1: ure14 plasmid, and then ure14 fragments were ligated into p KSV7, to get recombinant homologous recombination vector p KSV7: ure14, the structure was validated by PCR, and the results showed that its sequence homology with B.sutilis natto was 100%. The recombinant vector can be used to construct the ure deletion strain.The homology of B.subtilis natto(CGMCC No. 2801) and B. subtilis 168 on ure gene and its upstream and downstream sequences were 97%. In this paper, electricity transformation and protoplast transformation were used to transform recombinant vector p KSV7:ure14 into B.subtilis natto and B. subtilis 168. Both strains were cultured to OD600 1.1 and then was harvested and used to prepare competent cells.The solution containing 0.5 mol/L mannitol and 0.38 mol/L sorbitol was used as the electric shock buffer. When 25 μF, 200 Ω and 2.2 KV were chosed as electric shock parameters, positive transformants of B. subtilis168 were obtained. High quality protoplasts competent had been prepared with 5 mg/m L lysozyme, positive transformants of B. subtilis168 were obtained with 40% of PEG 6000-mediated transformation, and verified by PCR and sequencing. |
PDF Full Text Request