Effect Of Vitreoscilla Hemoglobin Expression And Nanobubbles On Bacterial Growth And Product Output

Poly-y-glutamic acid (y-PGA) is an unusual anionic, naturally occurring homo-polyamide that was made of D-and L-glutamic acid units connected by amide linkages between a-amino and γ-carboxylic acid groups. y-PGA is water soluble, biodegradable, edible and non-toxic toward humans and the environment. Therefore, potential applications of y-PGA and its derivatives have been of great interest in the past few years in a broad range of industrial fields such as medicine, food, conmetics and water treatment. The dissolved oxygen in the fermentation of γ-PGA is one of the key parameter. It was essential and made a great impact on microbial growth and product formation. Because of γ-PGA accumulation, the viscosity of culture medium became high and dissolved oxygen decreased.An homologous recombination expression vectors pET28a(+)-His(±)-vgb and pLJ-vgb were introduced into E.coil BL21(DE3) and B. subtilis DB403respectively by electric transformention, SDS-PAGE analysis indicated that vgb has been successfully expressed. The expression of vgb improved the growth of cell. Besides, nanobubbles water which growed the density of bubbles in culture medium can also improve y-PGA production.Our study included three parts as follows:(1) The optimization, cloning, expression and bioactivity of Vitreoscilla hemoglobin in E.coil.According to http://www.bioinformatics.org/codon/cgi-bin/codon.cgi, the Vitreoscilla hemoglobin(VHb) amino acid sequence was designed to express efficiently in B. subtilis.Two homologous recombination expression vectors pET28a(+)-His(+)-vgb which contained6His and pET28a(+)-His(-)-vgb were transferred into BL21(DE3) respectively forming two gene engineering strains pET28a(+)-His(+)-vgb/BL21(DE3) and pET28a(+)-His(-)-vgb/BL21(DE3). After IPTG induction, both VHb fusion protein and VHb protein were gained with apparent molecular weight18KDa and14KDa, respectively. The cell density of pET28a-His(-)-vgb/BL21(DE3) was4.3times higher than pET28a(+)/BL21(DE3) and was17.3%higher than pET28a(+)-His(+)-vgb/BL21(DE3) after induction at24h. L-Asparaginase bioactivity of recombinant strains were higher than their host E. coli BL21(DE3) by IPTG induction.(2) The cloning, expresson and bioactivity of Vitreoscilla hemoglobin in B.subtilisThree expression vectors pLJ-vgb,pLJ-p-vgb and pHT43-vgb were transferred into B.subtilis DB403respectively. VHb was gained with apparent molecular weight14KDa.We demonstrated the VHb biological activity by CO difference spectrum. It had an absorption peak at419nm. The cell dentiny after induction by maltose was7times higher than non-induction. The dentiny of recombinant strain was1.2times higher than wild stain. However, because it had original plasmid in the cell, the recombinant plasmid pLJ-vgb was introduced into B.subtilis DL unsuccessfully.(3) The effect of B.subtilis DL growth and y-PGA yield by nanobubblesB.subtilis DL was cultured in nanobubbles water medium. After cultivation at36h, the biomass increased51.1%at0r/min,37℃. The yield of y-PGA was improved from33.92±1.55g/L to37.21±0.39g/1at210r/min,37℃. However, the stability of nanobubbles was weak.In summary, on the basis of previous studies, the Vitreoscilla hemoglobin(VHb) were expressed in E.coil and B.subtilis, the biomass and production were improved by the vgb expression. The B.subtilis DL density and y-PGA production were improved by using nanobubbles water medium. It was a new way to use nanobubbles water in fermentation industry.