ABSTRACT
Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.
Subject(s)
Escherichia coli , Fermentation , Recombinant Proteins , beta-Mannosidase , beta-Mannosidase/metabolism , beta-Mannosidase/genetics , beta-Mannosidase/biosynthesis , beta-Mannosidase/chemistry , Escherichia coli/metabolism , Escherichia coli/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Mannans/metabolism , Mannans/chemistry , Mannans/biosynthesis , Bioreactors , Hydrogen-Ion Concentration , Aerobiosis , Galactans/metabolism , Galactans/biosynthesis , Galactans/chemistry , Culture Media/chemistry , Culture Media/metabolism , Plant Gums/chemistry , Plant Gums/metabolism , Actinobacteria/enzymology , Actinobacteria/metabolism , Actinobacteria/genetics , HydrolysisABSTRACT
A gene glu1 (WP_243232135.1) coding for ß-glucosidase from the genome of Microbacterium sp. CIAB417 was characterized for its cold adaptive nature and tolerance to high levels of glucose and ethanol. The phylogenetic analysis suggested the close association of glu1 with a similar gene from a mesophilic bacterium Microbacterium indicum. The purified recombinant GLU1 displayed its optimal activity and stability at pH 5 and temperature 30á´¼C. Additionally, the presence of L3 loop in GLU1 suggested its cold adaptive nature. The glucose tolerant Gate keeper residues (Leu 174 & Trp 169) with a distance of â¼ 6.953 Å between them was also predicted in GLU1. The GLU1 enzyme showed ≥ 95% and ≥ 40% relative activity in the presence of 5 M glucose and 20% ethanol. The Vmax, Km, and Kcat values of GLU1 for cellobiose substrate were observed to be 45.22 U/mg, 3.5 mM, and 41.0157 s-1, respectively. The GLU1 was found to be highly efficient in hydrolysis of celloologosaccharides (C2-C5), lactose and safranal picrocrocin into glucose. Hence, cold adaptive GLU1 with very high glucose and ethanol tolerance could be very useful in bio-refinery, dairy, and flavor industries.
Subject(s)
Microbacterium , beta-Glucosidase , beta-Glucosidase/metabolism , Microbacterium/metabolism , Phylogeny , Temperature , Hydrolysis , Glucose , Ethanol/chemistry , Hydrogen-Ion Concentration , Substrate Specificity , Enzyme StabilityABSTRACT
The rice Hybrid Proline Rich Protein (HyPRP) encoding gene, OsHyPRP16 expression exhibit early upregulation in response to Magnaporthe oryzae inoculation. Here, we functionally characterized the OsHyPRP16 promoter through deletion analysis in transgenic Arabidopsis using GUS (ß-glucuronidase) reporter assay. The promoter fragments, sequentially deleted from the 5' end could induce differential GUS activity in response to stresses induced by different hormones and abiotic stress conditions. In addition, a strong GUS induction was observed in M. oryzae inoculated transgenic Arabidopsis. Based on the insilico and stress-inducibility of D1 promoter fragment against various phytohormones and rice blast fungus, and with no basal activity under control conditions, we rationally selected D1 promoter fragment to drive the expression of a major rice blast resistance gene; Pi54 in the genetic background of blast susceptible TP309 rice line. The D1 promoter fragment was able to induce the expression of Pi54 at immediate-early stages of M. oryzae infection in transgenic rice. The transgenic plants with Pi54 under the control of D1 promoter fragment displayed complete resistance against M. oryzae infection as compared to control plants. The present study suggests that the D1 fragment of OsHyPRP16 promoter is a valuable tool for breeding and development of rice lines with early-inducible and pathogen-responsive enhanced disease resistance.
