High-Level Production of Bacillus subtilis Glycine Oxidase by Fed-Batch Cultivation of Recombinant Escherichia coli Rosetta (DE3)

Abstract

A fed-batch process for the high cell density cultivation of Escherichia coli Rosetta (DE3) and the production of the recombinant protein glycine oxidase (GOX) from Bacillus subtilis was developed. GOX is a deaminating enzyme that shares substrate specificity with d-amino acid oxidase and sarcosine oxidase and has great biotechnological potential. The B. subtilis gene coding for GOX was expressed in E. coli Rosetta under the strong inducible T7 promotor of the pET28a vector. Exponential feeding based on the specific growth rate and a starvation period for acetate utilization was used to control cell growth, acetate production, and reconsumption and glucose consumption during fed-batch cultivation. Expression of GOX was induced at three different cell densities (20, 40, and 60 g . L(-1)). When cells were induced at intermediate cell density, the amount of GOX produced was 20 U . g(-1) cell dry weight and 1154 U . L(-1) with a final intracellular protein concentration corresponding to approximately 37% of the total cell protein concentration. These values were higher than those previously published for GOX expression and also represent a drastic decrease of 26-fold in the cost of the culture medium.

Enhanced intracellular soluble production of 3‐ketosteroid‐Δ1‐dehydrogenase from Mycobacterium neoaurum in Escherichia coli and its application in the androst‐1,4‐diene‐3,17‐dione production

“…Although high temperature could improve the rate of protein synthesis, rapid KSDD synthesis rate may influence protein transport, leading to the accumulation of IBs. 24 When the induction temperature was at 37 ∘ C, the biomass of the recombinant E. coli BL21/pET28a-ksdd reached 9.6 g L −1 , which was 1.7-and 1.3-fold that at 20 and 25 ∘ C, respectively. But the intracellular KSDD activity at 37 ∘ C (0.2 U mg −1 ) was much lower than that at 20 and 25 ∘ C. Compared with the induction temperature at 20 ∘ C, the biomass and the intracellular KSDD activity of the recombinant strain induced at 25 ∘ C were higher and reached 7.4 g L −1 and 2.1 U mg −1 , respectively.…”

“…With the induction temperature increased from 25°C to 37°C, the adverse effect was more obvious. Although high temperature could improve the rate of protein synthesis, rapid KSDD synthesis rate may influence protein transport, leading to the accumulation of IBs . When the induction temperature was at 37°C, the biomass of the recombinant E. coli BL21/pET28a‐ ksdd reached 9.6 g L −1 , which was 1.7‐ and 1.3‐fold that at 20 and 25°C, respectively.…”

Calorimetric control for high cell density cultivation of a recombinant Escherichia coli strain

“…Different high cell density cultivation (HCDC) strategies have been described. In an open loop fed-batch process the feed rate is increased following a predefined exponential feeding profile which results in a more or less constant growth rate (Korz et al, 1995;Martinez-Martinez et al, 2007;Riesenberg et al, 1990). This strategy is easy to implement.…”

Characterization and structural modeling of a novel thermostable glycine oxidase from Geobacillus kaustophilus HTA426

“…These have focused on its overexpression, 12 its kinetic mechanism, 10 its possible physiological role in thiamine biosynthesis, 13 its structure-function relationships, 13,14 the maximization of its production and the study of its M261 mutants 15 and its high-level production by fed-batch cultivation. 16 Among the putative substrates of GOX, D-proline is of special interest. The replacement of proteinogenic amino acids with cyclic imino acids has been carried out in the search for new Abbreviations: DAAO, D-amino acid oxidase; DASPO, D-aspartate oxidase; GOXB, glycine oxidase from Bacillus subtilis strain ATCC 6633; GOXK, glycine oxidase from Geobacillus kaustophilus HTA 426; GR2, glutation reductase family 2; MSOX, monomeric sarcosine oxidase; T m , temperature of maximum transition rate.…”

“…Several reports have been published. These have focused on its overexpression,12 its kinetic mechanism,10 its possible physiological role in thiamine biosynthesis,13 its structure‐function relationships,13, 14 the maximization of its production and the study of its M261 mutants15 and its high‐level production by fed‐batch cultivation 16. Among the putative substrates of GOX, D ‐proline is of special interest.…”