Response surface optimization of the heparosan N-deacetylation in producing bioengineered heparin

Abstract

The chemical step in the chemoenzymatic synthesis of bioengineered heparin has been examined and optimized statistically using a response surface methodology. A four factor, two level full factorial design experiment and a three factor Box-Behnken design were carried out. The goal was to establish a method to prepare N-sulfo, N-acetyl heparosan of the desired N-acetyl content, number average molecular weight, and in maximum yield by controlling the reactant concentrations, reaction time and reaction temperature. The response surface models obtained were used to predict the reaction conditions required to optimally prepare N-sulfo, N-acetyl heparosan from E. coli generated heparosan starting material of different molecular weights.

Bioengineered heparins and heparan sulfates

“…The production of the structurally related chondroitin CPS from glucose using an engineered E. coli was also recently reported [98]. Control of the molecular weight of the heparosan CPS obtained from E. coli K5 may also be required [99,100]. Beside manipulation of culture conditions, chain termination and genetic optimization also can be possible to control the molecular weight of heparosan [101].…”

“…The midstream process involves the conversion of heparosan to N -sulfoheparosan. Base-catalyzed N -deacetylation can be carefully controlled using a response surface algorithm that gives the proper content of residual N -acetyl groups and the correct molecular weight properties [99]. The downstream process involves treating N -sulfoheparosan with recombinant C5-epimerase and OSTs in the presence of the cofactor 3'-phosphoadenosine-5'-phosphosulfate (PAPS).…”

Synergistic interactions of chemical additives on the strength development of silicate cement by a box‐behnken model optimization

“…15 The RSM is comprised of the central composite design (CCD), Doehlert matrix (DM), and Box-Behnken design (BBD). 16 BBD is one class of experimental designs for the response surface methodology.…”

“…Thus, these designs are useful in avoiding experiments performed under extreme conditions for which unsatisfactory results might occur. 15 The BBD was used to investigate the influence of four chemical additives on the compressive strength of cement mortar at 1, 3, and 28 days. It can not only make an accurate statistical analysis of experimental data but also provides image analysis with continuity characteristics, as well as intuitively understands the correspondence relationship between the factors and the response; it has been widely used in several fields such as biochemistry, agriculture, chemical engineering, material, etc.…”

“…It can not only make an accurate statistical analysis of experimental data but also provides image analysis with continuity characteristics, as well as intuitively understands the correspondence relationship between the factors and the response; it has been widely used in several fields such as biochemistry, agriculture, chemical engineering, material, etc. [15][16][17][18][19] Before designing the experiment, the levels of the factors must be coded; they were investigated at a low level (21), intermediate level (0), and high level (1). The ranges and levels of the chemicals used in the experiment are presented in Table III.…”