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Microbial Transglutaminase Separation by pH-Responsive Affinity Precipitation with Crocein Orange G as the Ligand

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Abstract

A microbial transglutaminase (MTG) was efficiently purified by using pH-responsive affinity precipitation with Crocein orange G (COG) as the affinity ligand. The docking method was used to identify the appropriate ligand. The molecular simulation results were compared with the label-free detection data analyzed by ForteBio’s Octet. A pH-responsive polymer, PMMDN, was polymerized and subsequently coupled with COG as the ligand. The isoelectric point (pI) and recovery of PMMDN and PMMDN–COG were 4.51, 99.8,% and 4.78, 98.1 %, respectively. The optimal adsorption conditions were found to be a ligand density of 60.0 μmol/g, pH 7.0, and 0.2 mol/L NaCl. The adsorption isotherm showed that the maximum adsorption capacity was 91.32 mg/g polymer and the dissociation constant was 0.021 mg/mL. Interaction information between PMMDN–COG and MTG in the whole process of affinity precipitation were obtained by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). PB (0.02 mol/L pH 7.0) in 20.0 % glycol was used to elute the binding MTG from PMMDN–COG. Under these conditions, electrophoretically pure MTG was obtained in only one step with elution recoveries of 98.96 % (protein) and 95.09 % (activity).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, Shanghai, 200237, China

    Zhaoyang Ding, Sipeng Li & Xuejun Cao

Authors
  1. Zhaoyang Ding
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  2. Sipeng Li
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  3. Xuejun Cao
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Corresponding author

Correspondence to Xuejun Cao.

Glossary

AM1-BCC

Semi-empirical (AM1) with bond charge correction (BCC)

ANTECHAMBER

Antechamber is a set of auxiliary programs for molecular mechanical studies

DMS

DMS is an open-source program written in C for computing the molecular surface of a molecule

GRID

Grid creates the grid files necessary for rapid score evaluation in DOCK

Mol2

The Mol2 file format is a popular method for specifying chemical structures, including atom types, positions, and bonding

SHOWBOX

Showbox is an interactive program for specifying the location and the size of the grids that will be calculated by the program grid

sphere_selector

Sphere_selector filters the output from sphgen selecting all spheres within a user-defined radius of a target molecule

SPHGEN

Sphgen generates sets of overlapping spheres to describe the shape of a molecule or molecular surface

sphgen_cpp

Sphgen_cpp has the same function as Sphgen

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Ding, Z., Li, S. & Cao, X. Microbial Transglutaminase Separation by pH-Responsive Affinity Precipitation with Crocein Orange G as the Ligand. Appl Biochem Biotechnol 177, 253–266 (2015). https://doi.org/10.1007/s12010-015-1742-8

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  • DOI: https://doi.org/10.1007/s12010-015-1742-8

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