Expression, Purification and Crystallization of Thermostable Mutant of Cutinase Est1 from &amp;lt;i&amp;gt;Thermobifida alba&amp;lt;/i&amp;gt;

Kengo Kitadokoro,Shigeki Kamitani,Kensuke Nakata,Shingo Matsui,Ryouhei Osokoshi

doi:10.4236/abb.2018.95015

Abstract

A double mutantEst1, which is a plastic degrading cutinase-type esterase in Thermobifida alba, has been over-expressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving immobilized metal affinity chromatography and cation-exchange chromatography, yielding 120 mg of protein per liter of bacterial culture. Crystals have been obtained by using the sitting-drop vapor-diffusion technique. Native diffraction data to 1.37 A resolution were obtained at the BL44XU beam line of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C2, with unit-cell parameters a = 127.2 A, b = 42.1 A, c = 63.2 A, β = 114.7°, likely containing one Est1 double mutant (296 residues) per asymmetric unit.

Highlights

Plastic degrading enzymes were very important for reducing plastic waste and various cutinases were capable to degrade plastic [1]
We present here the overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the thermostable mutant of cutinase Est1 from Thermobifida alba
The supernatant was purified by immobilized metal affinity chromatography (HIS-Select Nickel Affinity Gel, Sigma-Aldrich, Germany), which was equilibrated with lysis buffer, the sample was loaded and washed 5 times with lysis

Summary

Introduction

Plastic degrading enzymes were very important for reducing plastic waste and various cutinases were capable to degrade plastic [1]. We first determined the crystal structure of Est119 as the polyester hydrolyzing type cutinase [7]. We wanted to shed light on the so far undiscovered structural bases of Thermobifida cutinases, and to unveil the relationships between high activities and thermostable differences from comparing cutinase 3D structures of Est1DM with Est119. Toward this goal, we present here the overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the thermostable mutant of cutinase Est from Thermobifida alba

Protein Expression and Purification

Crystallization

Data Collection and Processing

Results and Discussion