Expression, characterization and structure elucidation of thermotolerant lipase with broad pH isolated from Antarctic Pseudomonas sp. strain AMS3
Lipase is an enzyme that plays an important role in detergent, cosmetic and pharmaceutical industries. Lipase from Antarctic region has gained huge interest for industrial applications but was impaired due to the limited crystallographic and structural information. To maximize its full biocatalyt...
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| Format: | Thesis |
| Language: | English |
| Published: |
2018
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| Online Access: | http://psasir.upm.edu.my/id/eprint/68522/1/FBSB%202018%2011%20IR.pdf http://psasir.upm.edu.my/id/eprint/68522/ |
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| Summary: | Lipase is an enzyme that plays an important role in detergent, cosmetic and
pharmaceutical industries. Lipase from Antarctic region has gained huge
interest for industrial applications but was impaired due to the limited
crystallographic and structural information. To maximize its full biocatalytic
potentials, the understanding on the biochemical and structural features of this
class of enzyme are imperative. Therefore, this research was conducted to
isolate, express, characterize and determine the structure of lipase from an
Antarctic bacteria via in silico and crystallographic approaches. In this study,
the best lipolytic producing bacterium was identified as Pseudomonas sp.
AMS3. The lipase gene was isolated from the reconstructed of genomic library.
The positive colonies were verified as recombinant lipase producer using
triolein and Rhodamine B agar plates. Recombinant plasmid analysis revealed
an open reading frame (ORF) of 1353 nucleotides encoding 450 amino acids.
The protein sequence has two domains, namely GST C and lipase domains at
the N and C terminal, respectively. The gene encoding the AMS3 lipase was
cloned into a pET 51b vector and heterologously expressed in E. coli BL21
(De3). The best expression condition for AMS3 lipase was at 0.5 mM IPTG,
incubated at 20 °C and 12 h of induction time. The recombinant AMS3 lipase
was purified using single step affinity chromatography with a 50% recovery and
1.52 fold purity. The molecular weight of the recombinant AMS3 lipase was
estimated at ~60 kDa. Biochemical characterization of the lipase showed the
enzyme work at a broad temperature profile of 10-70 °C and stable at 10-60
°C. The AMS3 lipase activity also exhibited a broad pH range of 5 to 10 and
was activated in the presence of metal ions. The lipase was able to hydrolyze
long chain lipid substrate with and without organic solvents. The AMS3 lipase
was successfully crystallized using sitting drop method. The X-ray diffraction of
the AMS3 lipase crystal was very poor, thus no crystal structure was able to be
determined. In order to obtain the crystal structure, the AMS3 lipase was
truncated at the N terminal domain based in the computational analysis. Molecular dynamic simulation revealed a flexible N-terminal domain possibly
interfering with the crystallization process. The truncated AMS3 lipase was
subcloned and express in E.coli BL21 (De3) .This truncated enzyme has an
approximate molecular weight of ~45 kDa. The truncated AMS3 lipase has
similar features with native except for pH profile whereby the lipase was most
active at alkaline condition (pH 8-pH 10). The truncated AMS3 lipase was
successfully crystallized using sitting drop method and the crystal was
diffracted to 2.7 Å. The 3D structure was refined, validated and deposited to
Protein Data Bank (PDB no. 5XPX). The refined truncated AMS3 lipase
structure revealed a common α/β hydrolase fold with bound calcium and zinc
ions. The protein active site contained serine hydrolase catalytic triad, namely
serine, aspartic acid and histidine. Superposition of the lipase thermostable
homologs showed structural differences that could be potentially important
towards the lipase temperature adaptation. It was generally concluded, the
reduction of hydrogen bond and changes to the secondary structure content of
the lipase allowed the enzyme to be active at broad temperatures. The
structural and biochemical information will be beneficial towards rational design
of lipase suitable for industrial application. |
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