Structural determination of improved methanol-tolerant mutant from Geobacillus zalihae T1 lipase by x-ray crystallography
Lipases are versatile enzymes that have been altered through various modification methods to improve their enzymatic properties to meet biotechnology industry requirement. Since decades ago, lipases from many sources have been study widely as potential biocatalyst to assist synthesis of biodiese...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/113000/1/113000.pdf |
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| Summary: | Lipases are versatile enzymes that have been altered through various
modification methods to improve their enzymatic properties to meet
biotechnology industry requirement. Since decades ago, lipases from many
sources have been study widely as potential biocatalyst to assist synthesis of
biodiesel. Some of them have been altered via protein engineering approach to
enhance their enzymatic performance and stability. The thermostable T1 lipase
from Geobacillus zalihae can also be a great biocatalyst candidate in biodiesel
production. However, inactivation of T1 lipase when the enzyme is surrounded
by high concentration of methanol solvent is limiting its uses in industrial
applications. Since introduction of non-bonded interactions hardly improved their
stability of T1 lipase in methanol, the introduction of disulphide bond could be
the best proposition to retain protein conformation and the enzyme stability in
the presence of methanol. Hence, current study aims to engineer a methanoltolerant
lipase by site-directed mutagenesis and divulge the interaction that
stabilizes the mutant by X-ray crystallography. The preliminary study on enzyme
stability was conducted by using online software ERIS, FoldX and MAESTRO.
The stability of the mutant 2DC lipase was tested virtually and molecular dynamic
simulation was performed in water and methanol solvent. Experimentally, the
purified protein of mutant 2DC lipase was used to screen protein crystal for
diffraction to elucidate and validate the mutant’s structure. It showed that the
substitution of amino acid S2 and A384 with cysteine could enhance the stability
of the enzyme by promoting the formation of disulphide bond to tighten the both
terminal ends of the protein structure. The substitution of amino acid cysteine
showed the changes on the active site distance (S113, D317, and H358),
however, it was not affected the lipase activity and folding of protein structure.
The 2DC mutant was successfully constructed and cloned into pET32-b and
transformed into Origami B (DE3) expression host. The expression and
purification using Ni2+-Sepharose affinity chromatography and gel filtration
chromatography S-200 of the protein yielding 4.0 mg/ml mutant 2DC lipase
suitable for protein crystallization. The mutant 2DC lipase was crystallized after
24-hour incubation at 20°C and diffracted by X-ray crystallography for deeper
evaluation in term of stability and rigidity. The crystal was diffracted at 2.04 Å
using in-house X-ray beam and the crystal belongs to monoclinic space group
C2, with unit cell parameter of a = 118.17, b = 81.5, c = 100.05. Details
information on structural elucidation of the mutant 2DC lipase has disclosed the
changes within the mutant structure which associated with the alteration of
enzyme activity and stability posed by the mutant. The increased in rigidity of the
structure as well as changes of interaction within the catalytic region of the
mutant 2DC lipase were suggested to be the factor influenced its enzymatic
activity, stability and tolerance towards methanol. Hence, this newly improved
mutant 2DC lipase could be the next potential biocatalyst in biodiesel production
industry. |
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