Skip to main content

LUP Student Papers

LUND UNIVERSITY LIBRARIES

Mutagenesis of the lipolytic enzyme LysinB-D29 to enhance its activity and thermostability.

Maochao, Mao LU and Fan, Yixin LU (2021) KBTM01 20211
Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
Abstract
Plastic has brought great benefits to human life, but it has also given rise to serious environmental problems as “microplastic” threatening the living organisms. Due to the promising properties, the polymer poly (ethylene) terephthalate (PET) has been widely used for packaging and textiles industries with large annual demand and low recycle rate. The imbalance between the production and recycling of PET has led to its accumulation in the environment. Thus, the development of bio-based polymers as well as developing recycling methods becomes a quite important approach. The engineered leaf-branch compost cutinase (LCC) enzyme, has been recently reported with promising thermostability and activity against PET. A phage-derived lipolytic... (More)
Plastic has brought great benefits to human life, but it has also given rise to serious environmental problems as “microplastic” threatening the living organisms. Due to the promising properties, the polymer poly (ethylene) terephthalate (PET) has been widely used for packaging and textiles industries with large annual demand and low recycle rate. The imbalance between the production and recycling of PET has led to its accumulation in the environment. Thus, the development of bio-based polymers as well as developing recycling methods becomes a quite important approach. The engineered leaf-branch compost cutinase (LCC) enzyme, has been recently reported with promising thermostability and activity against PET. A phage-derived lipolytic enzyme LysinB-D29 (LysB-D29) was revealed to be structurally similar to α/β hydrolase family of proteins with high similarity to cutinases with a melting temperature of 54.7 ℃. Rational design by site-directed mutagenesis and directed evolution by error-prone PCR were carried out to generate mutants with higher thermostability and activity. By screening the library generated from error-prone PCR, two mutants LysB-D29-S2I/I183V and LysB-D29-I212T showed higher thermostability and activity, respectively. Most of the constructed mutants (11 out of 16) were successfully expressed in E. coli BL21 (DE3) expression host and purified by Immobilized-metal Affinity Chromatography. Three mutants (A162CA169C, F29Y and D29CV248C) showed higher thermostability against Tween 80 than the wild type LysB-D29 when incubated at 60 ℃. (Less)
Popular Abstract
People cannot imagine life without plastics. The annual production has proliferated in modern world ever since people have realized the convenience of plastics. They have occupied every aspect in our daily life, from food packaging to pharmaceutical industry.
However, most of the plastics produced today are generally synthesized by chemicals derived from fossil-based fuels, which are not renewable. The unbiodegradable plastics accumulate in nature without being decomposed. Among those plastic wastes, only 9% has been recycled, while around 75% has been trashed in landfill, or leaked into the oceans. Scientific reports about the plastic pollution in marine environment started in early 1970s, however only in recent years the seriousness of... (More)
People cannot imagine life without plastics. The annual production has proliferated in modern world ever since people have realized the convenience of plastics. They have occupied every aspect in our daily life, from food packaging to pharmaceutical industry.
However, most of the plastics produced today are generally synthesized by chemicals derived from fossil-based fuels, which are not renewable. The unbiodegradable plastics accumulate in nature without being decomposed. Among those plastic wastes, only 9% has been recycled, while around 75% has been trashed in landfill, or leaked into the oceans. Scientific reports about the plastic pollution in marine environment started in early 1970s, however only in recent years the seriousness of the situation has been realized. Large amount of plastic debris enters the oceans, threatening marine animals and even public health. As a star product in packaging and textile field, poly (ethylene) terephthalate-based products, especially PET bottles, have an annual production of more than 50 million tons. The self-degradation of PET is extraordinarily slow; hence it can exist for decades or even hundreds of years in the environment.
In 2016, a Japanese scientist isolated a bacterium which could grow on PET film using PET as the carbon source. This bacterium, Ideonella sakaiensis, can produce an enzyme, named PETase, which is capable of degrading PET [1]. With the help of advanced biotechnology tools, the bio-degradation of PET with enzymes has been successively developed.
In this thesis, site-directed mutagenesis of an esterase enzyme LysB-D29 from a bacteriophage was performed to improve its thermostability as well as its activity. This was done by site-specific and random mutations of the amino acids in the enzyme followed by screening of the mutant library for selecting mutants with desirable properties. Purified enzymes were heated up to test the thermostability and tested for activity against synthetic ester substrates with different carbon chain length. They were also incubated with PET film and granules with different crystallinity to test the polymer degradability. An enzyme (LCC-WCCG) variant known to break down PET was also produced and used as a positive control of PET degrading assay.
This Master project provides an insight of engineered phage-derived lipolytic enzyme LysB-D29 for potential PET degradation and a basis for future research. (Less)
Please use this url to cite or link to this publication:
author
Maochao, Mao LU and Fan, Yixin LU
supervisor
organization
course
KBTM01 20211
year
type
H2 - Master's Degree (Two Years)
subject
keywords
LysB-D29, PET, Protein engineering, Site-directed Mutagenesis, Error-prone PCR, Biotechnology
language
English
id
9055557
date added to LUP
2022-04-28 09:42:48
date last changed
2022-04-28 09:42:48
@misc{9055557,
  abstract     = {{Plastic has brought great benefits to human life, but it has also given rise to serious environmental problems as “microplastic” threatening the living organisms. Due to the promising properties, the polymer poly (ethylene) terephthalate (PET) has been widely used for packaging and textiles industries with large annual demand and low recycle rate. The imbalance between the production and recycling of PET has led to its accumulation in the environment. Thus, the development of bio-based polymers as well as developing recycling methods becomes a quite important approach. The engineered leaf-branch compost cutinase (LCC) enzyme, has been recently reported with promising thermostability and activity against PET. A phage-derived lipolytic enzyme LysinB-D29 (LysB-D29) was revealed to be structurally similar to α/β hydrolase family of proteins with high similarity to cutinases with a melting temperature of 54.7 ℃. Rational design by site-directed mutagenesis and directed evolution by error-prone PCR were carried out to generate mutants with higher thermostability and activity. By screening the library generated from error-prone PCR, two mutants LysB-D29-S2I/I183V and LysB-D29-I212T showed higher thermostability and activity, respectively. Most of the constructed mutants (11 out of 16) were successfully expressed in E. coli BL21 (DE3) expression host and purified by Immobilized-metal Affinity Chromatography. Three mutants (A162CA169C, F29Y and D29CV248C) showed higher thermostability against Tween 80 than the wild type LysB-D29 when incubated at 60 ℃.}},
  author       = {{Maochao, Mao and Fan, Yixin}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Mutagenesis of the lipolytic enzyme LysinB-D29 to enhance its activity and thermostability.}},
  year         = {{2021}},
}