MINISTRY OF EDUCATION
AND TRAINING
VIETNAM ACADEMY OF
SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
-----------------------------
Nguyen Khanh Hoang Viet
ASSESSMENT OF THE DIVERSITY AND THE ROLE
OF SOME MODULES IN THE STRUCTURE OF CELLULOLYTIC
ENZYMES OF BACTERIA IN THEGOAT’S RUMEN
Major: Biotechnology
Code: 9.42.02.01
SUMMARY OF BIOLOGICAL DOCTORAL THESIS
Hanoi - 2020
The research was completed in
Graduate University of Science and Technology – Vietnam Academy of
Science and Technology
Scientific supervisor 1: Prof. Dr. Truong Nam Hai
Scientific supervisor 2: Assoc. Prof. Dr. Do Thi Huyen
Reviewer 1: …
Reviewer 2: …
Reviewer 3: ….
The thesis will be defended to the doctoral evaluation committee at
Graduate University of Science and Technology – Vietnam
Academy of Science and Technology at …….. on ………, 2020.
The thesis can be found at:
- Library of Academy of Science and Technology
- Vietnam National Library
1
PREAMBLE
1. The urgency of the subject
Microorganisms, as well as bacteria in particular have
demonstrated significant practical meaning to humans by their
application in medical, agriculture, industry and environmental
treatment. To find a large amount of new microbial genes and apply
them in life, the study of the genomic diversity of microbial
communities becomes an important issue that considerably gains
many interests from biologists. However, some recent discoveries
show that the vast majority of microbial species (approximately 99
%) have been uncultured. Therefore, the use of common culturing
techniques is unable to collect the entire genetic resources of all
microbiota. Presently, by using metagenomics - an effective tool to
sequence the whole genome of all organisms obtained directly from
an environmental sample, genes of both cultured and uncultured
microorganisms are fully studied and analyzed.
In Vietnam, the development of agriculture with large-scale
and concentrated production generates a large amount of wastes and
byproducts, which are commonly discarded by incineration. This
process not only causes a negative impact on the environment but
also wastes natural resources because the main component of
agricultural byproducts and wastes is lignocellulosic biomass.
Meanwhile, humankind is facing the shortage of fossil fuels as well
as consequences of the emission of greenhouse gases. Hence, it is
necessary to using carbohydrate energy created from abundant and
renewable resources such as lignocellulosic biomass to convert into
many valuable products to replace fossil fuels.
Lignocellulose or cellulose in specific, with extremely tough
and inflexible structure, has to be subjected to many steps, in which
saccharification is crucial to convert into the final product by the
2
action of cellulases. These enzymes play a key role in the biomass
conversion as well as the price of products. Thus, many researches
have been carried out to isolate and mine novel enzymes with high
activity and affinity to substrate for the efficiency of cellulose
conversion. Unlike other enzymes that only have the catalytic
domain, most cellulases poss additional modules which consist of
some discrete and unknown modules, such as FN3, Ig, CBM.
Currently, few investigations about the function of modules to
catalytic activity have been known. Some hypotheses suggest that
these modules not only act as a linker for the catalytic domain but
also display many biological functions such as stabilizing enzyme
structure or increasing the affinity of enzyme and substrate.
Therefore, the biological role of these modules in the cellulase
structure should be studied with the purpose of screening or
designing enzymes to enhance the efficiency of cellulolytic process.
From 2014 to 2017, with the financial support of the Project
code ĐTĐLCN.15/14, the researchers of the National Key
Laboratory of Gene Technology (Institute of Biotechnology, VAST)
have been sequenced the metagenomic DNA of bacteria in NinhBinh
and ThanhHoa goat’s rumen. From approximately 8.6 Gb of
metagenomic DNA sequence data, 816 genes encoding cellulase
were mined. In this study, we tend to analyze the diversity of
cellulase modularity sequences as well as discover novel specific
modular structure in order to determine the role of the module on
enzyme activity. Therefore, we carried out the Thesis: “Assessment
of the diversity and the role of some modules in the structure of
cellulolytic enzymes of bacteria in the goat’s rumen”.
2. Objectives of the study
- Analyzing the diversity of cellulases and modular cellulases
of microbial community in rumen goat by Metagenomics;
3
- Investigating the role of functionally unknown modules
(FN3 or Ig) on enzyme activity.
3. Research content
In order to accomplish the above objectives, we conducted
main research works:
1. Analyzing and evaluating the diversity of GH families, the
origins of cellulases and modular cellulases encoded by open reading
frames (ORFs) from metagenomic DNA data of bacteria in Vietnam
goat’s rumen.
2. Analyzing and selecting the sequence encoding the
modular enzyme in order to express and determine the impact of
functionally unknown domains.
3. Expression and purification of modular enzymes encoded
by selected sequence (XFn3Egc) in fusion form with SUMO partner.
4. Investigating the role of functionally unknown domains on
the cellulolytic activity of enzyme.
5. Determining some characteristics of recombinant enzyme
which is expressed by using selected sequence encoding modular
structure.
4. New contributes of the thesis
1. Based on 816 ORFs encoding cellulases of bacteria in
Vietnam goat’s rumen that were mined from in metagenomic DNA
data, 243 deduced modular cellulases had FN3 or Ig domains.
Among complete cellulases containing FN3, 99.2% FN3 domains
were found to be accompanied with betaglucosidase catalytic
domains GH3 while only a FN3 module was determined to be
collocated with endoglucanase catalytic domain GH5. Besides, all Ig
modules were associated with endoglucanase catalytic domains GH9.
It is rather uncommon to find endoglucanase GH5 collocated with
FN3 domain.