Nghiên cứu đánh giá sự đa dạng và vai trò của một số module trong cấu trúc enzyme thủy phân cellulose từ khu hệ vi sinh vật trong dạ cỏ của dê tt tiếng anh

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.


4
2. For the investigation of the FN3 function in enzyme
structure, genes encoding endoglucanase GH5 (XFn3Egc) was
artificial synthezised and whole gene, and different modular
structures (Fn3, XFn3, Fn3Egc, Egc) were expressed in E. coli,
purified and functional characterized. FN3 module was determined
to have ability to increase the solubility and stability of catalytic
domain as well as to loosen crystal cellulose in filter paper surface to
enable enzyme access on cellulose for hydrolysis. It also was found
to increase affinity of enzyme to the soluble substrate as CMC.
3. The SXFn3Egc has optimal activity at 40 oC, pH 4 and

stable below 60oC in 90 minutes. The Km and Vmax of SXFn3Egc
were 1.26 mg/ml and 148.12 µmol/min/ml respectively. This enzyme
showed a 2-fold increase in catalytic activity at a concentration of 40
mM Mn2+. In contrast, the activity decreased that caused by using metal
ions (Ca2+, Mg2+, Ni2+, K+, Co2+, Cu2+, Zn2+, Fe3+) or chemicals (SDS,
urea, 2-mercaptoethanol, EDTA, tween 80, triton X-100).
CHAPTER 1. OVERVIEW
1.1. Cellulose
Cellulose is a large molecular compound composed of a
linear chain of β-D-glucose units, which is the main component of
the plant cell walls. The use of cellulose as a renewable resource in
several industries such as food processing, manufacturing of
biofuels, pure chemicals has been a sustainable development
tendency in the economy and environment.
1.2. Cellulase
Cellulase is a primary group of enzymes, which are able to
cut the β-1,4-glycoside bond of cellulose to release the high-value
final product - glucose. Cellulase is often classified into three major
groups (endoglucanases, exoglucanases, β-glucosidases) with
different types of hydrolysis activity. Cellulase may have only


5
catalytic module in structure or contains extra domains such as CBM
or functionally unknown domains (FN3, Ig).
1.3. Metagenomics in gene mining
Metagenomics is described as a group of techniques in
molecular biology, bioinformatics that allows studying the genomic
diversity of most microbes recovered directly from environmental
samples. Metagenomics is proved as an effective method to discover

new enzymes, bioactive substances for many applications. In this
study, we are going to mine new cellulases, especially modular
cellulases (containing FN3, Ig modules) based on 816 ORFs
encoding cellulases. This data was analyzed from 164,644 ORFs and
assembled from 8.46 Gb of bacterial metagenomic DNA in Vietnam
goats rumen.
CHAPTER 2.MATERIALS AND METHODS
2.1. Materials, chemical and equipment



Materials: The 816 ORFs encoding cellulases from bacterial
metagenomic DNA in goat’s rumen.



Microorganisms, plasmids of Invitrogen (USA), PCR
primers of GenScript (USA); chemicals of Bio-Lab (USA),
Fermentas (USA), Sigma (USA), Merck (Germany).
2.2. Methods
2.2.1. Molecular biology techniques, microorganisms Transformation
of plasmid DNA into E. coli (Froger et al.,
2007); Extraction of plasmid DNA from E. coli and electrophoresis
on agarose gel (Sambrook et al., 2001); DNA was purified from
agarose gel by the DNA kit Qiagen - QIAquick Gel Extraction Kit;
Optimizing of triplet code was carried out based on online software
of Genscript (Rare Codon Analysis Tool).
2.2.2. Protein biochemical methods
Recombinant proteins were purified by affinity
chromatography column Ni-NTA (Invitrogen) and evaluated the

6
purity by Quantity One (Bio-Rad); protein was quantified by using
Bradford method (Bradford, 1976); determination of endoglucanase
activity on CMC substrate (Miller, 1959) and filter paper
(Camassolaet al.,2012) with some slightly modification;
determination of cellulase activity on agar-CMC agar plates (Teather
et al., 1982) and Zymogram analysis (Champasri et al., 2015); the
effect of the enzyme on the surface of filter paper was evaluated by
taking pictures on SEM scanning electron microscopy (Kataeva et
al., 2002).
2.2.3. Bioinformatics methods
The sequences containing Pfam and conservative regions
were studied using Pfam database ( />and BLASTP ( respectively.
For the prediction of tertiary structure of enzymes, two distinct
online software Phyre2 and Swiss model were used; AcalPred
software was applied to predict of acidic and alkaline enzymes; the
thermostability of a protein was estimated by TBI software.
2.2.4. Data processing
Statistical methods, Microsoft Excel were used to calculate
and show the results as ± SE (Standard Error)
CHAPTER 3.RESULTS AND DISCUSSION
3.1. The GH diversity and modular structure of cellulases
deduced from 816 open reading frames
3.1.1. Evaluation of the diversity and structure of GH cellulase
families
The 816 ORFs encoding cellulases were functionally
annotated that belonged to 11 distinct GH families (Table 3.1). In
particular, GH3 (400 ORFs) and GH5 (192 ORFs) were the most

popular families which accounted for 49% and 23.5%, respectively.
297 complete sequences were found to exist in the form that has
unique domain for the catalytic function or contains additional


7
functionally unknown modules (FN3, Ig). Specifically, 90.9% GH3
and 100% GH9 contained FN3 and Ig respectively. Besides, only one
FN3 module was collocated with GH5 domain. Therefore, FN3 and
Ig modules were not only basically linkers but also shown some
biological functions that have not been clearly defined.
Table 3.1. Summary of sequences encoding cellulases
based on COG và KEGG databank
GH

Module

ORFs

GH1
GH3

GH1
GH3
Fn3-GH3
GH5
Fn3-GH5
GH5-CBM2
GH5-CBM37
GH8

GH9
GH9-Ig
GH9-CBM3
GH9-CBM37
GH9-dockerin

16
198
202
189
1
1
1
48
11
30
2
1
1

GH5

GH8
GH9

GH

GH16

Module

GH16
GH16-CBM4
GH44
GH44
GH48
GH48
GH64
GH64-CBM6
GH74
GH74
GH94
GH94
CBM63 CBM63
FN3
FN3
-

ORFs

33
2
2
1
1
1
50
1
10
14

3.1.2. Evaluating the diversity of structures of completed modular
cellulase
The investigation of 243 ORFs encoding modular cellulases
showed that 148 ORFs had a completed structure (131 ORFs encoding
enzymes contain FN3 domain, 17 ORFs encoding enzymes have Ig
domain). Specifically, 17 ORFs encoding endoglucanases GH9
contained Ig module (Ig-GH9); 130 ORFs (in total 131 ORFs encoding
enzymes contain FN3 domain) are responsible for encoding betaglucosidase GH3 (GH3-Fn3) and only one endoglucanase GH5 domain
was accompanied with Fn3 domain (Fn3-GH5). The FN3 module
situating in front of catalytic domain of endoglucanase GH5 at Nterminal is known as an uncommon structure. Therefore, it is


8
necessary to study the role of this module in the efficiency of
enzymes hydrolysis.
3.1.3. Evaluation of diversity of the ORFs encoding for cellulase
For clearly understanding about the bacterial community and
their role in the digestion of cellulose in Vietnam goat's rumen, we
have identified the origin of 816 ORFs encoding cellulase. In
particular, 221 ORFs encoding cellulase mainly belonged to
Bacteroidetes (153 ORFs) and Firmicutes (53 ORFs) accounted for
69.2% and 24.0%, respectively. Bacteroidesuniformis (29 ORFs),
Prevotellabuccal (25 ORFs) were the most dominant species that
containing genes coding cellulase; Ruminococcus flavefaciens (7
ORF) was determined as a typical species belonged to the
cellulolytic bacterial group with high efficiency of cellulose biomass
hydrolysis.
3.1.4. Evaluation of the similarity of amino acid sequences deduced
from annotated ORFs encoding cellulase

Based on two NR and CAZy, 297 completed ORFs encoding
cellulases were demonstrated the similarity below 85% (new
sequence) accounted for 80.1% and 77.4% respectively. By
investigation of 148 completed ORFs encoding cellulase with
modular structure, 17 completed ORFs encoding endoglucanase
containing Ig module were firstly reported; 131 completed ORF
encoding cellulase having FN3 module, in which 90 sequences were
initially studied accounting for over 68% (89 ORFs encoding betaglucosidase, 01 ORF encoding endoglucanase). Thus, this data is
expected to exploit numerous new genes, especially the completed
sequences encoding cellulases containing modules such as FN3, Ig.
3.1.5. Prediction of properties of enzymes based on sequences
Rapid prediction of some optimal conditions for enzyme
activity such as pH range, temperature, pI value is necessary to


9
initially screen the prominent genes and study their application. The
investigation of some properties of modular cellulases based on 243
ORFs showed that most enzymes (from 130 ORFs) were stable at
55-65oC, meanwhile, cellulases encoded by 139 ORFs maintain
activity at alkaline pH and 146 enzymes have pI above 5-6. By the
survey of the pI values of 148 completed sequences containing FN3
and Ig-like domains, two sequences (an Ig-GH9 and a FN3-GH5)
were determined to have pI higher than 9.
3.2. Selection of sequences of the typical modular enzymes to
investigate the role of modules
3.2.1. Investigation of the three-dimension structure of enzyme
containing FN3 modules
The existence of FN3 module in GH5 endoglucanase was
found to be a rare structure compared to common Fn3-GH3

structure, which was selected for the study. The gene sequence
encoding for mature endoglucanase had a length of 1545 nucleotides.
The results of homologous comparative analysis by BLASTN and
classification by MEGAN software showed that this gene was
predicted to be derived from Ruminococcus bicirculans. The amino
acid sequence of endoglucanase GH5 analyzed using BLASTP
software exhibited the most similarity 60% with endoglucanase code
CDC67342.1, which is commonly found in Ruminococcus sp. CAG:
57, a bacterial species of goat rumen. By survey the conserved region
by SwissProt software, the sequence showed the highest similarity
(49%) with the frame of endoglucanase 3pzt.1.A with the recovery of
53%. It was also indicated as monomer structure with a ligand of
Mn++ (Figure 3.7). Using the Phyre2 tool, the sequence displayed the
highest similarity with the c3pzvB endoglucanase frame with the
confidence of 100 %. Besides, it had a separated functional region
and N-terminal region including separated FN3 structure (Fig 3.8).


10

Mn

Figure 3.7. Prediction of
Figure3.8. Prediction of conserved
conserved regions by regions by Phyre2 SwissProt
3.2.2. Prediction of pI and pH values of enzyme containing FN3
module based on sequences
Gene sequence encoding endoglucanase GH5 which contains
FN3 module was estimated to have pH optimum at acidic pH, be
stable at below 55°C and have high homogeneous pI values in both

unknown functionally regions (X domain, FN3 module) and the
active site (Egc). By using of pI values of general enzyme molecule
as well as each the homogeneous module, the expression and
optimization of some conditions for enzyme hydrolysis become more
convenient.
3.3. Cloning of XFn3Egc gene
3.3.1. Analysis of optimal triplet code of XFn3Egc sequence
The sequence encoding endoglucanase GH5 (XFn3Egc) was
optimized to have the best utilization rate of 97% compared to 46%
before optimization. After optimization, 86% of the sequence
showed relevance in the range of 91-100%, compared to the
sequence before optimization with only 49%. The gene sequences
before and after optimization for expression on E. coli are described
in Figure 3.10. The optimized XFn3Egc gene was artificially
synthesized and inserted into pET22b (+) at the NcoI+XhoI
restriction site to generate a vector named pET22-XFn3Egc.


11

Figure 3.10. Gene sequence of XFn3Egc before (A) and after
performing the codon optimization for expression in E. coli (B)
(yellow region is FN3 sequence; blue region is actived region; red
letters are optimal sequences)


12
3.3.2. Design of pETSUMO expression vectors containingFn3,
Egc, Fn3Egc, XFn3, XFn3Egc genes
The genes Fn3, Egc, Fn3Egc, XFn3, were amplified from the

pET22-XFn3Egc template BY PCR, then the target genes and
XFn3Egc were cut by two restriction enzymes NcoI-XhoI and
inserted into pET22b (+) at the same restriction sites to create
recombinant vectors respectively.After that, the genes in expression
vectors were sequenced and expressed in E. coli. However, they were
expressed at a low level and almost existed in inclusion body.
Therefore, the genes were transferred from pET22b(+) to pETSUMO
using NcoI and XhoI restriction enzymes to generate pET22SUMOFn3, pET22SUMO-Egc, pET22SUMO-Fn3Egc, pET22SUMOXFn3Egc and pET22SUMO-XFn3. All the plasmids then were
transformed into E. coli DH10B for cloning. The transformant
colonies were inoculated in LBA medium to screen E.coli strains
containing recombinant plasmids. These plasmids are linearized by
using single restriction enzymes,whereas, when they were cut by two
restriction enzymes, the generated genes showed the correct sizeas
calculated. Thus, the expression vectors carrying the target genes
have been successfully designed.
3.4. Expression of recombinant E. coli strains carrying the target genes

After determiningthe optimal conditions for expression, the
recombinant E. coli BL21 (DE3) strains were inoculated in LB
medium containingampicillin at 25°C, induced with 0.5 mM IPTG,
and cultured for 5 hours. The result of protein analyzing by
polyacrylamide gelelectrophoresis showed that all 5 types of
recombinant proteins were expressed at high level and had the
correct size as calculated. Almost proteins were found in the soluble


13
form except the Egc without FN3 existing in insoluble
fraction(Figure 3.18).
Total

Soluble

Insoluble

Figure 3.18. Analysis of SFn3, SEgc, SFn3Egc, SXFn3Egc, SXFn3
expressed in E. coli BL21 (DE3) strains in total, soluble and
insoluble fractionson 12.6% polyacrylamide gel containing SDS.
Negative control: pETSUMO; Marker: unstained protein standard

(A)

(B)

Figure 3.19. Analysis of proteinsin soluble fractions by nondenaturing polyarylamide gel electrophoresis (A)
andzymogram (B); Marker:

unstained protein standard (Thermo scientific);
cellulase: possitive control (Sigma)


14
The soluble fractions of recombinant proteinswere determined
cellulase activity on the CMC plates. The results illustrated that only SXFn3Egc
clearly exhibitied the hydrolysis activity on CMC substrate. On the other hand,
by using zymogram assays, all 4 proteins (SFn3, SFn3Egc, SXFn3Egc, SXFn3)
were migrated to the correct position on the polyacrylamide gel stained by
Coomassie brilliant blue despite they were separated in the gel under nonreducing conditions. On the gel stained by Congo red, a bright band can be
visualized in SXFn3Egc lane which was similar to the one in thelane of positive
control (Figure 3.19). Thus, XFn3Egc enzyme was successfully expressed by

XFn3Egc gene with the correct size as calculated and demonstrated the CMC
hydrolysis. The domain FN3 was determined to increase the solubility of
catalytic region. The X domainalso contributes to increasethe cellulase activity
of catalytic region.

3.5. Recombinant proteinspurification and determination of
cellulase activity
3.5.1. Purification of recombinant proteins
The recombinant proteins were completely elutedby elution
buffercontaining 400 mM imidazole. The results of analyzing the
eluted fractions by SDS-PAGE showed that only one band was
detected which was similar in size to the target proteins.The purity of
recombinant proteins evaluated by the Quantity One 1-D Analysis
softwarewas over 99%.

Figure 3.20. SDS-PAGE analysis of
the fractions collecting from SFn3
purification (F1-F9)

Figure 3.22. SDS-PAGE analysis of
the fractions collecting from
SFn3Egc purification (F1-F7)


15

Figure 3.24. SDS-PAGE analysis
of the fractions collecting
fromSXFn3Egc purification(F1F10)

Figure 3.26. SDS-PAGE analysis
of the fractions collecting
fromSXFn3 purification(F1-F8)

3.5.2. Evaluation of the enzyme activity after purification
3.5.2.1. Evaluation of hydrolysis activity of recombinant proteins on
CMC
After purification, SFn3, SFn3Egc, SXFn3Egc, SXFn3 were
evaluated their hydrolysis in CMC. In which, SXFn3Egc and
SFn3Egcclearly exhibited catalytic activity. The hydrolysis zones of
SXFn3Egc was larger than the hydrolysis zoneof SFn3Egc (having
higher enzyme activity).
3.5.2.2. The analysis of the ability of SFn3 and SXFn3 to promote
CMC hydrolysis activity
Mixing of functionally unknown proteins (SFn3, SXFn3)
and enzymes containing catalytic region increased CMC hydrolysis
activity. The combination of SFn3, SXFn3 with SFn3Egc led to the
increase in enzyme activity which accounted for 74.5% and 49.9%,
respectively while the CMCase activity illustrated an increase of 27
% by mixing SXFn3, SFn3 and SXFn3Egc (Figure 3.29). The results
showed that the FN3 domain significantly affected the hydrolysis
rate of enzymecompared to the single enzyme. This can be assumed


16
that the FN3 domain mayinteracttoCMC and help to increase the
affinity of enzymeforits substrate.

CMC substrate

Figure 3.29. Hydrolysis activities of single proteins and mixture of
proteins after purification in CMC; Possitive control (ĐC+):
Cellulase (Sigma)
3.5.2.3. The SFn3 and SXFn3 increased activity of enzymes to
hydrolysis of filter paper
On the filter paper, the hydrolysis activity of SXFn3Egc
mixing with SFn3, SXFn3 demonstrated an increase of 86.8% and
13.6% respectively, compared to single SXFn3Egc. The combination
of SFn3Egc with SFn3 or SXFn3 also help to increase the enzyme
activity in comparison with single SFn3Egc, however, the difference
was not statistically significant. This result showed that both SFn3
and SXFn3 helped to increase cellulose hydrolysis activity of
enzyme in filter paper. SFn3 and SXFn3 do not exhibit cellulase
activity, therefore, these proteins only help SXFn3Egc and SFn3Egc
to increase enzyme activity.
The domain FN3 increased catalytic activity of protein
containing FN3 module in filter paper by two hypothesized reasons:
(1) FN3 module loosedthe cellulose crystalline structures of filter


17
paper for enzymeseasilyaccessing into cellulose fibers then
hydrolysis; (2) FN3 module increased affinity between enzymes and
cellulose and helped the enzyme to anchor to substrate.

Filter paper substrate

Figure 3.30. Hydrolysis activities of single protein and mixture of
proteins after purificationinfilter paper;
Possitive control (ĐC+): Cellulase (Sigma)

3.5.3. FN3 module increased affinity of enzyme for the substrate
3.5.3.1. CMC
There was an increase in the cellulase activity of SXFn3Egc,
SFn3Egc in CMC treated by SFn3 and SXFn3(Figure 3.31). By
using CMC treated by SFn3 or SXFn3, SXFn3Egc exhibited the
catalytic activity stronger than SFn3Egc. The activity of SXFn3Egc
and SFn3Egc in CMC treated by SFn3 illustrated an increase of
31.5% and 23.8%, respectively. However, the activity of two
enzymes in CMC treated by SXFn3 slightly raised to7.3% and 5.9%,
respectively. Thus, SFn3 and SXFn3 demonstrated the ability to
promote catalytic activity by increasing the affinity of the enzyme for
CMC.
Based on protein bands visualized in the native gel with the
presence of CMC as substrate, both SFn3 and SXFn3 showed the


18

CMC adsorbedWithSXFn3

CMC adsorbedWithSFn3

CMC adsorbedWithSXFn3

CMC adsorbedWithSFn3

ability to bind and hydrolyze CMC. SFn3 and SXFn3 may increase
the affinity of enzyme for its substrate, so the hydrolysis activity of
enzyme was stronger than this of the single enzyme. The mixture of
SXFn3Egc and SFn3 completely reacted with CMC, soit was not

visualized on the gel compared to the mixture of SXFn3Egc with
SXFn3 (Figure 3.32). This is a reason why SFn3 increased the
catalytic activity of enzyme better than SXFn3.

Substrate

Figure 3.31. The role of SFn3 and SXFn3-treated CMCin
catalyticactivities of SFn3Egc and SXFn3Egc
In PBS

CMC 1% in PBS

Figure 3.32.Analysis of the ability of SFn3, SXFn3 to increase the
affinity between enzyme and CMC; M: Standard protein scale
(Thermo Scientific)


19

FP adsorbedwithSXFn3

Substrate

FP adsorbedưithSFn3

FP adsorbedwithSXFn3

FP adsorbedưithSFn3

3.5.3.2. Filter paper

The catalytic activity of SXFn3Egc revealed an increase by
using the filter paper pretreated by SFn3 and SXFn3. In particular, the
activity of SXFn3Egc in filter paper treated by SFn3 washigher than in
the filter paper treated by SXFn3 (Figure 3.33). However, the activity of
the SFn3Egc in filter paper treated by SFn3 and SXFn3 insignificantly
changed compared to native filter paper. This indicated that the
absorption of SFn3 and SXFn3 on the filter paper surface (especially
SFn3) may help to increase catalytic activity of SXFn3Egc.

Figure 3.33. The role of SFn3 and SXFn3-treated filter paper
incatalytic activitiyof SFn3Egc and SXFn3Egc; FP: Filter paper
(Whatman No. 1)
3.5.3.3. Analysis of the ability of SFn3 and SXFn3 to loose
crystalline structure in filter surface
The filter paper was treated by SFn3 and SXFn3 then
scanned by electron microscopy at 500x and 1000x magnifications.
Many loosen, separated and exfoliated cellulose fibers were seen in
the SNF3 and SXFn3-treated papers compared to the native filter
papers (Figure 3.34). At 5000x magnification, the surface of filter
papers treated by FN3 were not as smooth as the untreated samples.


20
Therefore, SFn3 and SXFn3 affected the catalytic activity of
enzymes by loosing the surface of the filter papers.

Figure 3.34. Scanning electron micrographs of filter papers (Whatman No.
1) untreated with SFn3 or SXFn3 (column 1) treated with SFn3 (column 2),
treated with SXFn3 (column 3) at the magnifications: 500 times (row A),
1000 times (row B) and 5000 times (row C)

3.5.3.4. The effect of temperature, pH on the adsorption of SFn3 and
SXFn3 on cellulose substrate
In order to increase the adsorption of FN3 on the filter paper
as well as cellulolytic activity, we assessed the effect of pH,
temperature on the filter paper absorption of the proteins. The result
showed that the adsorption of SFn3 and SXFn3 on filter paper
reached the highest rate at pH 4 and decreased at pH 6 and pH 8.
Besides, the absorption of both SFn3 and SXFn3 on filter paper at
40°C or 60°C was stronger than the absorptionat 20°C.
3.6. Evaluation of some properties of enzyme SXFn3Egc
3.6.1. Effect of pH


21
The optimum pH value for SXFn3Egc activity was 4. The
enzymes showed strong activity in the pH range 3.0-5.0. At pH 3 and
pH 5, the activity of SXFn3Egc was approximately 80% compared to
its activity at pH 4. The catalytic activity showed a gradual decrease
in the pH range 7.0-9.0.
3.6.2. Effect of temperature
In the temperature range 30 - 60 oC, SXFn3Egc had different
levels of activity: From 30oC to 40oC, enzyme activity increased and
reached the highest value at 40oC; in the range 45-50oC, the enzyme
still exhibited strong activity (accounted for 84-88% of activity at the
optimal temperature). However, SXFn3Egc activity decreased
gradually at 55-60oC.
3.6.3. Evaluation of the thermal stability
The SXFn3Egc demonstrated thermostability by retaining
the highest residual activity after 30 minutes and decreased gradually

from 60 to 90 minutes in the temperature range 30 - 60°C.
3.6.4. Effect of some metal ions and chemicals
The results of evaluating the effects of some metal ions
2+
(Ca ,Mg2+, Ni2+, K+, Co2+, Cu2+, Mn2+, Zn2+, Fe3+) and 6 common
chemicals (1% SDS, 1 µM urea, 1 µM, 2-mercaptoethanol, 1 µM
EDTA,1 mM tween 80,1 µM triton X-100) showed that only Mn 2+ at
a concentration of 10 mM helped to increase enzyme activity
insignificantly to 108%. When the concentration of Mn 2+ was
increased to 20 mM, 30 mM, 40 mM, the activity of the enzyme
revealed an increase of 202%, 215%, 222% respectively compared to
control samples.
3.6.5. Kinetic properties of XFn3Egc
The Km and Vmax values of XFn3Egc were 1.26 mg/ml and
148.12 µmol/min/ml respectively.


22
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
1. The 816 ORFs coding for cellulases of bacteria in
Vietnamese goat’s rumen were identified to belong to 11 different
GH families (GH1, 3, 5, 8, 9, 16, 44, 48, 64, 74 , 94) and annotated
mostly as beta-glucosidase of GH3 family and endoglucanase of
GH5, GH8, and GH9 families. The sequences were derived mainly
from Bacteroidetes and Firmicutes phylum and the most dominant
species were Bacteroides uniformis and Prevotella buccae.
2. The 243 ORFs (148 complete ORF) encoding cellulases
were determined as modular enzymes containing functionally
unknown domains namely FN3 or Ig. 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 are associated with
endoglucanase catalytic domains GH9. It is rather uncommon to find
endoglucanase GH5 collocated with FN3 domain.
3. The sequences encoding cellulase GH5 (XFn3Egc) and
different modular enzymes (containing Fn3, XFn3, Fn3Egc, Egc)
were synthesized and expressed in E. coli to study the role of FN3
module in the enzyme structure. FN3 module was determined to
increase the solubilization and stability of catalytic domain as well as
loose crystalline cellulose in filter paper surface for enzyme
accessing into cellulose for hydrolysis. It also was found to increase
the affinity of enzyme for the soluble substrate as CMC.
4. The SXFn3Egc had optimal activity at 40 oC, pH 4. This
enzyme was stable and durable at temperatures below 60 oC for 90


23
minutes. The Km and Vmax of SXFn3Egc were 1.26 mg/ml and
148.12 µmol/min/ml, respectively.
5. The endoglucanase activity of SXFn3Egc showed a 2fold increase in catalytic activity at a concentration of 40 mM Mn 2+,
whereas, the activity decreased that caused by using metal ions:
Ca2+, Mg2+, Ni2+, K+, Co2+, Cu2+, Zn2+, Fe3+ at the final
concentrations of 10 mM and 6 common chemicals including SDS
(1%), urea (1 µM), 2-mercaptoethanol (1 µM), EDTA (1 µM), tween
80 (1mM), triton X-100 (1 µM).
RECOMMENDATIONS FOR FUTHER RESEARCH
It is necessary to continue investigating and evaluating the
role of FN3 modules as well as other functionally unknown modules

such as Ig, X in the structure of cellulase with modular structure,
with the aim of producing the mixtures of enzymes that are suitable
for different types of substrate and purposes for application in
environmental treatment and biofuel production.