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: …

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


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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

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

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

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

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

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


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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

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)


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3.3.2. Design of pETSUMO expression vectors containingFn3,
Egc, Fn3Egc, XFn3, XFn3Egc genes

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)


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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


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

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


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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

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.


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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),

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.


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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

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.