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oderma Trich
longibrachiatum
Bio-polishing of jute fibers by using cellulase purified from locally
isolated Trichoderma longibrachiatum
Bio-polishing
Trichoderma longibrachiatum
AB1 NCBI
FT-IRSEM
1200
33 50 100 6-1 40
1.50 0.89 0.69
100 50 33
P<0.05)
40
26.5 100
120019.18 15.78
% 50 33
Summary
The Bio-polishing technique of jute fibers was achieved for the first time in Iraq
by using cellulase purified from locally isolated Trichoderma longibrachiatum which
recorded first in Iraq in NCBI site as AB1. Enzymatic treatmemt of jute fibers was
carried out and the released reducing sugars and percentage of loss in weight of jute
fibers were determined , then morphological and structural characteristic were examined
by using FT-IR spectroscopy and scanning electron microscope.
Enzymatic treatment was achieved by incubating 1200mg of jute fibers with
gradual concentrations of purified enzyme (33, 50 and 100%) for 1-6 hours at 40°C (the
optimum temperature of enzyme activity and stability). Results showed that the
maximum release of reducing sugars has been achieved after three hours of incubation
with the enzyme, where the concentration of released reducing sugars was 1.50, 0.89
and 0.69 mgml after the treatment with the enzyme in a concentrations of 100, 50 and
33% respectively.
The results of enzymatic treatment also indicated a significant decrease in P <0.05)
in the weights of jute fibers after enzymatic treatment for three hours at a temperature
of 40 ° C in all enzymatic concentrations. As the percentage of weight loss reached
26.5% after treatment with the concentrated enzyme 100% due to the presence of a
higher concentration of the enzyme relative to the fixed amount of jute fibers (1200 mg),
compared to the loss rate of 19.18% and 15.78% after the treatment with cellulase
enzyme at a concentration of 50% and 33%, respectively.
Structural and morphological characteristics of enzymatically treated jute fibers
were studied. Texture analysis by using scanning electron microscope showed that bio-
polishing caused the removal of the protruding hairs and micro fibrils, and loosened
fibers to give a smoother product compared with the control sample
Finally, The Bio-polishing method has demonstrated,through these criteria used
above, that jute fibers have been softened and biologically smoothed as it is considered
a cheep, safe and environmentally friendly method instead of using undesired chemicals
method that may cause harm to the environment and factory workers
63-58 24-211.5-0.8
14-12 1.2 – 0.6 0.8-0.4
(1
1
Bio-polishing)
1 1.9-1.7
(2
Trichoderma longibrachiatum
AB1
3
4 5
NCBI AB1
1-1Trichodrema
1
TSM agar
Trichoderma Elade4
50-45 2
PDAL- shape 3 2827 4 Trichoderma
(PDA)potato dextrose agarTrichoderma
2827 4
Trichoderma
Trichoderma TrichodermaSamuels Hebber 5
CMD)Corn Meal Dextrose Agar(CMA) Carboxy Methy Agar (SNA) Spezieller Nahrstoffarmer agar(MEA) Malt extract agar 282
Trichoderma DNA Extraction Mini Kit (8) ZYMO 1 150Trichoderma
2823200D.D.W
2 750 ( lysis solution )
vortex 5 3 . 5β- mercapto ethanol-8)
Zp Bashing bead lysis tube 4 10000 5 400 zymo spin iv spin filter
7000 6 1200 fungal DNA binding buffer 800
zymo spin iic column 7 10000 500) fungal DNA wash buffer (
zymo spin iic column 10000 . 8 zymo spin iic column micro centrifuge tube100 DNA elution buffer
10000 30 .DNA
Nano Drop
1
260
280
PCR1 Primer
) PCR(
) ITS( )rRNA (S ribosomal RNA 5.8 (9)1
Dnase
100
10
)ITS(S rRNA 5.8 ) Macrogen( ) DNA Sequenceing(
Blast NCBI Mega 6
Sequence analysis
NCBI National Center for Biotecnology and Information Query )
( Sbjct )
BLAST .
1-2
1 Trichoderma Mandle
2
HCl-Tris 3
4
) Ion exchange chromatography(elluloseC-DEAE 5
200-G
. 6 Standards G200-sephadex)d BSALysozyme, pepsin, Pronase, urease an(
3-1
T. Longibrachiatum
Jabasingh
Nachiyar 10 1 2
50 3 1200 5
T. longibrachiatum1005033 4 40
5 25
6 50
4-1
Otagevwo Aluyi11
1-4-1 1 110 10
3.0- 0.3
3 2 1.5 563
10 3 21.5
4 540 5 540
2
2-4-1
Fu
120.5 1.5
1.5 DNS
10
21.5
540
1-3-12
5-1
5072
(12
Weight loss (%)=W° W1 W°] x 100
°
W
1
W
6-1 1-6-1 FT-IR Spectral Analysis
Bagewadi
(13
º105
)500- 4000
1
2-6-1
Bagewadi13
Trichoderma
7 Trichoderma longibrachiatum 3
.1Genomic DNA Isolation
2.0-1.841.43 4
.2 Polymerase Chain Reaction
Trichoderma Internal transcribed spacer ( ITS) ITS1 ITS4
5.8S ribosomal RNA ( rRNA) ITS1 ITS 2
5
bp 550
ITS 5.85 rRNA
PCR
NCBIITS 5.85 rRNAITS1 , ITS4 18s rRNA
3. ication productsSequence analysis of amplif
ITS1 rRNA 5.8S
Trichoderma Macrogen 6
491) FASTA( 7
8
BLAST NCBI ITS1 rRNA
5.8S NCBI 100KY750397.1 ID: 4
9
Mega 6.0
ID: 401554.1 longibrachiatum Trichoderma
dermaTricho longibrachiatum10
NCBI AB1
KY750397.1 ID: 11
T. longibrachiatum
100 50 331200
40)
pH4
(pH4) .
(%100 )1.50 0.89 0.69
% 50 33P<0.05
5
P<0.05)
40
26.5 100
120019.18 15.78
% 50 33
100
FT-IR
3000-4000
1
800-900
1
C-O-C C-O
3500-3600
1
O-H
12
21
1730
1
4-O-methyl-glucanoacetyl xylan
xylan71 1500-1600
1
81 1200-1250
1
O-C
91 1107.17
1
O-C
(20)
897.12
1
O-C
Syringyl – Guaiacyl
– Hydrophenyl units of lignin)(21)
T. longibrachiatum
(SEM
13
1 2 3
1 T. longibrachiatum
NCBIAB1 2 T. longibrachiatum
AB1 3
T. longibrachiatum 4
5
6
7 8 9
1 T. longibrachiatum
2 1 T. longibrachiatum
Samuels Hebbar7
3 1
NCBI AB1 4 T. longibrachiatum
5
6 T. longibrachiatum100 50 3340
6 4 7 100
3 1.50 0.89 0.69 % 50 33
8 26.5
100
120019.18 15.78 % 50
33 9
10
1730
1
1500-1600
1
1250 -1200
1
1107.17
1
897.12
1
11
12
1) ITS( S ribosomal RNA 5.8
-
Primer
name
Sequence(5′→3′)
m
T
(ᵒC)
GC
content(
%)
Product
size(bp)
Company
Forward
ITS1
TCCGTAGGTGAACCTGCGG
60.3
50 %
650
IDT,
Canada
Reverse
ITS4
TCCTCCGCTTATTGATATGC
57.8
41 %
2PCR
No.
Phase
C)ᵒ(
m
T
Time
No. of cycles
1
Initial Denaturation
94
3 min.
1 cycle
2
Denaturation
94
45 sec
35 cycle
3
Annealing
52
45sec
4
Extension
72
45sec
5
Final extension
72
10 min.
1 cycle
3540
540
1
0
10
10
0
0
2
0.3
9.7
10
0.3
0.06
3
0.6
9.4
10
0.6
0.141
4
0.9
9.1
10
0.9
0.197
5
1.2
8.8
10
1.2
0.264
6
1.5
8.5
10
1.5
0.315
7
1.8
8.2
10
1.8
0.38
8
2.1
7.9
10
2.1
0.44
9
2.4
7.6
10
2.4
0.505
10
2.7
7.3
10
2.7
0.556
11
3.0
7.0
10
3.0
0.610
2
CMA -A-
y = 0.203x + 0.009
R² = 0.998
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2
OD (595nm)
Glucose concentration (mg/ml)
SNA -B -
MEA –C-
CMD–D-
-E-
-F -
3A-DTrichoderma longibrachiatum
EF100X
Genomic DNA
4
1% 5
5 ITS1 ITS4 PCR . S rRNA 5.825 M : 100100001 .Trichoderma
6Trichoderma longibrachiatum
1_ITS4.ab1 491
AAATTACAAAGGTCACTCCAACCCCATGTGAACGTTACCAATCTGTTGCC
TCGGCGGGATTCTCTTGCCCCGGGCGCGTCGCAGCCCCGGATCCCATGGC
GCCCGCCGGAGGACCAACTCCAAACTCTTTTTTCTCTCCGTCGCGGCTCC
CGTCGCGGCTCTGTTTTATTTTTGCTCTGAGCCTTTCTCGGCGACCCTAG
CGGGCGTCTCGAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTC
TGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAG
AATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATT
CTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGG
GGGGTCGGCGTTGGGGGATCGGCCCCTCACCGGGCCGCCCCCGAAATACA
GTGGCGGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGC
7 ( FASTA ) ITS1 5. 8 S rRNA Trichoderma longibrachiatum
1 cctggtgaag cggagggaca ttaccgagtt tacaactccc aaaccccaat gtgaacgtta
61 ccaatctgtt gcctcggcgg gattctcttg ccccgggcgc gtcgcagccc cggatcccat
121 ggcgcccgcc ggaggaccaa ctccaaactc ttttttctct ccgtcgcggc tcccgtcgcg
181 gctctgtttt atttttgctc tgagcctttc tcggcgaccc tagcgggcgt ctcgaaaatg
241 aatcaaaact ttcaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcgaaat
301 gcgataagta atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcacattgc
361 gcccgccagt attctggcgg gcatgcctgt ccgagcgtca tttcaaccct cgaacccctc
421 cggggggtcg gcgttggggg atcggcccct caccgggccg cccccgaaat acagtggcgg
481 tctcgccgca gcctctcctg cgcagtagtt tgcacactcg caccgggagc gcggcgcggc
541 cacagccgta aaacacccca aacttctgaa atgttgacct cggatcaggt aggaataccc
601 gctgaactta agcatatcaa aaagccggag gaaa
85 . 8 S rRNA Trichoderma longibrachiatum
4 100%score
Source
Identities
Expect
Score
Sequence ID
Region
Nucleotide
Location
Type of
substitution
No. of repeat
Trichoderma
longibrachiatum
100%
0.0
739
KY750397.1
74 to 473
None
1
Score
Expect
Identities
Gaps
Strand
739 bits(400)
0.0
400/400(100%)
0/400(0%)
Plus/Plus
Query 1 TCGGCGGGATTCTCTTGCCCCGGGCGCGTCGCAGCCCCGGATCCCATGGCGCCCGCCGGA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 74 TCGGCGGGATTCTCTTGCCCCGGGCGCGTCGCAGCCCCGGATCCCATGGCGCCCGCCGGA 133
Query 61 GGACCAACTCCAAACTCTTTTTTCTCTCCGTCGCGGCTCCCGTCGCGGCTCTGTTTTATT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 134 GGACCAACTCCAAACTCTTTTTTCTCTCCGTCGCGGCTCCCGTCGCGGCTCTGTTTTATT 193
Query 121 TTTGCTCTGAGCCTTTCTCGGCGACCCTAGCGGGCGTCTCGAAAATGAATCAAAACTTTC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 194 TTTGCTCTGAGCCTTTCTCGGCGACCCTAGCGGGCGTCTCGAAAATGAATCAAAACTTTC 253
Query 181 AACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 254 AACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATG 313
Query 241 TGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 314 TGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATT 373
Query 301 CTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 374 CTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCG 433
Query 361 TTGGGGGATCGGCCCCTCACCGGGCCGCCCCCGAAATACA 400
||||||||||||||||||||||||||||||||||||||||
Sbjct 434 TTGGGGGATCGGCCCCTCACCGGGCCGCCCCCGAAATACA 473
9ITS1 5 . 8 S rRNA
Trichoderma longibrachiatum
NCBI
10T. longibrachiatumMEGA v.6.0
11T.longibrachiatum AB1 NCBI https: //www.ncbi.nlm.nih.gov/nuccore/MF164044.1
5
AB1 T. longibrachiatum
40
100
50
33
1
0.90
0.50
0.33
2
1.10
0.69
0.51
3
1.50
0.89
0.69
4
1.25
0.76
0.56
5
0.76
0.45
0.31
6
0.55
0.35
0.20
A
B
12A
B
A
B
13A
B
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