الفهرس 
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Abstract
This study was conducted at Genetics and Cytology Department,
Genetic Engineering and Biotechnology division, National Research
Centre during the period from 2012 to 2016 with the objective of
improvement of bacterial xylanase production using different genetic
techniques to obtain strain(s) having the ability to produce more xylanase
than the original strains. Bacillus species are well known to produce
thermo-tolerant, alkaline tolerant xylanase, and high level of xylanase
which is poor or free of cellulase. These properties are required in many
industrial applications such as food processing, animal feeding, biofuel
production, textile and paper industries.
The study was divided into four parts: Evaluation and selection of the
best xylanase producing strains, induction of physical and chemical
mutation, protoplast fusion, cloning and expression of xylanase gene.
The results could be summarized as follows:
Firstly: Evaluation and selection of the best xylanase producing
strains.
1. Eight bacterial strains (seven Bacillus strains and one Geobacillus
strain) were collected from different sources, in adation to thirty
isolates were isolated from soil. Screening was done according to
four parameters, high level of xylanase, poor or free of cellulase,
alkaline and temperature tolerance.
2. The highest xylanase producing strains were Bacillus pumilus GH,
Geobacillus sterothermophilus 2027 and Bacillus pumilus 1794
which exhibited xylanase activity of 80 U/ml, 40 U/ml and 20
U/ml, respectively. 3. Negligible amounts of cellulase activity of 0.46, 0.45 and 0.37
were calculated for B. pumilus GH, G. sterothermophilus 2027 and
Bacillus pumilus 1794, respectively.
4. Studding the effect of temperature on xylanase activity for B.
pumilus GH and G. sterothermophilus 2027 indicating that both
strains have activity at a range of temperature from 40 to 100oC
with the optimum temperature of 60oC. Both Bacillus pumilus GH
and Geobacillus sterothermophilus 2027 maintain considerable
portion of its activity at 100oC, about 61 % (61.2 U/ml) and 60 %
(24 U/ml), respectively
5. The effect of pH on xylanase activity for both strains revealed that
they have activity at pH ranging from 7 to 11with optimum pH of
7. Bacillus pumilus GH maintains 70% (56 U/ml) of its activity at
pH 10 and 42.5 % (34 U/ml) at pH 11 while Geobacillus
sterothermophilus 2027 maintains about 71% (28.4 U/ml) of its
activity at pH 10 and about 27% (11 U/ml) at pH11. However, both
strains lost most of their activity at pH 12.
Secondly: Improvement of xylanase production using physical and
chemical mutagenesis
1. Bacillus pumilus GH and Geobacillus sterothermophilus 2027
were exposed to UV irradiation for different times (5, 10, 15, 20
minutes) at 20 cm distance from the UV lamp. As a result of UV
treatment, 25 mutants exhibited higher activity than their original
strain (B. pumilus GH). The best mutant M24/BU was obtained
after 15 minutes exposure to UV which increased by 18.49 % in
comparison with their parental strain. For G. sterothermophilus, 28
mutants gave activity more than the wild type strain. The best
mutant M20/GU was obtained after 15 minutes exposure to UV
light which increased by 28.22 % in comparison with the wild type.2. Bacillus pumilus GH and Geobacillus sterothermophilus 2027
were treated with EMS (40 μl/ml) for different periods (30, 60, 90
minutes). For B. pumilus, 49 mutants exhibited higher activity than
wild type. The superior mutant M 34/BE was obtained after 60 min
with xylanase activity of 148.82 U/ml which increased by 86.02 %
in comparison with the original strain. For G. sterothemophilus,
nine mutants exhibited higher activity than wild type. The best
mutant M29/GE was obtained after 60 min with xylanase activity
90.15 U/ml which increased by 125.37 % in comparison with their
parental strain.
3. It can be concluded that, Ethyl methane sulfonate (EMS) is more
effective as a mutagenic agent than UV irradiation for induction of
the improved mutants of xylanase production from both B. pumilus
GH and G. sterothermophilus 2027.
Thirdly: Enhancement of xylanase production through protoplast
fusion
1. In order to obtain selectable marker for protoplast fusion
experiments, auxotrophic mutants were isolated using limited
enrichment method. Bacillus pumilus M34/BE and Geobacillus
sterothermophilus M29/GE mutants which were the highest
xylanase producers mutants were treated with EMS (40 μl/ ml) for
60 minutes. For B. pumilus M34/BE, two auxotrophs were isolated,
arginine (Arg-) and histidine (His-), while two auxotrophs i.e.,
serine (Ser-) and valine (Val-), were isolated from G.
sterothermophilus M29/GE
2. Three fusion experiments were performed between four isolates
Bacillus pumilus M34/BE (Arg-), Bacillus pumilus M34/BE (His-),
G. sterothermophilus M29/GE (Ser-) and G. sterothermophilus
M29/GE (Val-).3. Cross 1: Intraspecific protoplast fusion between B. pumilus
M34/BE (Arg-) and B. pumilus M34/BE (His-)) revealed that F6/B
was the highest xylanase producing fusant exhibiting product
activity of 294.3 U/ml which increased by about 2 fold in
comparison to the two parents and 3.67 fold in comparison to B.
pumilus GH wild type.
4. Cross 2: Intraspecific protoplast fusion between G.
sterothermophilus M29/GE (Ser-) and G. sterothermophilus
M29/GE (Val-) showed that the highest xylanase producing fusant
was F2/G with activity of 234.8 U/ml which increased by 2.6 fold
in comparison to the parents and 6 fold in comparison to G.
sterothermophilus 2027 wild type.
5. Cross 3: Intergeneric protoplast fusion between Parent 1, B.
pumilus M34/BE (Arg-) and parent 2 G. sterothermophilus
M29/GE (Val-) exhibiting that the highest xylanase producing
fusant was F2/GB with xylanase activity 286.5U/ml increasing by
2 fold in comparison to parent 1 and 3.58 fold in comparison to B.
pumilus GH wild type. In addition xylanase activity increased by
3.17 fold in comparison to parent 2 and 7.16 fold in comparison to
G. sterothermophilus 2027 wild type.
6. These results indicated that protoplast fusion is a powerful method
for improving xylanase production which may be due to gene
dose.
Fourthly: Cloning and expression of xylanase gene
1. Xylanase gene from Bacillus pumilus GH was isolated from
chromosomal DNA and cloned to pET 29-a (+) plasmid vector
then transformed to E.coli DH5α host cell.
2. Xylanase gene from positive recombinant plasmid was sequenced.
The open reading frame (ORF) of xylanase gene has 687 bp
encoding a protein of 228 amino acids.3. The sequence was submitted to gene bank database at ncbi
website with the accession number of KT757524.1. Xylanase gene
from B.pumilus GH strain showed 99 % identity with other B.
pumilus strains of accession numbers AY887130.1, AY526092.1
and X00660.1. It differ only in two nucleotide bases at positions
579 and 600. The result showed 100% similarity according to
amino acids sequences because the difference in nucleotide
sequence occurred at the third position of the genetic code which
led to the same amino acids.
4. The recombinant plasmid containing xylanase gene was
transformed to expression host E.coli BL21 (DE3) and the
xylanase gene was successfully expressed.
5. SDS-PAGE analysis was performed for transformed E.coli BL21
(DE3). Profile pattern of IPTG induced and un-induced sample
were the same except one band for xylanase gene at a molecular
weight (MW) of about 23 KDa appeared in induced sample,
indicating that the recombinant xylanase gene was successfully
expressed in E.coli BL21 cells.
6. Activity was measured for both intracellular and extracellular
crude extractsfrom transformed E.coli BL21 (DE3) cells. The
total xylanase activity is 9 U/ml, 52 % of the activity was
extracellular (4.7 U/ml) and 48% of the activity was intracellular
(4.3 U/ml). This activity is lower than Bacillus pumilus GH wild
type strain. Lowering of protein expression in heterologous host
due to several reasons including protein toxicity, formation of
inclusion bodies and post translation modification.