Improvement the Activity and Specificity

of -L-arabinofuranosidase (Abfa)
from Bacillus thermoleovorans IT-08
expressed in Escherichia coli

Y. Sri Wulan Manuhara

Increasing optimum pH and pH stability of Abfa

(-L-arabinofuranosidase) by site directed
mutagenesis

INTRODUCTION
-L-Arabinofuranosidase (Abfa) is importance enzyme involved in
arabinose release from L-arabinofuranosyl.
There were 2 types of Abfa :
1. The exo-type Abfas (-L-Arabinofuranosidase
arabinofuranohydrolase, EC 3.2.1.55) hydrolyze the terminal
reducing from arabinose-containing polysaccharides. This
can hydrolyze (1
3)- and (1
5)- -arabinosyl
linkages of arabinan.

nonenzyme

2. The endo-type Abfas are called endo-1

5--arabinases (1
5-arabinan 1
5--arabinohydrolase EC 3.2.1.99) are endohrydrolisis
of 1,5-arabinofuanosidic linkages in 1,5- arabinan.

The Abfas have been classified into four families of

glycanases on the bases of amino acid sequence
similarities.
There are :
Families 43, 51, 54 and 62
Nyoman et al., have already determined the nucleotide
sequence of Abfa in pTP510 and pET-abfa.
The BLAST analysis revealed that Abfa of Bacillus
thermoleovorans IT-08 was belong to family 51 and showed
high homology with Abfa of Geobacillus thermoleuvorans T6 (90% similarities).

In recent year, several application of Abfa are:

1. bioconversion of lignocellulosic materials
to fermentable products,
2. delignification of paper pulp
3. digestibility enhancement of animal
feedstock,
4. clarification and thinning of juices
5. hydrolysis of grape monoterpenyl -Larabinofuranosylglucosides to increase
aroma during wine making.

The application of Abfa in delignification of paper pulp

industry is one of the benefit application of this enzyme,
because paper pulp industry still use chlorine as bleaching
agents that is very seriously problem in environment.
Abfa enzyme will be very useful as a bio-bleaching at least
to decrease the chlorine uses in paper-pulp bleaching
process.
The bleaching process needs alkali condition at 8-10.
Optimum pH of Abfa is 7 and pH stability is 5-8.

THE AIM OF THIS RESEARCH

Increasing the optimum pH and pH stability of
Abfa to decrease the chlorine uses in paper-pulp
bleaching process, so then we will use site
directed mutagenesis to obtain the Abfa mutants
which has more stability at alkali pH.

APPROACH
1. Mutation on site chain:
Type of Amino acid : Glutamate Glutamine
Position of amino acid : 29, 175, 294

2. Mutation on nucleophile site:

Tryptophan Arginine

Rationale
Compare to other site chain, glutamate acid play
important role in controlling the pH condition
since other amino acids attached are relatively
neutral
Replacement of glutamate acid to glutamine
considering the properties of the carboxylic
group (acid) to amide group (base) because of
the NH2 group
Position was varied to determine the active site
of the glutamate acid

NH3+
HOOC

CH2

CH2

CH

L - glutamat acid (Glu)

Acid

NH3+

O
H2N

COO-

CH2

CH2

CH

L - glutamine (Gln)
Amide-base

COO-

NH3+
CH2

COO-

CH

N
H

L - tryptophan (Trp)

Alkaline

Alkaline

NH
H 2N

NH3+
NH

(CH2)3

L - arginin (arg)

CH

COO-

METHOD
The mutagenic primers were (red lettering indicates mismatch
position):
E29Q 5-TGG-CTC-GTT-TAT-TCA-ACA-CCT-CGG-CCG-C-3
iE29Q 5-GCG-GCC-GAG-GTG-TTG-AAT-AAA-CGA-GCC-A-3
E175Q 5-GGT-GTC-TAG-GCA-ATC-AGA-TGG-ACG-GTC-C-3
iE175Q 5-GGA-CCG-TCC-ATC-TGA-TTG-CCT-AGA-CAC-C-3
E294Q 5-CAT-CTG-TCG-TTT-GAC-CAA-TGG-AAC-GTA-TG-3
iE294Q 5-CAT-ACG-TTC-CAT-TGG-TCA-AAC-GAC-AGA-TG-3
W298R 5-GAA-TGG-AAC-GTA-AGG-TAC-CAC-TCG-AAT-G-3
iW298R 5-CAT-TCG-AGT-GGT-ACC-TTA-CGT-TCC-ATT-C-3

PCR condition
First denaturation 95oC

30 sec

Denaturation 95oC
Annealing
55oC
Extension
68oC

30 sec
1 min
12 min

PCR reaction
Template
Primer forward
Primer reverse
dNTPs
pfu buffer
pfu polymerase
milliQ
Total volume

50x (0.5 l)
5 M (0.4 l)
5 M (0.4 l)
10 M (1.0 l)
10x (5.0 l)
1,25u (0.5 l)
42 l
50 l

18 cycle

Transformation of mutant to E. coliTOP10 competent cell

20 l of PCR result + 0.4 l DpNI
Incubation in 37oC for 1 hour
+ 100 l TOP10 competent cell
Place on ice for 30 min
Heat shock 42oC for 30 sec.
+ 500 l LB broth
Shaking in 37oC for 1 hour
Plate on LB agar + ampicilin
Incubation in 37oC overnight

Mutant plasmid isolation and characterization

Isolation of mutant plasmid were done by kit isolation
plasmid (Sigma) and then the mutant plasmid were
characterize by restriction enzyme (EcoRI)

RESULTS
PCR result
E29Q

W298R

E175Q

Double mutation
1 2 3

E294Q

1. E29Q/W298R
2. E175Q/W298R
3. E294Q/W298R

Characterization of Abfa mutant by EcoRI

E29Q

W298R

E175Q
1 2 3

E175Q/W298R

E294Q
1 2 3

E294Q/W298R