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 non-reducing from arabinose-containing polysaccharides. This enzyme can hydrolyze (1 3)- and (1 5)- -arabinosyl linkages of arabinan.

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 T-6 (90% similarities).

In recent year, several application of Abfa are:1. bioconversion of lignocellulosic materials to fermentable products, 2. delignification of paper pulp3. digestibility enhancement of animal feedstock, 4. clarification and thinning of juices5. hydrolysis of grape monoterpenyl -L- arabinofuranosylglucosides 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.

APPROACHMutation on site chain:Type of Amino acid : Glutamate GlutaminePosition of amino acid : 29, 175, 294Mutation on nucleophile site:Tryptophan Arginine

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

AcidAmide-base

METHOD

The mutagenic primers were (red lettering indicates mismatch position):

E29Q5-TGG-CTC-GTT-TAT-TCA-ACA-CCT-CGG-CCG-C-3iE29Q5-GCG-GCC-GAG-GTG-TTG-AAT-AAA-CGA-GCC-A-3

E175Q5-GGT-GTC-TAG-GCA-ATC-AGA-TGG-ACG-GTC-C-3iE175Q5-GGA-CCG-TCC-ATC-TGA-TTG-CCT-AGA-CAC-C-3

E294Q5-CAT-CTG-TCG-TTT-GAC-CAA-TGG-AAC-GTA-TG-3iE294Q5-CAT-ACG-TTC-CAT-TGG-TCA-AAC-GAC-AGA-TG-3

W298R5-GAA-TGG-AAC-GTA-AGG-TAC-CAC-TCG-AAT-G-3iW298R5-CAT-TCG-AGT-GGT-ACC-TTA-CGT-TCC-ATT-C-3

PCR conditionFirst denaturation95oC30 secDenaturation 95oC30 secAnnealing55oC1 min18 cycleExtension68oC12 min

PCR reactionTemplate50x (0.5 l)Primer forward5 M (0.4 l)Primer reverse5 M (0.4 l)dNTPs10 M (1.0 l)pfu buffer10x (5.0 l)pfu polymerase1,25u (0.5 l)milliQ42 lTotal volume 50 l

Transformation of mutant to E. coliTOP10 competent cell20 l of PCR result + 0.4 l DpNI

Incubation in 37oC for 1 hour

+ 100 l TOP10 competent cellPlace 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 + ampicilinIncubation 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)

RESULTSPCR result

E29Q E175Q E294Q

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

Characterization of Abfa mutant by EcoRI

E29Q E175Q E294Q 1 2 3 1 2 3

W298R E175Q/W298R E294Q/W298R