µ¦Ã ¨ ·É ¨³ µ¦Â °° ° ¥¸o Á µ µÂ¨ à »¨·¸¥r»¼¤·¼ ... · ´n ¥°£µ¬µÅ ¥hhhhhhhhhhhhhhhhhhhhhhhhhhhh ´n ¥°£µ¬µ°´ §¬hhhhhhhhhhhhhhhhhhhhhhhhhhhh

.

.

50401204 : MAJOR : BIOTECHNOLOGY KEY WORDS : CLONING/EXPRESSION/LACTOBACILLUS/RECOMBINANT BETA- GALACTOSIDASE

WITSANU SRILA : CLONING AND EXPRESSION OF BETA-GALACTOSIDASE GENE FROM THERMOPHILIC MICROORGANISM IN ESCHERICHIA COLI. THESIS ADVISORS : ASST.PROF.PHIMCHANOK JATURAPIREE, Dr.nat.tech., ASST.PROF.BUDSARAPRON NGAMPANYA, Ph.D., AND ASST.PROF.PITTAYA LIEWSAREE, Ph.D., 116 pp.

Thermophilic bacteria have been of interests for the commercial production of -galactosidase. However, most native thermostable enzyme is synthesized at very low levels

by the thermophilic bacteria. So, the objective of this thesis is to enhance the production of -galactosidase by cloning and expression in Escherichia coli. -Galactosidase gene (lacZ)

was cloned from Lactobacillus delbruckii subsp. bulgaricus TISTR 892 and expressed in E. coli TOP10F' (designated pHFgal17). Recombinant enzyme was purified one step by using Ni Sepharose 6 Fast Flow. The purified enzyme is a homodimer composed of two identical subunits of 115 kDa each. The optimum pH and temperature were 7.5 and 55 °C, respectively. Metal ions such as Mg2+, Mn2+ and Co2+ increased enzymatic activity of about 3-fold in comparison with that in the absence of metal ions, whereas Pb2+, Cu2+ and Ag2+

showed a complete inactivation. Enzyme was moderately inhibited by glucose, galactose and lactose (100 mM). Kinetic studies revealed a Km and Kcat of 1.2 mM and 252,000 S-1, respectively, using oNPG as substrate. In addition, this enzyme was also assayed for galactooligosaccharides (GOS) production. The enzyme produced GOS formation reached approximately 34.39 from 53.21 lactose hydrolysis at 2 h, When 250 gL-1 initial lactose concentration. It is a potential use for lactose hydrolysis in milk, whey and the production of GOS.

Department of Biotechnology Graduate School, Silpakorn University Academic Year 2010 Student's signature ........................................ Thesis Advisors' signature 1. ....................... 2. ....................... 3. .......................

..

.

.

Gal-OR E+Gal-OR ROH

E+Lac E-Gal+

Gal H2O

E-Gal-Glc

Glc

-

®

1 50 pGal14 T7 (1) ATGAGCAATAAGTTAGTAAAAGAAAAAAGAGTTGACCAGGCAGACCTGGCL.bulgaricus ATCC11842 (1) ATGAGCAATAAGTTAGTAAAAGAAAAAAGAGTTGACCAGGCAGACCTGGCConsensus (1) ATGAGCAATAAGTTAGTAAAAGAAAAAAGAGTTGACCAGGCAGACCTGGC 51 100 pGal14 T7 (51) CTGGCTGACTGACCCGGAAGTTTACGAAGTCAATACAATTCCCCCGCACTL.bulgaricus ATCC11842 (51) CTGGCTGACTGACCCGGAAGTTTACGAAGTCAATACAATTCCCCCGCACTConsensus (51) CTGGCTGACTGACCCGGAAGTTTACGAAGTCAATACAATTCCCCCGCACT 101 113 pGal14 T7 (101) CCGACCATGAGTCL.bulgaricus ATCC11842 (101) CCGACCATGAGTCConsensus (101) CCGACCATGAGTC

2973 3022 pGal14 SP6 (779) CTGGGGGCAGAAGGTCCACCCGGAATTCTGCCTGGATGCTCAAAAAGCCC L.bulgaricus ATCC11842 (2937) CTGGGGGCAGAAGGTCCACCCGGAATTCTGCCTGGATGCTCAAAAAGCCC Consensus (2973) CTGGGGGCAGAAGGTCCACCCGGAATTCTGCCTGGATGCTCAAAAAGCCC 3023 3063 pGal14 SP6 (829) GCCAGCTCCGCCTGGTGATTCAGCCCCTTTTACTAAAATAAL.bulgaricus ATCC11842 (2987) GCCAGCTCCGCCTGGTGATTCAGCCCCTTTTACTAAAATAAConsensus (3023) GCCAGCTCCGCCTGGTGATTCAGCCCCTTTTACTAAAATAA

0

1000

2000

3000

4000

5000

6000

Cytoplasm Periplasm Culture broth Empty vector

Comparment

Enzy

me

activ

ity(U

mL-1

)

á á

á á

á á

Lineweaver-Burk

1.0 -.8 -.6 -.4 -.2 0.0 .2 .4 .6

.01

.02

.03

Lineweaver-Burk

.4 -.2 0.0 .2 .4 .6 .8

.02

.04

.06

.08

%galactose%GOS%lactose%glucose

% lactose% glucose% galactose% GOS

% glucose% galactose% GOS% lactose

ł

â

of -galactosidase from Bacteroides polypragmatus.

Ö

ł

ç

&

&

from

®

Bgl2 Hind3 Kpn1 | Fsp1 | Xho1 EcoR1 BamH1 Acc65 | | Sal1 | | | | | | | | | ACCGTTGCGCAAGCTTCGCTCGAGGAATTCGGATCCGGTACCAGATCTGTCGAC

1 ---------+---------+---------+---------+---------+---- TGGCAACGCGTTCGAAGCGAGCTCCTTAAGCCTAGGCCATGGTCTAGACAGCTG

from OmpA > T V A Q A S L E E F G S G T R S V D

SpAcc | CACCATCACCATCACCATCACCATCATCACCTCGACTACAAGGACGATGACGACAAGTGA -----+---------+---------+---------+---------+---------+---- 114 GTGGTAGTGGTAGTGGTAGTGGTAGTAGTGGAGCTGATGTTCCTGCTACTGCTGTTCACT H H H H H H H H H H L D Y K D D D D K * FLAG >

®

®

®

factordilutionOD 50260

280

260

ODOD

®

®

®

insertofngratiomolarvectorinsertvectorofsizekb

insertofsizekbvectorofng :

420 480 20 750 500 15.7825 20 500 minOD dilution factor

factordilutionOD6558.0

595