Cloning and Expression of Exo-β-1,4-Cellobiohydrolase Gene From Bacillus Pumilus

Meng-jiao Yan1,a, Song Wei1,b, Ya-li Dai1,cand Lin Yuan*,1,d 1Institute of Life Sceince, Inner Mongolia University, Huhhot, China

ayanmengjiao207@126.com, b 930400232@qq.com, c1208031735@qq.com,dyuan0079@163.com

Keywords: Exo-β-1,4-cellobiohydrolase, Gene cloning, Expression, Bacillus pumilus

Abstract. The exo-β-1,4-cellobiohydrolase gene(cex) of Bacillus pumilus AC-6 was cloned. The cex

gene from AC-6 is 2100bp, and encodes 700 amino acids. The cex gene has 94% similarities to the

exo-β-1,4-cellobiohydrolase gene sequence from other Bacillus strains. The cex gene was linked with

the vector pGEX-4T-1, and transferred into the competent E. coli BL21 for expression. The result of

protein electrophoresis showed that the protein has expressed, whose molecular weight was about

104kDa. Measured the enzyme activity of the expression protein of the recombinant strains was

5.40U/mL, which is 1.20 times as the original strains.

Intruduction

Cellulose is the most abundant carbohydrate resource in the world. With the increasingly depletion of

non-renewable resources on Earth, it is a great significance to degradate the cellulose by

biotransformation. The exoglucanases is an important member of the cellulase system[1~3]. Now, the

research of cellulase is focus on the gene cloning and expression, site-directed mutagenesis, secretion

and so on[4~6]. We cloned the exo-β-1,4-cellobiohydrolase gene from Bacillus pumilus AC-6, and

expressed it in E. coli.

Materials and Methods

Strains and Plasmids. Bacillus pumilus AC-6 strain was isolated from a chemical fibre factory in

Hohhot, in China. The E.coli DH5α and BL21 were used for gene cloning and expression,

respectively. All E.coli strains were cultivated in Luria-Bertani (LB: 1.0% tryptone, 0.5% yeast

extract, and 1.0% NaCl) medium at 37°C. Ampicillin was added at a final concentration of

100µg/mL. The plasmid pMD19-T was used for gene cloning, and pGEX4T-1 was used as the

expression vector.

Genome DNA extraction.The strain AC-6 was cultured in LB medium at 37°C for 12-16 h with

shaking at 180rpm. The DNA extraction method was described by Wu Qiong[7]. The broth was

centrifuged at 10,000×g for 10 mins. Added 500µL of TE buffer (100mM Tris, 10mM EDTA) and

8µL of lysozyme into the tube with the collected cells. The mixture was incubated at 37°C for 30 mins.

Then added 2µL of proteinase K and 150µL of 10% SDS. The mixture was incubated at 50°C for 45

mins. Added 100µL of 5mol/L NaCl and 80µL of CTAB/NaCl. The mixture was incubated at 65°C

for 15 mins. Next, added 600µL of a mixture of phenol, chloroform and isoamyl alcohol (vol/vol/vol,

24:25:1), and waited for 5 mins, then centrifuged at 13,000×g for 10 mins. The supernatant was

transferred into a new tube, and added the equal volume of isoamylol. The mixture was cooled at

-20°C for 30mins and centrifuged at 12,000×g for 10 mins. Then, added 500µL of 70% ethanol to

precipitate the DNA, and the solution was centrifuged at 12,000×g for 5 mins. The DNA pellet was

washed by 200µL of cold 70% ethanol. After drying, the pellet was suspended in 30µL of 0.1×TE

buffer. The DNA was quantified by electrophoresis using 1% agarose gel.

Exo-β-1,4-cellobiohydrolase gene cloning and sequence analysis. Two primers, F1

[5’-TTGTCAAACAAAGAACGGTTTTTAACGC-3’] and R1 [5’-TTAGTTGATCA

GACGATGATATTCTGCGT-3’], were used for amplification of the cex gene. And two primers

which has the restriction sites sequence, F[5’-GAGGATCCTTGTCAAA CAAAGAACGGT-3’] and

Advanced Materials Research Vol. 647 (2013) pp 150-154Online available since 2013/Jan/11 at www.scientific.net© (2013) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AMR.647.150

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R [5’-ACGTCGACTTAGTTGATCAGACGATGA-3’], were used to add the restriction sites

sequence to the both sides of the cex gene. The underlined portions in the primer sequences represent

the recognition sites for the restriction enzymes BamHI and SalI, respectively. The PCR reaction was

performed in a 25µL volume, containing 20ng genomic DNA, 0.2mM dNTPs, 1.5mM MgCl2, 0.2µM

of each primer(Transgene,Beijing,China), and 0.5U Taq polymerase (Sangon, Shanghai, China). All

amplifications were performed with an initial denaturing at 95°C for 5 min, followed by 35 cycles of

95°C for 60 s, 54°C for 60 s and 72°C for 90 s, and a final elongation at 72°C for 5 min. Amplified

PCR products were purified by the DNA fragment purification kit (Takara, Dalian, China).The

purified PCR fragment was ligated with the vector pMD19-T, and the ligated product was

transformed into E.coli DH5α competent cells using a standard protocol. Positive transformants were

selected and confirmed by colony PCR. The final validated positive clone of pMD19-T-cex was sent

to Transgene Co. Ltd for sequence determination. The lipase gene sequence was submitted to

GenBank using the BLAST program[8].

Construction of Recombinant Expression Plasmid. Both the cloned cex gene and the plasmid

pGEX-4T-1 were digested with BamHI and SalI, the products were purified and ligated using T4

DNA ligase to form the expression vector pGEX-4T-1-cex. The recombinant expression vector was

transformed into E.coli BL21 competent cells. Positive clones were identified by colony PCR and

double restriction digestion. Detailed protocols were from Sambrook and Russell[9].

Expression of cex gene in E.coli BL21. A transformant of E.coli BL21 harboring pGEX-4T-1-cex

was cultured with vigorous shaking at 37°C overnight in 3mL LB broth containing 100µg/mL

ampicillin. 1ml of the culture was transferred into 100mL fresh LB broth containing the same

concentration of ampicillin. The culture was incubated with shaking at 37°C until the OD600 reached

0.6–0.8. IPTG (final concentration is 0.7mmol/L) was then added into the culture. The induced

protein product was analyzed by SDS-PAGE. Protein was visualized in the gel by Coomassie blue

staining.

The Optimization of Expression. The recombination strain PC211(pGEX-4T-1-cex) was

inoculated as the above. The optimal concentration of IPTG was determined between 100µg/mL and

2000µg/mL. The optimal induction time was determined between 1h and 6h after IPTG addition. The

optimal culture temperature for cellulose was determined between 28°C and 43°C. The induced

protein product was analyzed by SDS-PAGE.

Enzyme Assay.One unit of enzyme (U/mL) is the amount of 1µg of glucose produced from the

cellulose per minute at 50±0.1°C and pH 9.5. The measurement was done according to the Chinese

Industrial Standard (QB2583-2003) [10].

Results and Discussion

Cloning and Sequence Analysis of the cex Gene. A cex-encoding gene was amplified from Bacillus

pumilus AC-6 by PCR, the sequence of the cex gene was determined and submitted to GenBank

(accession no. CP000813).

The DNA fragments containing the exo-β-1,4-cellobiohydrolase gene was approximately 2100bp

(Fig.1), which encoded a protein of 700 amino acid residues and its predicted molecular weight was

about 104kDa. The BLAST results showed that the similarity between the cloned gene fragment and

the target gene sequences (Bacillus pumilus SAFR-032 exo-β-1,4-cellobiohydrolase gene) was 94%.

Fig. 1 The cex gene fragments amplified by PCR. Lane M: 250bp DNA Ladder Marker; Lane 1: the

cex gene.

Advanced Materials Research Vol. 647 151

Expression of the cex gene in E. coli BL21. The recombinant plasmid (pGEX-4T-1-cex) was

transformed into E. coli BL21. Expression of the exo-β-1,4-cellobiohydrolase gene was induced by

the addition of IPTG. Protein expression was monitored and analyzed by SDS-PAGE. SDS-PAGE

analysis revealed the presence of a new protein in the supernatant obtained after sonication treatment.

This new protein had an approximate molecular weight of 104 kDa, containing a tag-protein. The size

of the expressed protein agreed well with the predicted size of the

exo-β-1,4-cellobiohydrolase(Fig.2).

Fig. 2 The SDS-PAGE of the recombination protein in E.coli. Lane M: Protein Ruler III marker; Lane

1: empty pGEX-4T-1 vetctor; Lane 3 and 4: empty E.coli BL21(DE3); Lane 5: recombination strain

PC211. The arrow indicates the recombination protein.

The Effects of IPTG Concentration on the Expression of Target Protein. The recombinant

strains were cultured at different IPTG concentrations of 100µg/mL, 200µg/mL, 300µg/mL,

400µg/mL, 500µg/mL, 1000µg/mL, 1500µg/mL, 2000µg/mL. The broth was assayed by the method

of SDS-PAGE. As a result (Fig.3), the target protein can express to the maximum at IPTG

concentrations of 100µg/mL.

Fig. 3 The concentration of IPTG effects on the expression of target proteins. Lane 1: IPTG

concentration is 100µg/mL; lane 2: IPTG concentration is 200µg/mL; lane 3: IPTG concentration is

300µg/mL; lane 4: IPTG concentration is 400µg/mL; lane 5: IPTG concentration is 500µg/mL; lane 6:

IPTG concentration is 1000µg/mL; lane 7: IPTG concentration is 1500µg/mL; lane 8: IPTG

concentration is 2000µg/mL.

The Effects of Induce Time on the Expression of Target Protein. The recombinant strains were

cultured for different times after IPTG addition of 0h, 1h, 2h, 3h, 4h, 5h and 6h. The broth was

assayed by the method of SDS-PAGE. As a result (Fig.4), the target protein can express to the

maximum after induction for 6h.

Figure 4. Effects of induce time on the expression of target proteins. Line 1-7:induction

time:0h,1h,2h,3h,4h,5h,6h

Effect of induce time on the expression of target proteins. Lane 1: empty E.coli BL21; lane 2: induced

time is 6h; lane 3: induced time is 5h; lane 4: induced time is 4h; lane 5: induced time is 3h; lane 6:

induced time is 2h; lane 7: induced time is 1h; lane 8: induced time is 0h.

152 Biomaterial and Bioengineering

The Effects of Temperature on the Expression of Target Protein. The recombinant strains were

cultured at temperatures of 28°C, 31°C, 34°C, 37°C, 40°C and 42°C. The broth was assayed by the

method of SDS-PAGE. As a result (Fig.5), the target protein can express to the maximum at

temperature of 28°C.

Fig. 5 Effect of temperature on the expression of target proteins. Lane 1: induced temperature is 28°C;

lane 2: induced temperature is 31°C; lane 3: induced temperature is 34°C; lane 4: induced

temperature is 37°C; lane 5: induced temperature is 40°C; lane 6: induced temperature is 42°C.

Conclusion

The exo-β-1,4-cellobiohydrolase gene from Bacillus pumilus AC-6 has successfully expressed in E.

coli. Through the optimization of expression conditions, we have found that the recombinant strains

will express the target proteins to the maximum after an induced expression of 6h at 28℃. In addition,

the exo-β-1,4-cellobiohydrolase activity of recombinant strains is 1.20 times higher than that of the

original strain AC-6 by using DNS. Because of the lower enzyme activity of the original strain and

the engineering strain, it will be restricted in the industrial application. So we need to look for a better

expression system, for example, yeast expression system, in order to achieve higher expression and

activity.

Acknowledgement

This research was supported by “field rank college students training plan of the creation and

innovation fund” of Inner Mongolia university.

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