Part:BBa_K5136026

cellulase EG5C-His tag

Biology

Cellulase
Efficient hydrolysis of cellulose to glucose depends on the synchronized action of three classes of enzymes, endoglucanase, exoglucanase and β-glucosidase. Processivity is a common mode of action for many exoglucanases and plays a key role in the complete hydrolysis of crystalline cellulose. In contrast, classic endoglucanases randomly cleave β-1,4-glycosidic bonds in the interior of the cellulosic chains and have been considered to be non-processive. Studies have shown that in contrast to other cellulolytic systems that dependent on both endo and exoglucanases, a processive endoglucanase coupled with a β-glucosidase may be sufficient for the degradation of cellulose. Cellulase EG5C is a kind of proteins found from Bacillus subtilis BS-5 (1). It is hypothesized that B. subtilis BS-5 might produce a processive endoglucanase, which might substitute for the apparent deficiency in exoglucanase activity.

Usage and Design
Cellulase EG5C can achieve tight and fast binding of enzyme with pulp fiber through specific cellulose binding module, thus play a role in hydrolyzing pulp fiber to promote deinking. In order to verify if it is capable to deink waste paper, a His-tag (6×his) was added to the C-terminal of cellulase for purification. We constructed this part and assembled it on the expression vector pET-28a(+).

Characterization
Agarose gel electrophoresis (AGE) The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. Target bands (1518 bp) can be observed at the position between 1500 bp and 2000 bp. (Figure 1)

Figure 1 DNA gel electrophoresis of the colony PCR products of BBa_K5136026_pET-28a(+) in E. coli BL21(DE3). 

SDS-PAGE
The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 20 °C GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Figure 2), the target protein (56.1 kDa) can be observed at the position around 52 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.

Figure 2 SDS-PAGE analysis of cellulase EG5C protein. 

Pulp deinking
A certain amount of purified enzyme solution was added to the pulp, and the pulp was filtered after 1h reaction, and the gray value of the dried paper was measured to characterize the deinking effect of the enzyme. The higher the gray value, the better the deinking effect. As shown in the figure 3, cellulase EG5C alone can also show a better deinking effect.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 958
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 610
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 620