Part:BBa_K5136026

cellulase EG5C-His tag

Biology

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 are dependent on both endo and exoglucanases, a processive endoglucanase coupled with a β-glucosidase may be sufficient for the degradation of cellulose. Cellulase EG5C-His tag is a kind of protein 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-His tag can achieve tight and fast binding of the enzyme with pulp fiber through a specific cellulose binding module, thus playing a role in hydrolyzing pulp fiber to promote deinking. In order to verify if it is capable of deinking 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).

Figure 2 SDS-PAGE analysis of cellulase EG5C-His tag protein. 

Deinking Experiments

A certain amount of purified enzyme solution was added to the pulp, and the pulp was filtered after 1 h 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 Figure 3, cellulase EG5C-His tag alone can also show a better deinking effect.

Figure 3 The gray value of paper cake deinked with cellulase. 

Reference

[1] B. Wu et al., Processivity and enzymatic mechanism of a multifunctional family 5 endoglucanase from Bacillus subtilis BS-5 with potential applications in the saccharification of cellulosic substrates. Biotechnol Biofuels 11, 20 (2018).

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