Part:BBa_K4968002

Optimized CBM

Carbohydrate Binding Modules (CBMs) are components of several enzymes, and their primary function is to bind to specific carbohydrates. They are widely used in constructing bifunctional proteins. In the XJTLU-2023 project, we have chosen a family 2 CBM from endoglucanase A of C.fimi. This choice will contribute to enhancing the stability of cellulose nanocrystals and improving the methods for processing and utilizing cellulose.

Codon Optimization

In order to improve the expression efficiency of CBM in the target bacterial strain, we optimized its codons. Below are the DNA sequence changes in CBM before and after codon optimization:

CBM sequence before Codon-optimization:

       1 GCTCCCGGCT GCCGCGTCGA CTACGCCGTC ACCAACCAGT GGCCCGGCGG CTTCGGCGCC 
      61 AACGTCACGA TCACCAACCT CGGCGACCCC GTCTCGTCGT GGAAGCTCGA CTGGACCTAC 
     121 ACCGCAGGCC AGCGGATCCA GCAGCTGTGG AACGGCACCG CGTCGACCAA CGGCGGCCAG 
     181 GTCTCCGTCA CCAGCCTGCC CTGGAACGGC AGCATCCCGA CCGGCGGCAC GGCGTCGTTC 
     241 GGGTTCAACG GCTCGTGGGC CGGGTCCAAC CCGACGCCGG CGTCGTTCTC GCTCAACGGC 
     301 ACCACCTGCA CGGGC 
    

CBM sequence after Codon-optimization:

       1 GCACCAGGTT GTCGTGTAGA CTATGCCGTG ACAAACCAAT GGCCAGGAGG CTTCGGAGCG 
      61 AATGTGACGA TTACAAACTT GGGAGACCCG GTCTCATCCT GGAAATTAGA CTGGACATAT 
     121 ACCGCCGGGC AACGCATCCA ACAGTTATGG AATGGTACTG CCTCGACGAA CGGAGGACAA 
     181 GTCTCCGTGA CCAGTCTGCC TTGGAATGGA TCTATTCCAA CCGGCGGCAC CGCATCGTTT 
     241 GGCTTTAACG GCTCATGGGC TGGATCGAAT CCGACCCCGG CGTCCTTTTC GCTTAACGGC 
     301 ACCACATGCA CTGGG 

    

Usage & Biology

Carbohydrate Binding Modules (CBMs) are components of several enzymes that can bind to specific carbohydrates, and one of their primary functions is to be used in the construction of specific bifunctional proteins (Oliveira et al., 2015). Cellulose is an environmentally friendly material commonly used as an immobilization matrix. CBMs can recognize and selectively bind to characteristics on the crystal surface, providing possibilities for modifying cellulose.

Figure 1 |Scanning Electron Microscopy (SEM) analysis. A represents Microcrystalline cellulose that is not bound to any protein, B represents Microcrystalline cellulose bound to proteins containing CBM.

Research by Kevin Aïssa and others found that CBMs exhibit a strong affinity for crystalline cellulose. Introducing CBMs to modify the cellulose surface can provide powerful non-covalent modifications, enhancing the redispersibility of functionalized cellulose nanocrystals after drying and improving suspension stability based on spatial interactions (Aïssa et al., 2019).

In the XJTLU-2023 project, we have chosen a family 2 CBM from endoglucanase A of C.fimi, which is commonly used for enzyme immobilization.

Figure 2 |Protein concentration measured using BCA method. After the fusion protein containing CBM was fully contacted with microcrystalline cellulose for 1 hour, the content changes of the protein before and after adsorption were measured by the BCA method.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 90
  Illegal BsaI.rc site found at 83