Gene Cloning and Recombinant Expression of Chondroitinase AC from Pedobacter heparinus and Characterization of Recombinant Fusion Enzyme

doi:10.7506/spkx1002-6630-201309027

Abstract

Abstract:

Chondroitinase AC (ChonAC) is an important enzyme to reveal the biological function, structure and mechanism
of chondroitin sulfate, and it is important for low molecular weight chondroitin sulfate preparation and diseases treatment.
To achieve the efficient expression of ChonAC with high activity in recombinant Escherichia coli (E. coli), the ChonAC
gene (cslA) from Pedobacter heparinus was amplified by polymerase chain reaction (PCR), and the fusion expression vector
for expressing maltose-binding protein (MBP) fused ChonAC (MBP-ChonAC) was constructed. The results showed that the
fusion strategy using MBP was effective to enhance the solubility of the MBP-ChonAC when induced at low temperature of
15 ℃, the purity and the specific activity of MBP-ChonAC could reach 95% and 94.1 IU/mg-fusion protein (equivalent to
143.8 IU/mg ChonAC) by one-step MBPTrap HP purification, respectively. The enzyme characteristic study showed that the
optimum pH value, Ca2+concentration, NaCl concentration, and reaction temperature was 7.5—8.0 , 20 mmol/L, 50 mmol/L,
and 20—35 ℃, respectively. The half-life of MBP-ChonAC could reach 8.3 h at 30 ℃. The kinetic characterization showed
that the Km was higher while the kcat was a little bit lower than the reported native ChonAC for chondroitin sulfate A, while
the biocatalysis experiment for MBP-ChonAC indicated that the fusion of ChonAC with MBP did not affect the enzyme
function. Furthermore, the total activity of MBP-ChonAC by shake flask cultivation could reach 10800.5 IU/L, the highest
value reported so far, through the optimizations of host cells, IPTG induction concentration, and M9-based culture medium.

Key words: affinity purification, chondroitinase AC (ChonAC), enzymatic characteristics, expression level optimization, maltose-binding protein (MBP), recombinant expression

CLC Number:

Q814.1

WU Jing-jun1，LI Ye1,2，ZHANG Chong1，LI Mei1， XING Xin-hui1,*. Gene Cloning and Recombinant Expression of Chondroitinase AC from Pedobacter heparinus and Characterization of Recombinant Fusion Enzyme[J]. FOOD SCIENCE, 2013, 34(9): 127-134.

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