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. 2013 Oct 24;3:3043. doi: 10.1038/srep03043

Figure 2.

Figure 2

(a) Identification of the recombinant strain by PCR. Lane M: DNA marker DL2000. Lanes 1 and 3: PCR products obtained using primers 5′AOX1 and R1. Lanes 2 and 4: PCR products obtained using primers F1 and 3′AOX1. Lane 5: PCR result obtained with primers F1 and R1 using genomic DNA from the control strain as a template. (b) SDS-PAGE analysis of endochitinase expression. Lane M: Protein molecular weight standard. Lane 1: supernatant from the control strain. Lane 2: supernatant from the recombinant strain GS115 transformed with pPIC9K-ECH. Lane 3: supernatant from the recombinant strain GS115 transformed with pPIC9K-SECH. Plasmids pPIC9K-ECH and pPIC9K-SECH harbour the wild-type and codon-optimised cDNAs of endochitinase, respectively. (c) Endochitinase activities of the original and codon-optimised strains. (d) Degradable products of colloidal chitin. Lane M, the control sample without enzyme for 5 h; Lane S, standard samples (G1:G2:G3:G4 = 4:2:2:2 mM); 0, 1, 3 and 5 represent reaction times. (e) Degradable products of chito-oligomers. Lane M, standard samples (G1:G2:G3:G4:G5:G6 = 4:2:2:2:1:1 mM); Lane C, control reactions without enzyme; Lane S, endochitinase-degradable products of each chito-oligomer; G1–G6 represent (GlcNAc)1–6, respectively. (f) Schematic map of the assembly process of the codon-optimised endochitinase cDNAs by successive PCR.