Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168

Figures & data

Table 1. Sequences of primers used in this study.

Primer	Primer sequence (5′→3′)	Size (bp)
P1	ACACGGAGGATGAGGTTC	18
P2	TTGGCTTTACAATGAGTTCG	20
P3	GGGGTACCCAGACTCTCCTTGTGAGCTCGCTTG^a	33
P4	ATTAGCGGCCGCCTGCATGGAAGCATAATCTTCCAAGATAG^a	41
pGAP	GTCCCTATTTCAATCAATTGAA^b	22
3′AOX1	GCAAATGGCATTCTGACATCC^b	21

^aUnderlined sequences in P3 and P4 represent Kpn I and Not I restriction sites, respectively.

^b Primers pGAP and 3′AOX1 were synthesized according to Invitrogen User Manual of pGAPZαA.

Figure 1. Agarose gel electrophoresis of PCR amplification product from accc30161. M: DNA Marker DL2 000 (Takara Biotechnology Co., Ltd., Dalian, China); 1: PCR amplification products.

Figure 2. Identification of the recombinant plasmid pGAPZαA-GOD by agarose gel electrophoresis and its restriction digestion. Identification (A) of the recombinant plasmid pGAPZαA-GOD: lane M, DNA Marker DL5 000 (Takara Biotechnology Co., Ltd., Dalian, China); lane 1, pGAPZαA; lane 2, pGAPZαA-GOD. Restriction digestion (B) of the recombinant plasmid pGAPZαA-GOD with EcoR I: lane M, DNA Marker DL5 000; lane 1, digestion products of the plasmid pGAPZαA-GOD by EcoR I; lane 2, pGAPZαA-GOD plasmid; lane 3, pGAPZαA plasmid.

Figure 3. Verification of accc30161 GOD gene from SMD1168-GOD by PCR amplification and identification of GOD protein in the culture supernatant of SMD1168-GOD by SDS-PAGE analysis. PCR amplification analysis (A) of accc30161 GOD gene from SMD1168-GOD: M, DNA Marker DL2 000; 1, SMD1168-GOD; 2, SMD1168. SDS-PAGE analysis (B) of proteins in the culture supernatant of SMD1168-GOD: M, protein size marker (Fermentas, Burlington, Ontario, Canada); 1, SMD1168; 2, SMD1168-GOD.

Figure 4. Effect of temperature (A) and medium pH (B) on the expression of the GOD gene in SMD1168-GOD strain.

Figure 5. SDS-PAGE analysis of purified recombinant GOD protein expressed by SMD1168-GOD strain. M: Protein size marker (Fermentas, Burlington, Ontario, Canada); 1: purified recombinant GOD protein.

Figure 6. Thermal stability (A) and pH stability (B) of recombinant and A. niger GOD protein under the same conditions.