Expression of HpaI in Pichia pastoris and optimization of conditions for the heparinase I production.
Heparinase I has important applications in the fields of biomedicine and pharmaceuticals. The heparinase I gene (HpaI) from Flavobacterium heparinum was cloned and overexpressed in Pichia pastoris GS115, and the conditions for the heparinase I production were optimized by RSM. PCR analysis indicated that HpaI was integrated into the P. pastoris GS115 genome. The concentrations of key factors that affected the heparinase I activity were optimized, and were as follows: oleic acid, 0.07%, liquid volume in flask, 34.3 ml/L, and methanol, 0.96%. Under the optimal conditions, the activity of heparinase I was up to 323 U/L in shake flask. A maximal heparinase I activity of 398.5 U/L from the transformant 2 was achieved in a 5L fermentor. This study demonstrates the overproduction of heparinase I by recombinant P. pastoris.
College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou 310035, Zhejiang Province, People's Republic of China. Electronic address: yup9202@gmail.com.
Ping
P
Yu
College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou 310035, Zhejiang Province, People's Republic of China.
Jun
J
Yang
College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou 310035, Zhejiang Province, People's Republic of China.
Huifen
H
Gu
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019149:Bioreactors; D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media / Q000494:pharmacology; D018274:Electroporation; D005285:Fermentation; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005798:Genes, Bacterial; D016681:Genome, Fungal; D019763:Heparin Lyase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007218:Industrial Microbiology / Q000379:methods; D008954:Models, Biological; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D003470:Culture Media; D011993:Recombinant Fusion Proteins; D019763:Heparin Lyase
Fermentation; Gene expression; Heparinase I; Optimization; Pichia pastoris GS115