HIGH-LEVEL EXPRESSION β-GLUCOSIDASE GENE IN PACHIA PASTORIS
β-Glucosidase plays an important role in hydrolyzing lignocellulose to fermentable glucose in the preparation of fuel ethanol and in flavor formation in fruits. In order to further improve the gene expression level cloned in Pichia pastoris from a superior strain of Aspergillus niger, we have refined some vital factors of condition of fermentation and increased gene copy number and carry out high-density fermentation and other means to achieve a high level of expression. Inoculum OD600=10 in the shake flask with the suitable additive amount and time of methanol 0.5％, 24h and initial pH4～5 expressed the highest level. On this basis, we electroporate twice the recombinant of egll, then screen high-resistant strain from YPDS flat with 3000ug/L zeocin and its expression increased by 2.7 times of the primitive strain.SL fcrmentor was used for high-density fermentation. After 54h induction of methanol, the enzyme activity was 137.8U/ml which was 12.3 times of that in shake flask and the expression of protein reached 9.6mg/ml, at the same time, the production period was short down greatly. As a result, use multiple copies of gene and high-density fermentation methods can achieve a high-level expression of β-glucosidase which increased by 33.6 times. This study provides technical assurance for large-scale production of the enzyme.
β-glucosidase Pichia pastoris High-level production
ZHAO Lin-guo You Li-jin NI Hao LI Li-juan ZHOU Tanche YU Shi-yuan
College of Chemical Engineering,Nanjing Forestry University,Nanjing 210037,China
国际会议
广州
英文
761-767
2008-12-03（万方平台首次上网日期，不代表论文的发表时间）