High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae.
A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1-603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1-511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.
Tsuchiya
K
K
Research Institute of Brewing Resources Co., Ltd., Tokyo, Japan.
Nagashima
T
T
Yamamoto
Y
Y
Gomi
K
K
Kitamoto
K
K
Kumagai
C
C
Tamura
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001236:Aspergillus oryzae / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D002417:Cattle; D002478:Cells, Cultured; D012085:Chymosin / Q000235:genetics* / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014908:Triticum
D004274:DNA, Recombinant; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D005087:Glucan 1,4-alpha-Glucosidase; C032443:prorennin; D012085:Chymosin