ABSTRACT
In the title compound, C13H15NO2, the acetate group [C-C(=O)-O] makes a dihedral angle of 62.35â (13)° with the mean plane of the indole ring system [maximum deviation = 0.011â (3)â Å]. In the crystal, mol-ecules are linked by N-Hâ¯O hydrogen bonds, forming helical chains propagating along [010].
ABSTRACT
An integration plasmid pMW1-pepB for the pepB gene disruption in Aspergillus was constructed. The plasmid contained the pepB gene upstream (P) 1.4kb and downstream (T) 1.3kb homologous fragments with insertion of the expression unit of the hygromycin resistance gene (hph) between them. P and T DNA fragments were synthesized by PCR from Aspergillus niger chromosomal DNA. The integration plasmid was digested with the Hpa I restriction enzyme, the resultant 4.2kb linear fragment was introduced into the Aspergillus niger strain GICC2773 which expressing the glucoamylase/laccase fusion protein by PEG-mediated transformation. 62 Hygromycin resistance transformants were screened, and from them one strain named pepB29 was identified to be the pepB disruptant by PCR analysis. Data of functional assay of the pepB29 strain indicated that the disruption of the pepB gene secreted reduced acid proteolytic activity, and improved the heterologous protein laccase production.
Subject(s)
Aspartic Acid Endopeptidases/genetics , Aspergillus niger/enzymology , Aspartic Acid Endopeptidases/physiology , Aspergillus niger/genetics , Cinnamates/pharmacology , Drug Resistance, Bacterial , Hygromycin B/analogs & derivatives , Hygromycin B/pharmacology , Laccase/biosynthesis , Plasmids , Polymerase Chain ReactionABSTRACT
Glucoamylase overproducing A. niger T21 was mutated by UV mutagensis. An extracellular acid protease-deficient mutant, A. niger T21-201, which produced only 0.76% extracellular acid protease activity of the parent strain, was screened by casein-degradating plate and determination of protease activity. Moreover, the growth properties and the ability to secrete glucoamylase of A. niger T21-201 are identical to these of starting strain T21. The comparison of expression-secretion levels of heterologous gene in A. niger T21-201 and T21 was carried out with bacterial vhb as reporter, the level of expression-secretion of VHb in A. niger T21-201 was 6-7 times higher than that in T21, but the transcriptional levels of vhb gene in both strains were similar revealed by Northern blot. Therefore, it was demonstrated that the deficiency of acid protease of recipient T21-201 has significant effect on the protection of heterologous protein.
