Prochymosin and chymosin (prorennin and rennin)

Foltmann, Bent

doi:10.1042/bj1150003p

Cited by 35 publications

(45 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In order to investigate whether the production levels of heterologous proteins were improved in the Aovps10 gene disruptant NSlDv10, we constructed strains expressing CHY, as a model protein. The initial translation product of CHY, prochymosin, contains a 42-amino-acid Nterminal sequence which is automatically cleaved at low pH to yield active chymosin (7,8). A. oryzae ␣-amylase (AmyB) was used as the carrier protein for fusion with CHY since such fusions have been shown to aid the successful production of heterologous proteins (15,16,31).…”

Section: Resultsmentioning

confidence: 99%

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Yoon

Aishan

Maruyama

et al. 2010

Appl Environ Microbiol

View full text Add to dashboard Cite

Filamentous fungi have received attention as hosts for heterologous protein production because of their high secretion capability and eukaryotic posttranslational modifications. However, despite these positive attributes, a bottleneck in posttranscriptional processing limits protein yields. The vacuolar protein sorting gene VPS10 encodes a sorting receptor for the recognition and delivery of several yeast vacuolar proteins. Although it can also target recombinant and aberrant proteins for vacuolar degradation, there is limited knowledge of the effect of its disruption on heterologous protein production. In this study, cDNA encoding AoVps10 from the filamentous fungus Aspergillus oryzae was cloned and sequenced. Microscopic observation of the transformant expressing AoVps10 fused with enhanced green fluorescent protein showed that the fusion protein localized at the Golgi and prevacuolar compartments. Moreover, disruption of the Aovps10 gene resulted in missorting and secretion of vacuolar carboxypeptidase AoCpyA into the medium, indicating that AoVps10 is required for sorting of vacuolar proteins to vacuoles. To investigate the extracellular production levels of heterologous proteins, ⌬Aovps10 mutants expressing either bovine chymosin (CHY) or human lysozyme (HLY) were constructed. Interestingly, the ⌬Aovps10 mutation increased the maximum extracellular production levels of CHY and HLY by 3-and 2.2-fold, respectively. Western blot analysis of extracellular heterologous proteins also demonstrated an improvement in productivity. These results suggest that AoVps10 plays a role in the regulation of heterologous protein secretion in A. oryzae and may be involved in the vacuolar protein degradation through the Golgi apparatus.

show abstract

Section: Resultsmentioning

confidence: 99%

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Yoon

Aishan

Maruyama

et al. 2010

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…More recently the inherent catalytic activity of bovine procarboxypeptidase A S5 has been probed with substrates and active site-directed reagents (23,24). The zymogens of rennin (25) and pepsin (26) exhibit a propensity for self-activation by limited proteolysis. It is apparent, therefore, that zymogens are neither inactive nor inert, but rather constitute a chemically and conformationally defined class of "biologically modified" enzymes.…”

Section: Methodsmentioning

confidence: 99%

Inactivation of Bovine Trypsinogen and Chymotrypsinogen by Diisopropylphosphorofluoridate

Morgan

Robinson

Walsh

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Diisopropylphosphorofluoridate reacts with trypsinogen and chymotrypsinogen and inhibits the potential activity of both zymogens. The reactions follow pseudo first-order kinetics and proceed approximately four orders of magnitude slower than diisopropylphosphorylation of the corresponding enzymes. Correlation of initial rates of inactivation with incorporation of the reagent indicates that zymogen inactivation results from incorporation of 1 mol of organic phosphate per mol of protein. Peptides isolated from the active-site region of trypsinogen account for more than 60% of the label originally present in the ['4Cjdiisopropylphosphoryl zymogen. It is concluded that loss of activation of trypsinogen is due to alkylphosphorylation of Ser183. It is proposed that reduced reactivity of the zymogen, as compared to the enzyme, primarily reflects inefficient binding of substrates and inhibitors, and that Ser183 of the active site exists in trypsinogen in an activated state.Diisopropylphosphorofluoridate (DFP) inhibits serine proteases by alkylphosphorylation of a specific serine residue of the active site (1). In contrast, and by definition, the parent zymogens are believed to be unreactive toward this and other active-site reagents (2-4). Recently, Robinson (5) reported that carbamylation of the a-amino group of E-guanidinated trypsin destroys esterase and amidase activity without abolishing reactivity toward DFP. Control experiments with native and guanidinated trypsinogen revealed unexpected reactivity of the zymogens toward DFP. It was concluded, therefore, that in trypsinogen (Robinson, N. C., Neurath, H. & Walsh, K. A., submitted for publication), as in trypsin (6), a serine residue of the actives site had been alkylphosphorylated.The present investigation was undertaken to measure the rate of zymogen inactivation, to confirm the site of interaction between DFP and trypsinogen, and to extend the applicability of these findings to chymotrypsinogen. 4-fold with 1.0 M sodium formate (pH 3.0) in order to quench the reaction with DFP. Excess DFP was removed from each aliquot by gel filtration on 1.5 X 6.0-cm columns of Sephadex G-25 equilibrated with 0.1 M sodium formate (pH 3.5). Protein concentration was determined by measuring the absorbance of each aliquot at 280 nm.Suitable portions of the desalted aliquots were removed, assayed for activatability, and counted on a scintillation counter. Trypsinogen (0.5 rng) in 1.0 ml of 0.1 M sodium formate (pH 3.5) was activated with 50 units of the acid protease from Aspergillus oryzae as described by Robinson (5).After 1 hr. the activation mixture was assayed for tryptic activity toward N-a-benzoyl-L-arginine ethyl ester as described by Walsh and Wilcox (7). "Activatability" was expressed in units of tryptic activity per mg of trypsinogen.Chymotrypsinogen (0.1 mg) was activated with 0.27 mg of trypsin after dilution to 2.0 ml with 1.0 M Tris HC1 (pH The oxidized protein (150 mg) was incubated with trypsin (3 mg) at 40°while the pH was maintained at 8.0. After base u...

show abstract

“…Approximately 2 10 5 conidia of the CHY-expressing transformants were inoculated in 20 ml 5 DPY (pH 5.5) medium and then cultured at 30 C for 3 to 6 days. The CHY activity was measured by a modifi cation of the method described by Foltmann (1970). The culture medium (100 μl) obtained every 24 h was added to 1 ml of 12% skim milk solution containing 10 mM CaCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Improved heterologous protein production by a tripeptidyl peptidase gene (AosedD) disruptant of the filamentous fungus Aspergillus oryzae

Zhu

Nemoto

Yoon

et al. 2012

J. Gen. Appl. Microbiol.

View full text Add to dashboard Cite

Prochymosin and chymosin (prorennin and rennin)

Cited by 35 publications

References 5 publications

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Inactivation of Bovine Trypsinogen and Chymotrypsinogen by Diisopropylphosphorofluoridate

Improved heterologous protein production by a tripeptidyl peptidase gene (AosedD) disruptant of the filamentous fungus Aspergillus oryzae

Contact Info

Product

Resources

About