The Biochemistry of Prorennin (Prochymosin) and Rennin (Chymosin)

Foltmann, Bent

doi:10.1016/b978-0-12-485202-0.50011-7

Cited by 20 publications

(9 citation statements)

References 70 publications

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“…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

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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.

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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

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“…Chymosin, an aspartyl protease produced in the abomasum of young ruminants, is the preferred milk clotting protease for cheese manufacture (Foltmann, 1971). The isoelectric point is 4.6 and chymosin stability is greatest at about pH 2 and between pH 5 and 6.5.…”

Section: Introductionmentioning

confidence: 99%

“…Chymosin catalyses specific cleavage of the PHE 105 -MET 106 bond of -casein, destabi-lizing the casein micelle and causing precipitation. The initial translation product, prochymosin, contains a 42 amino acid N terminal sequence which is cleaved to yield active chymosin (Foltmann, 1971). Both recombinant prochymosin (Emtage et al, 1983;Goff et al, 1984) and chymosin (Dunn-Colemann et al, 1991;Harkki et al, 1989) have been produced.…”

Section: Introductionmentioning

confidence: 99%

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One step purification of chymosin by mixed mode chromatography

Burton¹,

Haggarty²,

Harding³

1997

Biotechnol. Bioeng.

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Mixed mode Sepharose and Perloza bead cellulose matrices were prepared using various chemistries. These matrices contained hydrophobic (aliphatic and/or aromatic) and ionic (carboxylate or alkylamine) groups. Hydrophobic amine ligands were attached to epichlorohydrin activated Sepharose (mixed mode amine matrices). Hexylamine, aminophenylpropanediol and phenylethylamine were the preferred ligands, on the basis of cost and performance. Other mixed mode matrices were produced by incomplete attachment (0-80%) of the same amine ligands to carboxylate matrices. The best results were obtained using unmodified or partially ligandmodified aminocaproic acid Sepharose and Perloza. High ligand densities were used, resulting in high capacity. Furthermore, chymosin was adsorbed at high and low ionic strengths, which reduced sample preparation requirements. Chymosin, essentially homogeneous by electrophoresis, was recovered by a small pH change. The methods described were simple, efficient, inexpensive and provided very good resolution of chymosin from a crude recombinant source. The carboxylate matrices had the best combination of capacity and regeneration properties. The performance of Sepharose and Perloza carboxylate matrices was similar, but higher capacities were found for the latter. Because it is cheaper and can be used at higher flow rates, Perloza should be better suited to large scale application. High capacity chymosin adsorption was found with carboxymethyl ion exchange matrices, but low ionic strength was essential for adsorption and the purity was inferior to that of the mixed mode matrices.

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“…The sequence of the remaining part of the molecule was established after a second tryptic digestion of the demaleylated peptide, from which the two basic peptides Tm-2, t-2 and Tm-2, t-3 were isolated. An overlapping sequence of the peptides Tm-1 and Tm-2 has eventually been provided by a peptide (AP-Cl) which has been isolated from a chymotryptic digest of activation peptides liberated during the conversion of prochymosin into chymosin [9].…”

Section: Methodsmentioning

confidence: 99%