Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter

Noseda, Diego Gabriel; Recúpero, Matías Nicolás; Blasco, Martín; Ortiz, Gastón Ezequiel; Galvagno, Miguel A.

doi:10.1016/j.pep.2013.08.018

Cited by 34 publications

(33 citation statements)

References 38 publications

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“…The chymosin production values obtained in Erlenmeyer flasks here were in accordance to the productions reported by Noseda et al. () at this scale in different growth conditions.…”

Section: Resultssupporting

confidence: 92%

“…As chymosin induction occured optimally at pH 4.0 (Noseda et al. ), pH adjustment would not be necessary to induce enzyme production.…”

Section: Resultsmentioning

confidence: 99%

“…Growth at different pH values in the presence of GRE was analyzed to find the optimal range for biomass production of the recombinant K. pastoris strain. On the other hand, as chymosin production by this strain is strongly dependent on the pH of the induction medium (Noseda et al 2013), the selection of an appropriate value for biomass production has to be taken into account in order to maintain a constant pH along the chymosin production bioprocess. As Figure 1C shows, the optimal pH values were found to be between pH 4.0 and pH 5.0 units, meanwhile the extreme pH values tested (3.0 and 6.0 units) resulted in lower biomass productions.…”

Section: Effect Of Ph On Yeast Growthmentioning

confidence: 99%

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Use of grape pomaces to produce biomass of a Komagataella pastoris strain expressing a bovine chymosin activity

Kingston

Novelli

Cerrutti

et al. 2014

Food Science & Nutrition

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The use of agroindustrial wastes not only decreases bioprocesses and disposal costs but also contributes to the upgrading of the residues. An active recombinant methanol-inducible bovine chymosin has been expressed in our laboratory in the yeastKomagataella pastoris, and grape pomace extracts (GRE) were proposed as a convenient C-energy source for the biomass production of the genetically engineered strain. Carbon and nitrogen sources, growth factors, and initial pH conditions were selected by classical methodology; thereafter, growth conditions optimization was performed using statistical designed experiments (DoEs). In the presence of (in g·L−1) 67.0 monosaccharides (glucose and fructose) from GRE, 5.0 (NH4)2SO4, and 10.0 sugar cane molasses (CMz), a yield of 20.0 g·L−1 cell dry weight (CDW) was obtained aerobically after 60 h incubation at 28°C and pH 4.0. Applying a fed-batch strategy with methanol:sorbitol as the enzyme inducers, a chymosin production of 8.53 International Milk Clotting Units (IMCU) per mg protein was obtained in the supernatant. The results presented show that through a statistical design, a simple, cheap, and easy to prepare culture medium could be developed using two agroindustrial derivatives (GRE and CMz) to obtain a higher value added product.

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“…The chymosin production values obtained in Erlenmeyer flasks here were in accordance to the productions reported by Noseda et al. () at this scale in different growth conditions.…”

Section: Resultssupporting

confidence: 92%

“…As chymosin induction occured optimally at pH 4.0 (Noseda et al. ), pH adjustment would not be necessary to induce enzyme production.…”

Section: Resultsmentioning

confidence: 99%

Section: Effect Of Ph On Yeast Growthmentioning

confidence: 99%

See 1 more Smart Citation

Use of grape pomaces to produce biomass of a Komagataella pastoris strain expressing a bovine chymosin activity

Kingston

Novelli

Cerrutti

et al. 2014

Food Science & Nutrition

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show abstract

“…This value was in the range of molecular weights normally determined for other aspartyl proteinases and for recombinant bovine chymosin as well (Noseda et al . ). Fox et al .…”

Section: Resultsmentioning

confidence: 97%

Purification and characterisation of milk‐clotting and caseinolytic activities of pepsin isolated from adult sheep abomasa

Slamani

Labadi

Errahmani

et al. 2018

Int J of Dairy Tech

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Ovine pepsin was isolated and assessed for its milk‐clotting properties and caseinolytic activity in comparison with commercial chymosin. Ovine pepsin showed similar responses to variations in pH, temperature and CaCl2 concentration of milk compared with chymosin, although its pH sensitivity was higher. SDS‐PAGE electrophoretic analysis of the casein fractions treated with ovine pepsin showed that alpha‐casein was more susceptible to proteolysis than beta‐casein, in contrast to chymosin. Curd‐firming properties of skim milk gels obtained with ovine pepsin and chymosin were evaluated by Gelograph under the same conditions. Curd produced using ovine pepsin was less firm than that made with chymosin.

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“…Pichia pastors has been successfully used to produce chymosin from different sources using different promoters and expression systems. [25][26][27] Considering these results, new expression system tools are being developed to improve the secretion of chymosin by the L. lactis NCDO2118 strain.…”

mentioning

confidence: 99%

RecombinantLactococcus lactisfails to secrete bovine chymosine

et al. 2014

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Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.

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Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter

Cited by 34 publications

References 38 publications

Use of grape pomaces to produce biomass of a Komagataella pastoris strain expressing a bovine chymosin activity

Use of grape pomaces to produce biomass of a Komagataella pastoris strain expressing a bovine chymosin activity

Purification and characterisation of milk‐clotting and caseinolytic activities of pepsin isolated from adult sheep abomasa

RecombinantLactococcus lactisfails to secrete bovine chymosine

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