Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris
Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively exp...
Main Authors: | , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Elsevier
2021-05-01
|
Series: | Heliyon |
Subjects: | |
Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844021012408 |
id |
doaj-083ad6865982468b86dbc27cfc8ba583 |
---|---|
record_format |
Article |
spelling |
doaj-083ad6865982468b86dbc27cfc8ba5832021-06-03T14:45:44ZengElsevierHeliyon2405-84402021-05-0175e07137Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastorisZhiger Akishev0Assel Kiribayeva1Arman Mussakhmetov2Kairat Baltin3Yerlan Ramankulov4Bekbolat Khassenov5National Center for Biotechnology, 13/5 Kurgalzhynskoye Road, Nur-Sultan, 010000, Kazakhstan; L.N.Gumilyov Eurasian National University, 2 Kanysh Satpayev Street, Nur-Sultan, 010008, KazakhstanNational Center for Biotechnology, 13/5 Kurgalzhynskoye Road, Nur-Sultan, 010000, Kazakhstan; L.N.Gumilyov Eurasian National University, 2 Kanysh Satpayev Street, Nur-Sultan, 010008, KazakhstanNational Center for Biotechnology, 13/5 Kurgalzhynskoye Road, Nur-Sultan, 010000, KazakhstanNational Center for Biotechnology, 13/5 Kurgalzhynskoye Road, Nur-Sultan, 010000, KazakhstanNational Center for Biotechnology, 13/5 Kurgalzhynskoye Road, Nur-Sultan, 010000, KazakhstanNational Center for Biotechnology, 13/5 Kurgalzhynskoye Road, Nur-Sultan, 010000, Kazakhstan; Corresponding author.Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in Pichia pastoris cells under glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. It was purified by sequential anion and cation exchange chromatography. SDS-PAGE analysis resulted in two bands, approximately 42 and 35 kDa. The 42 kDa band vanished when the sample was treated with endoglycosidase H, indicating that the recombinant protein is partially glycosylated. Optimal pH for the activity of the highest-purity recombinant chymosin was pH 4.5 for cow's milk and pH 4.0 for mare's milk. The range 45–50 °C and 70 °C for cow's and mare's milk types, respectively, was found to be the most appropriate for maximal relative milk-clotting activity. Concentration of CaCl2 that ensured the stability of the chymosin milk-clotting activity was between 20 and 50 mM with an optimum at 30 mM. Milk-clotting activity of camel recombinant chymosin and ability to make curd was successfully tested on fresh mare's milk. Pichia pastoris strain with integrated camel chymosin gene showed high productivity of submerged fermentation in bioreactor with milk-clotting activity 1412 U/mL and 80 mg/L enzyme yield. These results suggest that the constitutive expression of the camel chymosin Camelus bactrianus in the yeast Pichia pastoris has good prospects for practical applications.http://www.sciencedirect.com/science/article/pii/S2405844021012408Recombinant DNAProtein purificationCamel chymosinConstitutive expressionMilk coagulationGlycosylation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Zhiger Akishev Assel Kiribayeva Arman Mussakhmetov Kairat Baltin Yerlan Ramankulov Bekbolat Khassenov |
spellingShingle |
Zhiger Akishev Assel Kiribayeva Arman Mussakhmetov Kairat Baltin Yerlan Ramankulov Bekbolat Khassenov Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris Heliyon Recombinant DNA Protein purification Camel chymosin Constitutive expression Milk coagulation Glycosylation |
author_facet |
Zhiger Akishev Assel Kiribayeva Arman Mussakhmetov Kairat Baltin Yerlan Ramankulov Bekbolat Khassenov |
author_sort |
Zhiger Akishev |
title |
Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris |
title_short |
Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris |
title_full |
Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris |
title_fullStr |
Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris |
title_full_unstemmed |
Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris |
title_sort |
constitutive expression of camelus bactrianus prochymosin b in pichia pastoris |
publisher |
Elsevier |
series |
Heliyon |
issn |
2405-8440 |
publishDate |
2021-05-01 |
description |
Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in Pichia pastoris cells under glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. It was purified by sequential anion and cation exchange chromatography. SDS-PAGE analysis resulted in two bands, approximately 42 and 35 kDa. The 42 kDa band vanished when the sample was treated with endoglycosidase H, indicating that the recombinant protein is partially glycosylated. Optimal pH for the activity of the highest-purity recombinant chymosin was pH 4.5 for cow's milk and pH 4.0 for mare's milk. The range 45–50 °C and 70 °C for cow's and mare's milk types, respectively, was found to be the most appropriate for maximal relative milk-clotting activity. Concentration of CaCl2 that ensured the stability of the chymosin milk-clotting activity was between 20 and 50 mM with an optimum at 30 mM. Milk-clotting activity of camel recombinant chymosin and ability to make curd was successfully tested on fresh mare's milk. Pichia pastoris strain with integrated camel chymosin gene showed high productivity of submerged fermentation in bioreactor with milk-clotting activity 1412 U/mL and 80 mg/L enzyme yield. These results suggest that the constitutive expression of the camel chymosin Camelus bactrianus in the yeast Pichia pastoris has good prospects for practical applications. |
topic |
Recombinant DNA Protein purification Camel chymosin Constitutive expression Milk coagulation Glycosylation |
url |
http://www.sciencedirect.com/science/article/pii/S2405844021012408 |
work_keys_str_mv |
AT zhigerakishev constitutiveexpressionofcamelusbactrianusprochymosinbinpichiapastoris AT asselkiribayeva constitutiveexpressionofcamelusbactrianusprochymosinbinpichiapastoris AT armanmussakhmetov constitutiveexpressionofcamelusbactrianusprochymosinbinpichiapastoris AT kairatbaltin constitutiveexpressionofcamelusbactrianusprochymosinbinpichiapastoris AT yerlanramankulov constitutiveexpressionofcamelusbactrianusprochymosinbinpichiapastoris AT bekbolatkhassenov constitutiveexpressionofcamelusbactrianusprochymosinbinpichiapastoris |
_version_ |
1721399087333376000 |