Effect of Pichia pastoris host strain on the properties of recombinant Aspergillus niger endoglucanase, EglB

Shazilah Kamaruddin, Nor Muhammad Mahadi, Rosli Md Illias, Osman Hassan, Suhaila Sulaiman, William Broughton, Izwan Bharudin, Farah Diba Abu Bakar, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

Abstract

Aims: The methylotrophic yeast Pichia pastoris is widely used to express foreign proteins fused to secretion signals. As the effect of the expression host on the final protein product is unclear, we compared the properties of an endoglucanase (eglB of Aspergillus niger) expressed in two different P. pastoris strains. Methodology and results: Full-length cDNA encoding endoglucanase of A. niger strain ATCC10574 was isolated and expressed in P. pastoris X33 (the methanol utilisation plus phenotype, Mut + ) and P. pastoris GS115 (slow methanol utilisation, Mut S ). EglB-GS115 showed the highest activity and stability at 60 °C while EglB-X33 was most active at 50 °C. EglB-X33 was active towards other substrates such as arabinogalactan, guar gum and locust bean gum besides its specific substrate, carboxymethyl cellulose (CMC). However, EglB-GS115 was only active on CMC. The affinity of EglBX33 towards CMC (K m = 7.5 mg/mL and specific activity 658 U/mg) was higher than that of EglB-GS115 (K m = 11.57 mg/mL, specific activity 144 U/mg). Conclusion, significance and impact of study: Although eglB was cloned in the same expression vector (pPICZαC), two different characteristics of enzymes were recovered from the supernatant of the different hosts. Thus, expression of recombinant enzyme in different P. pastoris strains greatly affects the physical structure and biochemical properties of the enzyme.

Original languageEnglish
Pages (from-to)554-562
Number of pages9
JournalMalaysian Journal of Microbiology
Volume14
Issue numberSpecialissue6
DOIs
Publication statusPublished - 1 Jan 2018

Fingerprint

Carboxymethylcellulose Sodium
Pichia
Aspergillus niger
Cellulase
guar gum
Methanol
Enzymes
Proteins
Complementary DNA
Yeasts
Phenotype

Keywords

  • Cellulase
  • Endoglucanase
  • Glycosylation
  • Methanol utilisation phenotype
  • Recombinant enzyme

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases

Cite this

Effect of Pichia pastoris host strain on the properties of recombinant Aspergillus niger endoglucanase, EglB. / Kamaruddin, Shazilah; Mahadi, Nor Muhammad; Md Illias, Rosli; Hassan, Osman; Sulaiman, Suhaila; Broughton, William; Bharudin, Izwan; Abu Bakar, Farah Diba; Abd. Murad, Abdul Munir.

In: Malaysian Journal of Microbiology, Vol. 14, No. Specialissue6, 01.01.2018, p. 554-562.

Research output: Contribution to journalArticle

Kamaruddin, Shazilah ; Mahadi, Nor Muhammad ; Md Illias, Rosli ; Hassan, Osman ; Sulaiman, Suhaila ; Broughton, William ; Bharudin, Izwan ; Abu Bakar, Farah Diba ; Abd. Murad, Abdul Munir. / Effect of Pichia pastoris host strain on the properties of recombinant Aspergillus niger endoglucanase, EglB. In: Malaysian Journal of Microbiology. 2018 ; Vol. 14, No. Specialissue6. pp. 554-562.
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abstract = "Aims: The methylotrophic yeast Pichia pastoris is widely used to express foreign proteins fused to secretion signals. As the effect of the expression host on the final protein product is unclear, we compared the properties of an endoglucanase (eglB of Aspergillus niger) expressed in two different P. pastoris strains. Methodology and results: Full-length cDNA encoding endoglucanase of A. niger strain ATCC10574 was isolated and expressed in P. pastoris X33 (the methanol utilisation plus phenotype, Mut + ) and P. pastoris GS115 (slow methanol utilisation, Mut S ). EglB-GS115 showed the highest activity and stability at 60 °C while EglB-X33 was most active at 50 °C. EglB-X33 was active towards other substrates such as arabinogalactan, guar gum and locust bean gum besides its specific substrate, carboxymethyl cellulose (CMC). However, EglB-GS115 was only active on CMC. The affinity of EglBX33 towards CMC (K m = 7.5 mg/mL and specific activity 658 U/mg) was higher than that of EglB-GS115 (K m = 11.57 mg/mL, specific activity 144 U/mg). Conclusion, significance and impact of study: Although eglB was cloned in the same expression vector (pPICZαC), two different characteristics of enzymes were recovered from the supernatant of the different hosts. Thus, expression of recombinant enzyme in different P. pastoris strains greatly affects the physical structure and biochemical properties of the enzyme.",
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AU - Kamaruddin, Shazilah

AU - Mahadi, Nor Muhammad

AU - Md Illias, Rosli

AU - Hassan, Osman

AU - Sulaiman, Suhaila

AU - Broughton, William

AU - Bharudin, Izwan

AU - Abu Bakar, Farah Diba

AU - Abd. Murad, Abdul Munir

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N2 - Aims: The methylotrophic yeast Pichia pastoris is widely used to express foreign proteins fused to secretion signals. As the effect of the expression host on the final protein product is unclear, we compared the properties of an endoglucanase (eglB of Aspergillus niger) expressed in two different P. pastoris strains. Methodology and results: Full-length cDNA encoding endoglucanase of A. niger strain ATCC10574 was isolated and expressed in P. pastoris X33 (the methanol utilisation plus phenotype, Mut + ) and P. pastoris GS115 (slow methanol utilisation, Mut S ). EglB-GS115 showed the highest activity and stability at 60 °C while EglB-X33 was most active at 50 °C. EglB-X33 was active towards other substrates such as arabinogalactan, guar gum and locust bean gum besides its specific substrate, carboxymethyl cellulose (CMC). However, EglB-GS115 was only active on CMC. The affinity of EglBX33 towards CMC (K m = 7.5 mg/mL and specific activity 658 U/mg) was higher than that of EglB-GS115 (K m = 11.57 mg/mL, specific activity 144 U/mg). Conclusion, significance and impact of study: Although eglB was cloned in the same expression vector (pPICZαC), two different characteristics of enzymes were recovered from the supernatant of the different hosts. Thus, expression of recombinant enzyme in different P. pastoris strains greatly affects the physical structure and biochemical properties of the enzyme.

AB - Aims: The methylotrophic yeast Pichia pastoris is widely used to express foreign proteins fused to secretion signals. As the effect of the expression host on the final protein product is unclear, we compared the properties of an endoglucanase (eglB of Aspergillus niger) expressed in two different P. pastoris strains. Methodology and results: Full-length cDNA encoding endoglucanase of A. niger strain ATCC10574 was isolated and expressed in P. pastoris X33 (the methanol utilisation plus phenotype, Mut + ) and P. pastoris GS115 (slow methanol utilisation, Mut S ). EglB-GS115 showed the highest activity and stability at 60 °C while EglB-X33 was most active at 50 °C. EglB-X33 was active towards other substrates such as arabinogalactan, guar gum and locust bean gum besides its specific substrate, carboxymethyl cellulose (CMC). However, EglB-GS115 was only active on CMC. The affinity of EglBX33 towards CMC (K m = 7.5 mg/mL and specific activity 658 U/mg) was higher than that of EglB-GS115 (K m = 11.57 mg/mL, specific activity 144 U/mg). Conclusion, significance and impact of study: Although eglB was cloned in the same expression vector (pPICZαC), two different characteristics of enzymes were recovered from the supernatant of the different hosts. Thus, expression of recombinant enzyme in different P. pastoris strains greatly affects the physical structure and biochemical properties of the enzyme.

KW - Cellulase

KW - Endoglucanase

KW - Glycosylation

KW - Methanol utilisation phenotype

KW - Recombinant enzyme

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