Screening of signal sequences for extracellular production of Aspergillus niger xylanase in Pichia pastoris

Karaoglan M., Yildiz H., İNAN M.

BIOCHEMICAL ENGINEERING JOURNAL, cilt.92, ss.16-21, 2014 (SCI-Expanded) identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 92
  • Basım Tarihi: 2014
  • Doi Numarası: 10.1016/j.bej.2014.07.005
  • Dergi Adı: BIOCHEMICAL ENGINEERING JOURNAL
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.16-21
  • Anahtar Kelimeler: Yeast, Enzyme production, Purification, Optimization, Pichia pastoris, Xylanase, EXPRESSION, PROTEIN, PERFORMANCE, SECRETION, CLONING, GENES, ENDO-BETA-1,4-XYLANASE, SUPPLEMENTATION
  • Akdeniz Üniversitesi Adresli: Evet

Özet

In this study four secretion signal sequences were evaluated for extracellular production of Aspergillus niger xylanase (XylB) in Pichia pastoris. These sequences were namely; Saccharomyces cerevisiae a-mating factor (alpha-MF), Human Serum Albumin (HSA), P. pastoris Acid Phosphatase (PHO1) and P. pastoris Protein with Internal Repeats (PIR1) secretion signals. The codon optimized XylB gene fused to secretion sequences was expressed under the AOX1 promoter of P. pastoris in KM71H strain. Xylanase enzyme activities for single-copy clones with a-MF, HSA, PHO1 and PIR1 as secretion sequence were 170, 123, 139 and 250 U/mL, respectively, after 48 h induction period. P. pastoris PIR1 leader sequence resulted highest xylanase enzyme activity at shake-flask scale among the tested secretion signal sequences. The xylanase was purified with a single step Ni-NTA purification method. The optimum temperature and pH for recombinant xylanase was 40-50 degrees C and pH 5, respectively. Additionally, xylanase activity in purified enzyme was stable over a wide pH range (pH 3-7) and temperature of up to 50 degrees C. Specific activity of purified xylanase was 13,271 U/mg. (C) 2014 Elsevier B.V. All rights reserved.