Recombinant expression and characterization of the endochitinase Chit36-TA from Trichoderma asperellum in Komagataella phaffii for chitin degradation of black soldier fly exuviae

Bioprocess Biosyst Eng. 2024 Oct;47(10):1751-1766. doi: 10.1007/s00449-024-03067-4. Epub 2024 Aug 8.

Abstract

The natural polymer chitin is an abundant source for valuable N-acetylchitooligosaccharides and N-acetylglucosamine applicable in several industries. The endochitinase Chit36-TA from Trichoderma asperellum was recombinantly expressed in Komagataella phaffii for the enzymatic degradation of chitin from unused insect exuviae into N-acetylchitooligosaccharides. Chit36-TA was purified by Ni-NTA affinity chromatography and subsequently biochemically characterized. After deglycosylation, the endochitinase had a molecular weight of 36 kDa. The optimum pH for Chit36-TA was 4.5. The temperature maximum of Chit36-TA was determined to be 50 °C, while it maintained > 93% activity up to 60 °C. The chitinase was thermostable up to 45 °C and exhibited ~ 50% activity after a 15 min incubation at 57 °C. Chit36-TA had a maximum specific enzyme activity of 50 nkat/mg with a Km value of 289 µM with 4-methylumbelliferyl-N,N',N″-triacetyl-β-chitotrioside as substrate. Most tested cations, organic solvents and reagents were well-tolerated by the endochitinase, except for SDS (1 mM), Cu2+ (10 mM) and Mn2+ (10 mM), which had stronger inhibitory effects with residual activities of 3, 41 and 28%, respectively. With a degree of hydrolysis of 32% applying colloidal shrimp chitin (1% (w/v)) and 12% on insect larvae (1% (w/v)) after 24 h, the endochitinase was found to be suitable for the conversion of colloidal chitin as well as chitin from black soldier fly larvae into water-soluble N-acetylchitooligosaccharides. To prove scalability, a bioreactor process was developed in which a 55-fold higher enzyme activity of 49 µkat/l and a tenfold higher protein expression of 1258 mg/l were achieved.

Keywords: Komagataella phaffii; Trichoderma asperellum; Biopolymer; Black soldier fly larvae; Chitin; Chitinases.

MeSH terms

  • Animals
  • Chitin* / chemistry
  • Chitin* / metabolism
  • Chitinases* / biosynthesis
  • Chitinases* / chemistry
  • Chitinases* / genetics
  • Chitinases* / metabolism
  • Fungal Proteins / biosynthesis
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Hydrogen-Ion Concentration
  • Hypocreales* / enzymology
  • Recombinant Proteins* / biosynthesis
  • Recombinant Proteins* / chemistry
  • Recombinant Proteins* / genetics
  • Recombinant Proteins* / metabolism
  • Saccharomycetales / enzymology
  • Simuliidae

Substances

  • Chitin
  • Chitinases
  • Recombinant Proteins
  • Fungal Proteins

Supplementary concepts

  • Trichoderma asperellum