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

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-ß-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.

Ion chromatography coupled with optical emission spectrometry (IC-ICP-OES) methodology for the analysis of inositol phosphates in food and feed.

This study presents the development of ion chromatography coupled with inductively coupled plasma optical emission spectrometry (IC-ICP-OES) for the simultaneous determination and quantification of inositol phosphates (InsPx). Using a CarboPac PA100 column with a nitric acid-water gradient, 28 InsPx isomers (InsP6 to InsP2) were separated within 33 min. The method eliminates baseline drift and post-column derivatization thereby simplifying detection and quantification. It achieves low detection limits of 63 µg/L P across a range of 63-3200 µg/L P. Various extraction and sample preparation methods for food and feed matrices were tested, including acidic and alkaline agents, C18 SPE and spin concentrators. The analysis shows intra-day and intra-laboratory reproducibility with deviations smaller than 1 % for standard solutions and under 4 % for feed samples (80 % recovery rate of phytate). This methodology is applicable to explore enzymatic degradation pathways and the analysis of InsPx in complex food and animal feed matrices.