Effect of Trichoderma reesei Proteinases on the Affinity of an Inorganic-Binding Peptide Andrew Care & Helena Nevalainen & Peter L. Bergquist & Anwar Sunna Received: 25 December 2013 /Accepted: 19 June 2014 / Published online: 27 June 2014 # Springer Science+Business Media New York 2014 Abstract An inorganic-binding peptide sequence with high affinity to silica-containing ma- terials was fused to a glycoside hydrolase GH26 mannanase, ManA, from the extremely thermophilic bacterium Dictyoglomus thermophilum. The resulting recombinant enzyme pro- duced in Escherichia coli, ManA-Linker, displayed high binding affinity towards synthetic zeolite while retaining its catalytic activity at 80 °C. ManA-Linker was able to bind to the zeolite at different pH levels, indicating a true pH-independent binding. However, complete degradation of the peptide linker was observed when the recombinant ManA-Linker was exposed to the supernatant from the filamentous fungus Trichoderma reesei. This degradation was caused by extracellular proteinases produced by T. reesei during its growth phase. Several derivatives of ManA-Linker were designed and expressed in E. coli. All the derivatives carrying a single sequence of the linker were still susceptible to T. reesei proteinase degrada- tion. Complete substitution of the linker sequence by (GGGGS) 16 resulted in a proteinase- resistant ManA derivative, ManA-Linker-(GGGGS) 16 , which was able to bind to zeolite in a pH-dependent manner. Keywords Trichoderma . Proteinases . Affinity . Inorganic-binding peptide Introduction Immobilisation of proteins on solid matrices is integral to numerous technologies, e.g. affinity chromatography, protein microarrays, biosensors and biocatalysis [ 1]. Conventional methods of immobilisation have relied on non-specific adsorption or on the covalent reactions between chemical groups within proteins and those on the matrix surface [2]. In both circumstances, proteins attach to the surfaces in non-uniform Appl Biochem Biotechnol (2014) 173:2225–2240 DOI 10.1007/s12010-014-1027-7 A. Care : H. Nevalainen : P. L. Bergquist : A. Sunna (*) Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, North Ryde, Sydney NSW 2109, Australia e-mail: anwar.sunna@mq.edu.au P. L. Bergquist Department of Molecular Medicine and Pathology, Medical School, University of Auckland, Auckland, New Zealand