Abstract: A mutant hydrolase optionally fused to a protein of interest is provided. The mutant hydrolase is capable of forming a bond with a substrate for the corresponding nonmutant (wild-type) hydrolase which is more stable than the bond formed between the wild-type hydrolase and the substrate. Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein.

Abstract: The invention pertains to a method for preparing a product for fermentation comprising the steps: i. sterilizing and expanding a substrate to obtain a sterile expanded substrate, ii. cooling and inoculating the sterile expanded substrate by contacting the sterile expanded substrate with a cooling medium and an inoculum to obtain a cooled inoculated substrate. The expansion step can be performed using techniques from the state of the art known as popping or puffing. The method can be performed in standard equipment and is very flexible in its use. Furthermore, the method allows reducing the time and energy necessary to obtain a product for fermentation, such as solid state fermentation.