More than a million tons of soybean meal is imported from South America to Denmark every year to use as feed.To reduce the import and to reduce transportation, alternative locally grown crops are researched. A potentialprotein source is perennial legumes. Green biorefinery concept is a concept that converts different greensubstrates into multiple products including a protein concentrate. While this concept extracts some of theprotein present in the substrate, there are still structure bound proteins left. This project focus on optimizingenzyme production and enzymatic hydrolysis, which is used to break the lignocellulosic structure and extract theproteins. Four different experimental parts are performed. Alfalfa press cake is used as the main substrate inthree of the experimental parts and fresh clover grass is used in the last part. The first experimental part is thesubstrate composition, where moisture, ash, protein, sugar, and lignin content are measured. The secondexperimental part is the optimization of enzyme production, where the parameters time of incubation, lightavailability, inoculation temperature, initial moisture content, and inoculum size are analyzed together withtesting four different substrates. The third experimental part is the optimization of enzymatic hydrolysis, wherethe parameters incubation time, enzyme units per substrate, pH of inoculum, and incubation temperature aretested together with analyzing the potential of using diluted brown juice as a buffer. The last experimental partis the effect of enzymatic hydrolysis, where the created enzyme cocktail is used on fresh substrate and the effectof the enzymatic hydrolysis is tested by analyzing the supernatant and solids using different methods. Alfalfapress cake contains a dry matter on 34.6 ± 0.7 g/100g substrate, with a protein content on 13.8 ± 0.08 g/100gDM substrate and a cellulose concentration on 31.7 ± 0.4 g/100g substrate. The optimal parameters for enzymeproduction are incubation in darkness for 9 days at 25-30°C with a moisture content on 75% and at least 5*106spores/g-DM substrate. Of the tested substrates, alfalfa press cake is the substrate producing the least amountof FPA activity. The optimal parameters for enzymatic hydrolysis are 7.5 FPU/g-DM substrate at 50°C and pH inthe range of 4-5.5. Incubation at 24 hours yields 8% of total released sugars. It is possible to use diluted brownjuice as buffer. In the effect of enzymatic hydrolysis, substrate is degraded. Unfortunately, the proteinconcentration results do not indicate a higher protein extraction after an enzymatic hydrolysis. It is thereforeunclear if the hydrolysis affects the extracted protein concentration.