Different techniques and strategies to produce and partial purificaton of mannanase

β-mannanases are mainly products of filamentous fungi including different Aspergillus species, and can degrade the β-1,4-mannose linkages of galactomannans. It could be easily produced by solid-state fermentation or flask fermentation. However amount of the products are limited. Moreover these microorganisms need air to produce these value-added products. So bioreactors must be used for Aspergillus fermentation. However controlling of the filamentous fungi in a broth is very difficult because of high hyphae development. This study was undertaken to enhance β-mannanase production using magnesium silicate and aluminum oxide as the microparticles, which control cell morphology of recombinant Aspergillus sojae in carob extract medium. Both microparticles improved and controlled fungal growth in carob pod extract medium in shake flask and stirred tank bioreactor fermentations. It was also carried out that the partial purification of β-mannanase from fermentation broth, lyophilization, and characterization of the enzyme (working conditions, Km and Vmax values of some substrates, reagent inhibition etc.). First of all, shake flask fermentations were performed by different amounts of aluminum oxide or magnesium silicate in the fermentation media. The highest β-mannanase activity was found as 568,7 U/ml with 5 g/L of magnesium silicate for shake flask fermentations. Increase in microparticle concentration resulted in decrease at the pellet size diameter. Furthermore, more than 10 g/L of magnesium silicate addition changed the filamentous fungi growth type from pellet to pellet/mycelium mixture. The highest β-mannanase activity for bioreactor fermentations was 643,16 U/ml for 3 g/L of magnesium silicate. And all of the microparticle bioreactor assays were resulted higher β-mannanase activity than control fermentation. All of the fermentation assays except control fermentation could easily control the hyphae development until the end of the fermentation. None of the microparticle concentration changed the microorganism growth type from pellet to pellet/mycelium mix type for bioreactor assays. Partial purification was completed by ultrafiltration system, and carefully lyophilized. Enzyme characterization was carefully done, and optimum working pH and temperature range were determined to be from pH 5 to pH 6, and 50°C to 60°C respectively. It was also found that locust bean gum was the best substrate for β-mannanase, Vmax and Km values for locust bean gum was calculated to be 2719 U/mg, and 2,26 µmole/ml. Finally effects of reagents on working on β-mannanase enzyme were determined. As the result, this research was clearly showed that β-mannanase enzyme could be produced in a stirred tank bioreactor with microparticle addition and partial purification could be performed by ultrafiltration system. So results showed that carob pod extract medium with different microparticle agents was a good substrate for recombinant Aspergillus sojae to produce β-mannanase enzyme. This technique can also be used for the other filamentous fungi microorganisms to produce the valuable enzymes in higher bioreactor plant systems.