A thermostable-endo-β-(1,4)-mannanase from Pediococcus acidilactici (M17): purification, characterization and its application in fruit juice clarification

Abstract

A Turkish fermented sausage was bought from a local market in Erzurum and was used for isolation and characterization of lactic acid bacteria. As the result of 16S rRNA analysis, this bacterium was determined as Pediococcus acidilactici (Genbank number KF895384) at a rate of 99 %. The mannanase from P. acidilactici (M17) was purified using the (NH4)2SO4 precipitation, DEAE-cellulose ion exchange and Sephacryl S200 gel filtration chromatography techniques. The yield and purification fold of mannanase were 10.6 % and 106.1, respectively. The molecular mass of the enzyme (38 kDa) was determined by SDS-PAGE and gel filtration chromatography. In order to identify, the resistance of the purified mannanase to Mn2+, Co2+, Cu2+, Zn2+ and Ca2+ was examined, and all the metal ions activated the enzyme at a significant level. The V max and K m values for locust bean gum were calculated as 78.74 mmol/min mg and 0.592 mM, by Lineweaver–Burk. Purified mannanase was also used to clarify some fruit juices. The studied variables were, the incubation time (from 0 to 60 min), the enzyme concentration (17 EU/L), and the temperature (60 °C). It was observed that the purified mannanase had maximum yield on orange juice with 161.1 %. Also maximal clarification was obtained as 84.65 %, and the clarification for grape juice and sugar reduction was obtained as 19.26 mg/mL for apple juice. Consequently, it was concluded that the P. acidilactici (M17) mannanase could be successfully used in the fruit juice industry.