Puirifcation And Characteirzation Of8-poly-L-lysine-degrading Enzyme From Streptomyces Sp. M-Z18

ε-poly-L-lysine-degrading enzyme (Pld) has been found in ε-poly-L-lysine (ε-PL)producing strains and ε-PL-tolerant bacteria. Pld derived from different microorganismsexhibit different characterizations. Overall, Pld degrades ε-PL through exo-type andendo-type. The exo-type Pld acts at the N-terminus of ε-PL and releases L-lysine one by one,while the endo-type Pld catalyzes the hydrolysis of internal peptide bonds of ε-PL andproduces ε-PL with low degree polymerization.In the present study, the Pld derived from Streptomyces sp. M-Z18was purified and itsenzymatic characteristics were subsequently clarified. Furthermore, this Pld was employed toproduce the low polymerization of ε-PL. The main results were as follows:1. The Pld was purified to electrophoretical homogeneity through HiTrapTMButyl HPhydrophobic chromatography after pretreated by ultrasonic and NaSCN dissolving. As a result,the purification fold was80.4and the yield was59.3%.2. The Pld was characterized. The optimal temperature and pH for the purified Pld were37℃and7.0, respectively. The Pld was stable in the range of pH7.0-10.0, and temperatureup to50℃, respectively. Moreover, the Kmwith L-lysyl-p-nitroanilide as substrate wascalculated to be0.621mmol·L-1, and the V-1maxwas701.16nmol·min·mg-1. The activity ofPld was activated by Ca2+, Li+and was inhibited by Zn2+, Co2+and Ni2+. Additionally, theactivity of Pld was strongly inhibited by EDTA, indicating Pld is a metalloenzyme probably.Time profile of ε-PL degradation by the purified Pld indicated that the Pld catalyzedendo-type degradation of ε-PL.3. ε-PL with low degree of polymerization was achieved through degrading commodityε-PL by Pld. ε-PL with high degree of polymerization was preparated by ultrafiltration with1kDa Molecular Weight Cutoff. The experiments of minimal inhibitory concentration showedthat ε-PL with high degree of polymerization (30-35) had a superior antibacterial effect onbacteria and the low degree of polymerization ε-PL (8-20) had a better antibacterial effect onyeasts. However, ε-PL with various degrees of polymerization had a poor antibacterial effecton mould.