Cloning, Charaterization And Domain Evolution Of Pullulanase From Paenibacillus Sp. And Geobacillus Sp.

In this research, we have selected three strains Paenibacillus lautus (DSMZ 3035), Paenibacillus mucilaginosus (DSMZ 24461) and Geobacillus kaustophilus (DSMZ 7263) from Paenibacillus sp. and Geobacillus sp. for further research. First, the original strains were determined secreting activity of pullulanase on the pullulan screening plates. Then the pullulanase gene of 3035,24461 and 7263 were cloned by PCR. The sizes of the sequences were 2355 bp,1965 bp and 2157 bp respectively. And the GeneBank numbers are KT 876382, KT 899326 and KT 899327. All of them belong to type I pullulanase by conserved sequence analysis.The recombinant strain BL21-pET-28a-PulPL was constructed successfully, and the enzyme activity of recombinant pullulanase 3035-28a-PulPL was 13.56 U/mg. The optimum temperature and pH were 40℃ and 7.0 respectively. The recombinant bacteria BL21-pET-28a-PulPM could realize soluble expression of pullulanase 24461-28a-PulPM with the enzyme activity of 220 U/mg. The optimum temperature and pH is 40℃ and 6.0 respectively. The enzyme activity was stable under pH range of 6.0 to 9.0. Although the recombinant enzyme 24461-28a-PulPM shows higher enzyme activity, the enzyme lost most activity at high temperatures. Compared with industrialized enzyme 2wan, the N-terminus of 24461-28a-PulPM lacks one domain X45-X25 through 3D-structure analysis. Therefore we spliced X45-X25 to 24461-28a-PulPM, and constructed protein 2w-24461-Pul successfully. The chimeric enzyme (2w-24461-Pul) which containing 955 amino acids showed optimum temperature improvement from 40℃ to 45℃, and the optimum pH was changed from 6.0 to 7.0. Meanwhile, the T1/2 of 2w-24461-Pul increase 10 min at 45℃.The Bacillus subtilis WB800N-pNW-PulGK could secrete protein 7263-pnw-PulGK into fermentation supernatants. The activity of 7263-pnw-PulGK was 5.18 U/mL and the optimum temperature/pH were 65℃ and 6.0 respectively.7263-pnw-PulGK maintained most activity under high temperature (55-70℃). The sodium alginate entrapment was used to immobilizating 7263-pnw-PulGK. The immobilized enzyme maintained more actvity at high temperature and can be used multiple times.