High-Level Expression Of Alkaline Protease In Recombinant Bacillus Strains

Alkaline protease, commercially produced by Bacillus licheniformis B5102in China, havebeen widely used in the leather industry. Three recombinant strains expressiong the alkalineprotease gene were designed and constructed, respectively. These strains were characterizedby improved alkaline protease production and decreased fermentation time. The main resultswere as follows:1. A pair of primer was designed according to the sequence of the alkaline protease gene ofB. licheniformis. The apr gene encoding alkaline protease was obtained by PCR using thegenomic DNA of B. licheniformis B5102as a template. The PCR product was ligated withpLY and transformed into B. subtilis WB600. The recombinant plasmid was abstracted fromtransformants and sequenced. Sequence analysis revealed that the transformant contained therecombinant plasmid, with a1.8kb DNA fragment inserted into the BamHI site of pLY. Thefragment contained the full-length gene apr. The sequence of this fragment was deposited inGenBank under accession number JQ285995. The sequence of the amplified fragmentshowed the highest homology with a previously published alkaline protease-encoding genefrom B. licheniformis ATCC14580. The61st base pair upstream from the start codon featureda substitution of A to G compared with the apr gene from B. licheniformis ATCC14580.Another substitution of T to C occurred at the556th base pair, and showed a synonymousmutation of codons CTG to TTG. Recombinant B. subtilis was aerobically cultured, and theprotease activity in the medium was679u/mL. SDS-PAGE electrophoresis showed that theapparent molecular mass of the recombinant alkline protease was about31kDa.2. The recombinant plasmid pLY-apr was transformed into B. amyloliquefaciens B4803viaelectroporation, one transformants designated as B. amyloliquefaciens B4803/pLY-aprexhibited the highest protease production. The maximum protease activity of B.amyloliquefaciens B4803/pLY-apr was5122u/mL after64h, compared to B. licheniformisB5102, the maximum activity increased46%and the fermentaion cycle decreased32h.Results obtained from25L fermentation tanker tests showed that the fermentation cycle of B.amyloliquefaciens B4803/pLY-apr was60h, or a36h reduction compared with B.licheniformis B5102. The maximun alkaline protease activity of B. amyloliquefaciensB4803/pLY-apr reached9847u/mL, similar to that of B. licheniformis B5102.3. The recombinant plasmid pLY-apr was transformed into B. licheniformis B5102viaelectroporation. One strain exhibited the highest protease production was selected and it wasdesignated as B. licheniformis B5102/pLY-apr. The maximum protease activity of B.licheniformis B5102/pLY-apr was5477u/mL. Under the same conditions, B. licheniformisB5102yielded a maximum activity of3505u/mL. Results obtained from25L fermentationtanker tests showed that the maximun alkaline protease activity of B. licheniformis B5102was9658u/mL after96h, whereas the maximun enzymatic activity of B. licheniformisB5102/pLY-apr reached14700u/mL after104h.4. The fermentation conditions of B. licheniformis B5102/pLY-apr were optimized. Theoptimum medium contained80g/L xylose,45g/L soybean cake powder,3g/L ammoniumchloride,15mmol/L sodium phosphate, and0.2g/L calcium chloride. Results obtained from 25L fermentation tanker tests showed that the alkaline protease activity reached26500u/mLunder the optimal conditions, which was1.8times that reached under pre-optimizationconditions.5. The recombinant alkaline protease produced by B. licheniformis B5102/pLY-apr wascharacterized. The maximum protease activity was observed at pH9.5and50°C.Recombinant enzyme was stable at50°C and pH8-11.