High-efficient Extracellular Expression Of Recombinant Bacillus Deramificans Pullulanase And Its Appilication

Pullulanase(EC 3.2.1.41) is a debranching enzyme that specifically hydrolyzed the ?-1,6-glucosidic bond in pullulan, amylopectin, and related dextrin. It’s mainly used in starch sugar processing industry to improve the utilization of raw material, reduce the proportion of side product, and reduce reaction time. Besides, pullulanase are also used widely in the field of pharmaceutical, brewing, and feed. However, due to the low yield that leads to high production cost, domestic large-scale production of pullulanase in industry has not been achieved and showed a long-term dependence on imports.In our previous study, recombinant strain expressing pullulanase from Bacillus deramificans was constructed. An N-terminus truncated mutant with improved thermostability and enhanced acid-resistance was constructed and displayed potential value in application. However, its limited ability in expressing soluble protein and extracellular secretion has impeded its application in industrial production. This work focuses on these issues, aiming to enhance soluble extracellular expression of pullulanase through fermentation optimization of E. coli. The Brevibacillus choshinensis, with a high efficiency of protein secretion, was selected to be a host for pullulanase expression. The fermentation process was optimized and the mechanism of magnesium ion on improved “enzyme activity” was studied. In addition, application of pullulanase in the preparation of maltosyl-?-cyclodextrin was also studied. The main results are listed as follows:(1) The effects of surfactants on the disaggregation and secretion of pullulanase aggregates in vivo were investigated. Bacillus deramificans pullulanase was found to be an aggregationprone protein that can be solubilized from the insoluble fraction by the addition of surfactants in vitro. Studying the effects of various surfactants on pullulanase production in Escherichia coli in shake flasks revealed that the addition of Triton X-100 not only increased the soluble expression of pullulanase, but also improved the secretion efficiency of the recombinant protein. A modified fed-batch fermentation strategy was then applied to the production of pullulanase in a 3-L fermentor. When supplemented with 0.5% Triton X-100 at 40 h, the maximal extracellular pullulanase production and secretion ratio were 812.4 U/m L and 86.0%, which were 46.2- and 45.5-fold that of the control, respectively.(2) The extracellular production of pullulanase in E. coli was enhanced by optimized induction and glycine supplement. Cell growth and pullulanase production in shake-flask cultures were investigated as a function of the type and concentration of inducer, and the concentration of added glycine. It turned out that lower induction density benifited the soluble expression of pullulanase, and the glycine facilitated its extracellular expression. From the results of these experiments, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 3-L fermentor. The gradual addition of lactose was utilized for the induction of protein expression. The optimal lactose feeding rate and induction point were 0.4 g/L/h and a dry cell weight(DCW) of 15 g/L, respectively. Furthermore, a glycine feeding strategy was formulated to promote the secretion of recombinant protein. The optimal extracellular pullulanase activity and protein secretion ratio were 1567.9 U/m L and 62.1%, which were 21.5 and 9.5 times higher than those observed under unoptimized conditions, respectively. The yield of pullulanase represents the highest yield reported so far.(3) Heterologous expression and characterization of pullulanase in B. choshinensis were investigated. A recombinant strain of Brevibacillus choshinensis(p NCMO2/pul A-d2) was constructed. When expressed using the TM medium, the pullulanase activity reached 50.1 U/m L with negligible amount of intracellular soluble protein and inclusion bodies. The optimal medium and culture condition for protein expressionwas determined through a combination of single-factor experiments and response surface methodology. The pullulanase activity reached 543.4 U/m L, which was 9.8-fold of that under the specified conditions. The thermostability and specific enzyme activity of pullulanase expressed by B. choshinensis or E. coli showed minor discrepancies.(4) Addition of magnesium ion to the culture medium has been found to dramatically increase the activity of Bacillus deramificans pullulanase expressed by B. choshinensis. The effects of magnesium ion on the expression of pullulanse were unique, and the mechanism of this increase was studied. When cultured in medium without added magnesium ions, B. choshinensis mainly produced a thermolabile, inactive form of pullulanase. The addition of magnesium ions led to the production of a thermostable, active form of pullulanase. Compared with the pullulanase expressed without added magnesium ion, the pullulanase expressed under the effects of magnesium ion showed no difference in the aggregation condition, but its specific enzyme activity was improved by 2.9-fold and its secondary structure showed obvious difference. The effects of magnesium ion on the morphological alterations of cells and the gene transcription level were studied. When cultured in medium without added magnesium ion, the cell wall protein(HWP) was shed from the bacterial surface in the stationary phase. This derepresses transcription from P2, one of the major promoters shared by the cell wall proteins and pullulanase, permitting increased amount of m RNA. This may accelerate the rate of protein synthesis and result in hasty protein folding, which may be inadequate for pullulanase. On the contrary, the added magnesium ion represses the shed of HWP from the cell wall. The P2 promoter was hence repressed and slowed the process of synthesis. Therefore, more time was allowed for the correct folding of pullulanase after translocation.(5) Based on the shake flasks, the production of pullulanase expressed by B. choshinensis was further studied in a 3-L fermentor. Results of a batch strategy showed that low DO level or acidic conditions could result in both poor cell growth and disastrous enzyme production. The effects of p H and dissolved oxygen were studied in a fed batch fermentation. The optimal p H and DOwere 7.0 and 30%, respectively. The enzyme activity reached 627.5 U/m L. The effects of inorganic nitrogen sources were studied. It was found that the inorganic nitrogen sources benefited the cell growth but worsened the enzyme production. When the B. choshinensis cells grow at an accelerated rate, it transformed from a rod to a sphere and showed a severe plasmid loss. The effects of beef extract were investigated. The highest pullulanase activity of 1164.8 U/m L was obtained at a beef extract concentration of 40 g/L, which was 5.3-fold of that of the batch fermentation.(6) The pullulanase was used to produce maltosyl-?-cyclodextrin with its reverse synthetic activity, catalyzing on maltose and ?-cyclodextrin. The production conditions, including temperature, p H, organic solvent type and concentration, substrate concentration, enzyme dosage, and reaction time were optimized. The organic solvents were proven to increase the substrate concentration and shorten the reaction time drastically. Under optimal conditions, a yield of 48.5% of maltosyl-?-cyclodextrin was obtained, which improved 75.7% compared with what it was before optimization.