Gene Cloning And Expression, Characterization Of Pul28，a New Pullulanase From Anoxybacillus Sp.

As a starch debranching enzyme, pullulanase can specifically hydrolyze α-1,6glucosidic linkagesin polysaccharide to change amylopectin into amylose. The utilization efficiency of starch can beimproved through the cooperation of pullulanase, α-amylase and β-amylase. Due to their specificproperties, pullulanases have been widely applied in food, chemistry and medical industries. Recently,microbial pullulanases have become the main resources of the industrial pullulanases.Seven pullulanase-yielded strains were isolated from hot spring sand by the screening plates usingpullulan as the carbon source. Three of them with the higher pullulanse activities were identified as thesame strain by morphological characteristics and phylogenetic comparisons of their16S rDNA. Thesethree strains were identified as Anoxybacillus sp., and the No.28strain was selected for the furtherresearch and named Anoxybacillus sp.P-28.The paticial nucleotide sequences of the pullulanase gene from Anoxybacillus sp.P-28wasamplified using touch-down PCR with the degenerate primers designed based on the conservativedomains of pullulanase from Bacillaceae. The full-length sequences were obtained by TAIL-PCR usingspecific primers designed according to the partial nucleotide sequences. The pullulanase gene pul28contained2121nucletides, comprising one open reading frame encoding a polypeptide of706aminoacids. Its deduced amino acid sequence showed95.5%identity to the probable pullulanase ofAnoxybacillus flavithermus WK1using Blastp program in NCBI. The calculated molecular mass of thisprotein was81.83kDa with a theoretical pI of5.60. The Pul28contained four conservative amino acidsfragments of the glycoside hydrolase family13（GH13） and a highly conserved motif （YNWGYDP）,and thus the Pul28was speculated to belonged to GH13as a type I pullulanase.The pul28gene was cloned and expressed in E. coli BL21（DE3） under the control of inducible T7promoter. The recombinant protein Pul28was expressed in the soluble form and then purified byNi-NTA column and characterized. The optimal pH and temperature of the purified recombinant Pul28were5.0and50°C. The enzyme showed stability at60°C and over a pH range of pH5.0⁷.0. Theenzyme activity was remarkably improved by divalent cations such as Ca²⁺, Ni²⁺, Ba²⁺and Cu²⁺, butinhibited by EDTA and SDS. The Km, Vmaxand kcatof the purified Pul28with pullulan as substrate wereapproximately0.43±0.044mg/mL,23.23U/mg and499.75±14.64/s, respectively. According to the HighPerformance Liquid Chromatography （HPLC） analysis, the recombinant Pul28was confirmed as type Ipullulanase, because the hydrolyzed products of pullulan only contain maltotriose.The shuttle plasmid containing pul28was transformed into Bacillus subtilis WB800byelectroporation. The pul28gene was expressed under the control of the inducible promoter of amylasewith soluble starch as the inducer. The Bacillus subtilis WB800containing the expression plasmid cansecrete extracellular recombinant Pul28. The enzyme yield was increased with the extension ofincubation time and had a highest enzyme activity of0.06U/mL at72h. HPLC analysis confirmed that Pul28can be expressed by Bacillus subtilis WB800. The extracellular Pul28was convenient for thefurther treatment and application of this enzyme.