| Pullulanase can specifically hydrolyze α-1,6-linkages in amylopectin and plays important roles in starch saccharification. In this study, a chimera of pullulanase was obtained after multiple rounds of DNA shuffling. The chimera encoding gene named as WXP03 was expressed in Pichia pastoris GS115. An indirect selection method was used to screen high yield strains. The main content of this research is as follows:（1） According to the codon bias of P. pastoris, the pullulanase encoding gene from Bacillus deramificans and Bacillus naganoensis were optimized, artificially synthesized, and designated as Bd P8 and Bn P2, respectively. Bd P8 and Bn P2 were inserted into the vector p PIC9 K to obtain the recombinant plasmids p PIC9K-Bd P8 and p PIC9K-Bn P2. These plasmids were transformed into P. pastoris GS115. Recombinant pullulanase encoded by Bd P8 and Bn P2 were expressed actively, and relatively high yield strains BD15 and BN17 were screened for production of Bd P8 and Bn P2, respectively. The specific activity of the purified Bd P8 was 1.9 U·mg-1. The optimal temperature and p H of this recombinant enzyme were 55°C and 4.0, respectively. The specific activity of the Bn P2 was 247.1 U·mg-1. The optimal temperature and p H of this purified recombinant enzyme were 55°C and 5.0, respectively.（2） The novel chimera gene WXP03 was obtained by DNA shuffling of Bd P8 and Bn P2. The chimera enzyme WXP03 encoded by the gene WXP03 was expressed in P. pastoris GS115. A transformant with relatively high yield of pullulanase was screened and designated as P. pastoris GS115/p PIC9K-WXP03 M8. The optimal temperature and p H of the purified recombinant enzyme WXP03 were 55°C and 4.5, respectively, and the specific activity was 213.3 U mg-1. WXP03 is a pullulanase chimera, with characteristics of acido-resistant Bd P8 and high specific activity Bn P2.（3） Bd P4, the gene encoding a mutant of pullulanase from B. deramificans, was optimized and expressed in P. pastoris. A transformant with relatively high yield of pullulanase was screened and designated as P. pastoris GS115/p PIC9K-Bd P4 WB54. Fermentation conditions of WB54 were optimized in a 5 L fermentor. The relatively optimized conditions were determined as follows: p H, 4.5; start of methanol induction, OD600=360; and feeding rate of methanol, 9.9 m L·h-1·L-1. Under optimized conditions, WB54 produced 2031.0 U·m L-1 pullulanase activities.（4） P. pastoris GS115/p PIC9K-WXP03 M8 was mutagenized, and ura3 auxotrophic strain, designated as E107, was screened. The recombinant plasmid p PIC9K-Sf A-TT-URA3-RP, with the amylase encoding gene Sf A controlled by promoter AOX and the prototroph marker gene URA3, was transformed into E107. The amylase encoded by Sf A was secretory expressed in transformants and the strain designated as H11 was selected for further study. The strain H11 was mutagenized, and 17 mutants with relatively high yield of amylase were screened. Those 17 mutants were employed in fermentation and intracellular pullulanase assay carefully. A total of 7 strains, about 1/3 of 17 mutants, exhibited the increase of pullulanase yield. The strain J359, with a relatively high pullulanase yield, was used in further study. The culture of J359 was spread to the SM+5-FOA plates to obtain a ura3 auxotrophic mutant. The ura3 auxotrophic mutant J359 C was screened, and PCR test showed that the Sf A gene was also deleted in J359 C. The ura3 auxotrophic marker was then covered and transformed by plasmid harbor Pp URA3 gene. Then, a prototrophic transformant WBB359 was selected. In flask shaking process, pullulanase yield of WBB359 was increased by 15.6% than the starting strain M8. Under optimized conditions, WBB359 produced 2101.3 U·m L-1 pullulanase activities. |
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