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Cloning and Expression of Exoinulinase Gene from Penicillium Janthinell Um and Construcion of Gene Engineering Strain for Ethanol Production from Inulin

Author: WangLin
Tutor: LiuZhaoPu
School: Nanjing Agricultural College
Course: Application of marine biology
Keywords: Inulinase Exoinulinase gene Inulin High-level expression Saccharamyes cerevisia Bioethanol
CLC: Q939.97
Type: PhD thesis
Year: 2012
Downloads: 17
Quote: 0
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Abstract


Inlinase has a broad application on food, pharmacy and biofuel industry and plays an important role in high valuable utiliazation of inulin plants such as Jerusalem artichoke (Helianthus tuberosus L). In this study, exoinulinase gene was cloned from Penicillium janthinellum B01and expressed in Pichia pastoris. Production of ethanol from inulin by Saccharomyces cerevisiae harboring inulinase gene was investigated. Results are as follows:Based on a fragment of inulinase gene cloned by degenerate primers, a full-length cDNA of exoinulinase gene (inuAl) was cloned from P. janthinellum strain B01using RACE PCR technique. An open reading frame (ORF) of2115bp is interrupted by a single intron of67bp. The fragment encodes a signal peptide with20amino acids and a mature protein with684amino acids. The protein sequence deduced from the inulinase gene contained the consensus sequence of N terminal of glycosyl hydrolases family32and seven putative N-glycosylation sites.The inuAl was subcloned to the pPICZaC expression vector and succesfully over-expressed in Pichia pastoris X-33. The highest activity of exoinlinase reached272.8U/ml in the fermentation liquid, that is appromately11-fold of wild-strain B01. The recombinant exoinulinase was purified and characterized. The molecular weight of the purified recombinant exoinulianse was100kDa. The mass spectrometry fingerprinting indicated that the purified protein was indeed recombinant exoinulinase. A large amount of fructose was identified by TLC after the hydrolysis of inulin with the recombinant exoinulinase, which indicated that the recombinant enzyme is mostly exo-inulinase. The optium temperation and pH of purified recombinant exoinulinase is50℃and4.5respectively. The activity of enzyme remains stable below50℃and in pH between5and9. The recombinant exoinulinase activity was inhibited by several metal ions. However, Ca2+enhanced the activity of the enzyme strongly. The purified recombinant inulinase also had high exoinulinase activity. This genetically engineered P. pastoris X-33is potential candidate for industrial production of fructose due to its relatively low culture cost, high production and simple purification.Saccharemyces cereviasia6525can produce high concentration of ethanol, while could not utilize inulin directly by itself. In order to modify strain6525to produce both inulinase and ethanol in one step fermentation, a expression vector pUG-PICS with inulinase gene and PGK1promoter ligated into the18SrDNA was constructed, which was integrated with chromosome of S.cereviasiae6525by elecroporation transformation. The obtained transformant BR8produced1.1U/mL inulinase activity within72h. Although the inulinase activity was not as high as expected, BR8showed good ability of inulin fermentation to produce ethanol. In100mL fermentator,9.5%v/v ethanol was produced, three times of S. cereviasia6525without inulinase gene. In5-L fermentator, ethanol concentration reached14.0%(v/v) and the conversation efficiency of total sugar was94.5%, indicating that most of inulin in the fermentation media was utilized for ethanol production.Since Br8had realatively low inulinase activity, another S. cereviasia2805and BY4741were also used to express same inulinase, and transformants AR34and R5were obtained respectively. The activities were examined as4.4U/mL and4.5U/mL individually. Ethanol production from inulin was carried out by AR34and R5each with S. cereviasia6525repectively. The result showed highest ethanol concentration were11.0%v/v and10.8%v/v for the mixed population respectively, much higher than the single population.

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