Constuction And Expression Of High-level Endoinulinase Production Engineered Multicopy Strains

Jerusalem artichoke has many good properties such as cold resistance,drought tolerance,fast-growing,high product of tuber and less plant diseases and insect pests,which can be grown in wastelands,saline and desert regions.It does not need fertile farmland and helps to improve soil and control desertification.Jerusalem artichoke is widely grown in various areas in our country and its tuber is rich in inulin,which can be used to product high value-added high-fructose syrup,fructo-oligosaccharide,biodiesel and other products with bioconversion methods of microbial fermentation and enzymatic hydrolysis.Inulooligosaccharides(IOS)is a functional oligosaccharide,which has a great application in food and medicine.IOS has a lot of advantages such as difficult to digest,a role as dietary fiber,low caries,promoting bifidobacterium proliferation,improving lipid metabolism and so on,which meets the demand of morden people for safety,health and functionality of food.Thus,IOS becomes an more important product of inulin biotransformation.There are two main methods for producing fructooligosaccharides in industry.The traditional way is the application of frucofranosidase in transferase action using sucrose as a raw material.The downside of this method is that it will generate a large amount of glucose as a by-product that typically accounts for more than 35%and is difficult to remove,which increases the cost.The other method is the application of endoinulinase from microorganisms using inulin as the raw material.The advantage of this method is that it needs only a single enzymatic step and can generate more than 80%of high purity IOS while glucose in the product is very low.What's more,the source of inulin is rich and cheap,which is attracting more and more attention to study it.The application of endoinulinase to produce IOS has high limitations for the enzyme-producing microorganism.First of all,the microorganism should only produce endoinulinase without exoinulinase and transferase.Second,the enzyme should have high endoinulinase activity and good stability.Thus,the key of the application depends on obtaining excellent endoinulinase-producing microorganisms.However,the vast majority of inulinases produced by microbe in nature contain both endo-type and exo-type inulinases,and few microorganisms only.produce endoinulinase which is always too low to meet the industrial application.Nowadays,enzyme gene can be cloned and heterologously expressed in industrial microorganism with gene recombination technology,which is a good way to obtain efficient singly endoinulinase-producing microorganisms.In this study,an endoinulinase gene inu2 from Aspergillus ficuum ATCC16882 was cloned and expressed in Pichia GS115 with the help of the pPIC9K plasmid.The recombinants with different copies of endoinulinase INU2 were isolated and screened using different pressure levels of antibiotics G418.The recombinants of INU2-1,INU2-2,INU2-3 and INU2-5 were identified to be 1 copy,2 copies,3 copies and 5 copies,respectively,determined by using real-time fluorescence quantitative PCR.The results of methanol induction fermentation of the recombinants show that the multi-copy recombinants yielded more enzyme production than the single copy one while the highest endoinulinase activity reached 570 U/ml for both INU2-3 and INU2-5 by shake flask culture.High-density fed-batch fermentation in 3 L fermentor was further studied for INU2-3 and the highest inulinase activity reached 2190 U/ml after methanol induction for 7 days.Three copies of inu2 expression cassette which was regulated by GAP promoter were strung together in the plasmid pGAPZaA and the recombinant plasmid pGAPZ?A-3INU2 was transformed into INU2-3 strain.One recombinant was isolated and screened with antibiotics Zeocin,and the real-time fluorescent quantitative PCR analysis showed that 2 copies of INU2 expression cassette were integrated.Thus,the recombinant INU2-A3-G2 had 5 copies of inu2 in the genome and 3 copies were regulated by AOX1 promoter while 2 copies by GAP promoter.High-density fed-batch fermentation in 3 L fermentor for INU2-A3-G2 showed that its inulinase activity could reach as high as 3006 U/ml afer methanol induction for 204 hours,which was 2.2 times of the highest endoinulinase in the literature reports.The crude recombinant INU2 enzyme acted on the substrate inulin and the products containing 12.4%F,10.6%F2 and 77.0%F3 determined by thin-layer chromatography.It showed that this enzyme could be applied to produce IOS.The results of the enzyme precipitation by using ammonium sulfate from crude enzyme solution show that the total recovery of the recombinant protein could be reached at 65.4%with the ammonium sulfate saturation of 50%and the pH of 4.The study of protective agent for the crude recombinant INU2 enzyme showed that the best compositions of the protective agent are 15 mmol/L CaCl2,40%sucrose and 50%sorbitol,which could be used for long term storage of the endoinulinase agent.