Fermentation And Preparation Of Agar Sulfataseand The Application In Enzymatic Agar Extraction

Agar sulfatase,belonging to a conservative family which can specifically hydrolyze sulfate and provide many kinds of purposes,was cloned by Pseudoalteromonas carrageenovora for the sulfate removal from agar.In this article,Fermentation and preparation of agar sulfatase and the application in enzymatic agar extraction was studied.First,the induct on conditions of recombinant Escherichia coli in the flask culture was optimized by single parameter,and the 5 L,20 L,200 L pilot-scale experiment was proceed in terms of the optimum consequence.Second,the solid preparation of agar sulfatase was prepared by the spray drying method,and then the storage stability of enzyme preparation was investigated to find the best storage method.Finally,the pattern of agar extraction was studied by adding cellulase and agar sulfatase,meanwhile,the extracting technology of agar extraction with compound enzyme was designed.This experiment regarded the strain of recombinant Escherichia coli?E.coli-ars?which produced recombinant agar sulfatase as the research object.At first,the optimization of induction conditions in shaking flask were implemented by single factor experiment to enhance enzyme production,and then the optimal induction conditions were obtained:induction temperature21?,induction time 24 h,inducing agent?-lactose 4 g/L,inducing initial biomass(OD₆₀₀)0.6.whereafter,the fermented experiment of E.coli-ars in 5 L bioreactor was carried out.Research indicated that additional medium significantly improved cell density,while it had had no efficiency on the enzyme activity.Finally,the pilot enlarge experiment of 20 L and 200 L were conducted on the basis of the optimized fermentation conditions of shaking flask and 5 L bioreactor.The enzyme activity of the agar sulfatase in 20 L and 200 L bioreactor reached 157.5U/mL and 110.4 U/mL,respectively.The solid enzyme preparation of agar sulfatase was prepared by spray drying.The process parameters were investigated by single factor experiment.Under the optimal process parameters,?maltodextrin 25%,inlet air temperature 130?,hot air flow rate 3.5 m³/min,feeding speed 400mL/h,atomizer pressure 0.25 Mpa?,the enzyme recovery of agar sulfatase reached 52.2%,which increased by 48.7%compared with the intial enzyme recovery?26.7%?.Storage stability of enzyme preparations were studied,experimental results showed that temperature,air,light and humidity had a large impact on the storage stability of agar sulfatase preparation.According to the results of the above mentioned,the agar sulfatase enzyme preparation was proposed to store at the condition of 4?,sealed,dark,dry condition.The optimum conditions of enzyme extraction of agar were obtained through single factor experiment.The optimum extraction conditions of cellulase processing were temperature 50?,enzyme processing time 2 h,enzyme amount of 7200 U,the ratio of material to water for 1:20.On this basis,the yield of agar was 14%.In addition,the results of agar extracted in 200 L showed that enzymatic extraction technology had a good reproducibility and stability.Based on the previous small laboratory and pilot tests,the agar production workshop of annual output of 300 tons was preliminary designed.The program of material balance,equipment selection and economic benefit estimation were applied to the whole workshop.