Study On Separation And Purification Of Poly-γ-glutamtic Acid From Broth

Poly-γ-glutamic acid(γ-PGA)is a kind of extracellular polypeptide produced by Bacillus species as an important component of microorganism capsule.Owing to its excellent characters including biocompatibility,biodegradability,non-toxicity and non-pollution,γ-PGA,as an ideal biomaterial,has broad application in many areas such as medicinal delivery material,moisturizing agent of soil and sandlot, water-condensing agent of food and high-strength fiber.In this dissertation,the process of separation and purification ofγ-PGA is mainly studied.Cell removal fromγ-PGA broth is very difficult by using common centrifugal separation due to high viscosity ofγ-PGA broth,so an effective cell removal method must be established in place of centrifugation.In this paper,centrifugation, pressured-filtration and micro-filtration method are evaluated by comparing their cell removal rate and other relevant factors,micro-filtration is confirmed as suitable separation method and cell removal rate can reach 97.46%.Meanwhile, micro-filtration operating conditions are optimized as follows:dilution ratio(v/v) between broth and water 1:2,temperature 40℃,pressure 0.3～0.4Mpa,membrane module 1.0μm spiral micro-filtration membrane.Furthermore,reverse impulse is adopted to increase permeation flux and reduce membrane obstruction.Then,the broth without cell is condensed by ultrafiltration,which can decrease the quantity of ethanol used in the subsequent precipitation step.By optimization experiment,the optimal operating condition is confirmed as follows:temperature 40℃,pressure 0.3Mpa,concentration rate 5.In the whole process,steady filtration region is not obtained in that membrane obstruction is obvious,therefore,reverse impulse can be used to improve permeation flux which is increased by 16.1%,but less than that of micro-filtration.Moreover,decoloration efficiency of two kinds of diafiltration is evaluated when diluting and ultrafiltratingγ-PGA broth.DD is confirmed as suitable decoloration operation.The result shows:DD times is 3～4, decoloration rates of concentrated liquor and penetrant liquor reach 83.89%and 99.90%respectively,which are more than CD only with 72.11%and 91.05% decoloration rates respectively.Ethanol is optimal precipitator andγ-PGA precipitation reaches 61.99g/L by comparing with some other organic solvent and chemical precipitators.The type of ethanol has no obvious influence on precipitation efficiency.The maximum ofγ-PGA precipitation can reach 103.69g/L by using ethanol and ammonium sulfate as a codeposition additive,but the negative factors of codeposition still need to be further investigated.Moreover,the optimal precipitation condition is:pH7.0,ethanol multiple 6.In this condition,γ-PGA precipitation is 75.44g/L.The method of broth pigment detection is established and the result shows:the relationship between relative pigment concentration and OD is most suitable linear function in wavelength 440nm.Through static selection of different decolorants, macroporous resin D303 is confirmed to be most effective with 81%decoloration rate and 67%γ-PGA recovery efficiency,but still less than that of DD process.Hence, optimizations of decolorants need to be deeply studied.Finally,freeze drying is most suitable approach to attainγ-PGA product with 93.58%recovery efficiency and higher appearance quality than the other two drying operations.