Fermentation Optimization And Production In Pilot Scale Of Microbial Transglutaminase Using Streptomyces Hygroscopicus WSH03-13

Microbial transglutaminase (MTG;protein-glutamine γ-glutamyltransferase, EC2.3.2.13) is a recently developed enzyme and is capable of catalyzing acyl transferreactions, which introduces covalent cross-links between proteins, peptides andvarious primary amines. So it has potential application in food, cosmetic, pharmacyand other industries.A strain WSH03-13 secreting MTG was identified as Streptomyceshygroscopicus based on morphological, physiological, biochemical and phylogeneticanalysis. By using Streptomyces hygroscopicus WSH03-13, optimization of MTGfermentation was performed by chosen proper carbon and nitrogen sources. Aimed toindustrial production, the MTG fermentation scale-up was carried out from lab scaleto pilot scale. Effects of hydroxylamine addition on the accumulation of MTG byStreptomyces hygroscopicus WSH03-13 were investigated. The main research resultswere shown as follows:1. Based on morphological and physiological characteristics and biochemical andphylogenetic analysis, the strain WSH03-13 was identified as Streptomyceshygroscopicus, which belonged to Bacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Streptomycineae;Streptomycetaceae;Streptomyces.2. By choosing appropriate carbon and nitrogen sources and optimization of theircombinations, MTG fermentation media by Streptomyces hygroscopicusWSH03-13 was determined.3. After MTG fermentation condition was determined, fermentation was performedin 7L fermentor. When agitator speed was 400 rpm and aeration rate was 1.25vvm, the dissolved oxygen (DO) stayed between 30～25%, which was able tomake Streptomyces hygroscopicus WSH03-13 grow and secrete MTG. MTGactivity reached 4.48 u/ml. It was showed that DO was the key factor during thefermentation process in 7L fermentor.4. The fermentation results in 30 L and 150 L fermentor were campared.Fermentation in 30 L fermentor could be carried out at the same conditions in 7 Lfermentor, for scale-up level was not high. MTG activity reached 4.26 u/ml.When fermentation in 150 L fermentor, the conditions have to be changed, for thedifferent size and equipments of fermentor. Based on DO concentration, the keyfactor of process, agitator speed and aeration rate were changed to be 250 rpmand 1.5 vvm, respectively. MTG activity reached 4.20 u/ml.5. Effects of hydroxylamine addition on the accumulation of MTG by Streptomyceshygroscopicus WSH03-13 were investigated. The results showed that theaddition of hydroxylamine induced MTG accumulation, optimum additionconcentration were 0.5 mM and the addition time of hydroxylamine was 38h.The maximum MTG activity in the culture broth was 5.3 u/ml and increased by32.5% compared with that of the control.