Secretion Of Cyclodextrin Glycosyltransferase In Komagataella Phaffii And Its Characterization Analysis

Cyclodextrin（CD）is applied in food,pharmaceutical,cosmetic industry widely.Generally,CD is prepared by CD glycosyltransferase CGTase process.Heterologous expression become the major approach of CGTase production because low productivity of CGTase from wild strains.In this research,cgt gene was high-level expressed in Komagataella phaffii.The process of CGTase expression was optimzed in flask.And the characters of CGTase was investigated.The chemical stabilizer was developed to enhance the stability of CGTase.Finally,the bioconversion process was carried out for CD production.The main results are stated as following:（1）In this study,cgt gene from Bacillus pseudalcaliphilus was expressed in K.phaffii after codon optimization and expression vector selection.Theβ-CGTase activity in the transformant reached 3885.09 U/ml,which is the highest value reported so far,at28℃,6%inoculum ratio,and 1.5%methanol addition per 24 h of incubation.The highestβ-CGTase activity is 25.1 fold of initial K.phaffii CGTase,27.8 fold CGTase activity of wild starin,3.0 fold of recombinant E.coli.The highestγ-CGTase activity is7.3 fold of initial K.phaffii CGTase,15.1 fold CGTase activity of wild starin,8.7 fold of recombinant E.coli.The specific activity ofβ-CGTase andγ-CGTase reached 29505.13U/mg and 580.15 U/mg after ammonium sulfate precipitation.（2）The optimal temperature and pH ofβ-CGTase was 50℃,pH6.0 and 9.0.The optimal temperature and pHγ-CGTase was 50℃and pH 8.0.Both theβ-CGTase andγ-CGTase activity decreased sharply when temperature was higher than 50℃.The glycosylation was considered as the reason of the difference between Komagataella-based CGTase and wild CGTase.（3）The mixture of chemical stabilizer was developed as 25%glycerol,15%PEG400,0.5 mmol/L CaCl₂.The optimal temperature increased from 50℃to 60℃.The activity ofβ-CGTase andγ-CGTase with stabilizer was 7.5 and 8.4 fold of CGTase without stabilizer.With the stabilizer at 4℃,CGTase activity kept 100%activity within37 d in this research.With the stabilizer at 37℃,the activities ofβ-CGTase andγ-CGTase was 2.6 fold and 3.3 fold of CGTase without stabilizer.Finally,bioconversion process of CD production was carried by CGTase with stabilizer.The total yield of CD produced by CGTase at 90℃was 100%.