The Site-Directed Mutation Of β-Cyclodextrin Glycosyltransferase And Optimization Of Mutant Strains Fermentation Conditions

β-Cyclodextrin glucanotransferase (β-CGTase, EC2.4.1.19) belongs to the family of a-amylase, which is a large family of starch processing enzymes. β-CGTase is able to catalyze the intramolecular or intermolecular transglycosylation reaction:cyclization, coupling, hydrolysis and disproportionation. Due to its hydrophilic outer surface and nonpolar hole, the product of starch cyclization, β-cyclodextrin, has functions of stabilization, solubilization, slow-release and odors-masked. And as molecular capsule and emulsifier, β-cyclodextrin is widely used in food, harmaceutical, cosmetic, agricultural and chemical industries.As the key enzyme of β-CD synthesizing, β-CGTase can hydrolyse starch into β-cyclodextrin by its transglycosylation activity. The present researches focus on site-directed mutation of β-CGTase in vitro to increase the yield of β-CD. In the present study, we systematically investigated the in vitro site-directed mutation of β-CGTase, optimization of Mutant Strains fermentation conditions and zymologic characteristics of site-directed mutation enzyme. And, we further carried out fermenter scaling-up to meet industrialized production. These results provided rationale technique supporting for industrialized production of β-CD by enzymic method.The main study subjects as follows:1. The crystal structure of β-CGTase has already been determined in its native form. On the basis of this, we analyze the β-CGTase by bio-informatics, and then performed Y127F, H167C,R254R and D355R mutation within the conserved domain of gene by Site-directed Mutagenesis to obtain the recombinant plasmids (p-T::CGTasey127F, p-T::CGTaseH167C p-T::CGTaseR254F and p-T::CGTaseD355R) on the basis of recombinant engineering strain pUC-18::CGTase, through primer design the gene with His6-Tag at5’end. Based on these studies, we obtained four mutated β-CGTase genes with His6’Tag, and then integrate them into the prokaryotic expression vector pUC-18. Moreover, the recombined plasmids (pUy127F, pUH167C, pUR254F and pUD355R) were transformed into E.coli BL21(DE3) competen cell and induced by lactose. The mutant strains were named p-YF,p-HC,p-RF and p-DR,respectively. The fusion proteins His6·Tag of four mutated genes with a relative molecularmass of about70KDa were detected by SDS-PAGE and western blotting, the same as the theory value of β-CGTase. The mutant enzyme activity was assayed by the blue value method. Measured by this method, the activity of mutant strains p-YF,p-HC,p-RF and p-DRare1224U/mg,1228U/mg,1147U/mg,976U/mg,respectively.They are obviously higher than original strain pET-28b::CGTase(621U/mg)。2. The properties of the P-CGTase from mutant strains were studied, including temperature,pH and tempurature or pH on the stability of the β-CGTase. The experimental result shows the mutant enzymes have the same properties with the initial enzyme. The optimization temperature and pH of the mutant enzymes were60℃and pH6.0. They were not thermostable as they nearly lost activity after being incubated at60℃for60min,yet they were quite stable in wide pH range of5.0～8.0. By analyzing the dynamics parameters of mutant enzymes, results showed that the values of Km were lower than original strain (0.019mg/mL) and the Vmax were higher than original strain. The value of Km are0.015mg/mL and0.017mg/mL of p-HC、 p-YF and p-RF、p-DR, respectively. The improvement of the activity of enzyme is realized by strengthening the affinity between enzyme and substrate3. Corn starch was selected as the best carbon source for the β-CGTase production of mutant strains. The complex nitrogen sources of Peptone and corn steep liquor was also selected as the most suitable nitrogen sources. Cell growth and (3-CGTase synthesis were significantly promoted by the addition of magnesium and phosphates. The initial pH, fermentation temperature, inoculation proportion and rotation speed of shaker also directly or indirectly effect the growth of strains and the production of P-CGTase. The best composition of fermentation medium and the parameters of fermentation were confirmed through single factor experiment,4. The optimal medium and conditions of fermentation which were determined by Box-Benhnken’s central composite design and response surface analysis. the importance order of factors on enzyme production was obtained as such, corn starch (X1)> Peptone and com steep liquor (X2)>MgSO4·7H2O (X3)>K2HPO4·3H2O (X4); fermentation temperature (X2)> initial pH (X1) rotation speed of shaker (X4)> inoculation proportion (X3)。 Compared to the result obtained before optimization, the optimal fermentation medium (%, W/V) of YF:corn starch1.1, Peptone1.1, com steep liquor4.6, MgSO4·7H2O0.02, K2HPO4·3H2O0.12; and the optimal fermentation conditions:initial pH of fermentation medium8.7, fermentation temperature37℃, inoculation proportion5.3%, rotation speed of shaker201r/min,the maximal yield of β-CGTase from p-YF reached4235U/mL, which was increased by1.99-folds; the optimal fermentation medium (%, W/V) of HC:corn starch1.1, Peptone1.1, com steep liquor4.4, MgSO4·7H2O0.02, K2HPO4·3H2O0.10; and the optimal fermentation conditions: initial pH of fermentation medium8.4, fermentation temperature37.4 ℃, inoculation proportion5.0%, rotation speed of shaker202r/min, the maximal yield of β-CGTase from p-HC reached4355U/mL, which was increased by1.93-folds; the optimal fermentation medium (%, W/V) of RF:corn starch1.0, Peptone1.1, corn steep liquor4.3, MgSO4·7H2O0.02, K2HPO413H2O0.10; and the optimal fermentation conditions:initial pH of fermentation medium8.6, fermentation temperature37.3℃, inoculation proportion5.3%, rotation speed of shaker202r/min, the maximal yield of P-CGTase from p-RF reached3935U/mL, which was increased by1.98-folds; the optimal fermentation medium(%, W/V) of DR:corn starch1.2, Peptone1.1, corn steep liquor4.4, MgSO4·7H2O0.02, K2HPO4·3H2O0.10; and the optimal fermentation conditions:initial pH of fermentation medium8.4, fermentation temperature37.6℃, inoculation proportion5.0%, rotation speed of shaker201r/min, the maximal yield of β-CGTase from p-DR reached3640U/mL, which was increased by2.19-folds.5. The influences of various agitation speeds, dissolved oxygen(DO)levels, different temperature at constant pH on β-CGTase production by mutant strains were investigated in a5L fermentor.The results show that,when pH was controlled at8.7with feeding1.0mol/L NaOH or0.2mol/LHC1, the agitation speed was controlled at300rpm, Do was controlled at20%, temperature was controlled at37℃, and fed corn starch at50g/L, corn steep liquor at20g/L lactose at50g/L, the production of β-CGTase and cell concentration were both improved obviously, the maximal yield of β-CGTase reached2536U/mg, which was increased by4.08-folds of pUC-18::CGTase621U/mg.6. The cyclization activity of the P-CGTase was detected using HPLC-MS, the molecular weight of1134.4, with the β-CD standard consistent with the molecular weight substances, the β-CGTase had the transfer Glucosyl activity to β-CD from the spectrum of a graph.