Catalytic Mechanism And Rational Transformation Of β-Cyclodextrin Glycosyltransferase

Cyclodextrins(CDs)are well known as pharmaceutical excipients and are a family of cyclic oligosaccharides with hydrophilic external and hydrophobic central cavities.It has the ability to form water-soluble inclusion complexes with many insoluble lipophilic molecules.Therefore,CDs are widely used in pharmaceutical,textile,agriculture,cosmetic,chemical and food industries.CDs are usually produced from starch or starch derivatives by the catalytic action of cyclodextrin glycosyltransferase(CGTase,EC 2.4.1.19).CGTase,an important member of the alpha-amylase family,is an extracellular enzyme that catalyzes the cleavage of α-1,4 linkages in starch or polysaccharides into CDs.CGTase catalyzes four reactions: cyclization,coupling,disproportionation and hydrolysis.In the above four reactions,CDs are produced by cyclization as the characteristic reaction of CGTase,that is,CGTase catalyzes the α-glycosidic bond cleavage of starch,and some donors are separated to form CDs as the receptor.CGTase catalyzes the formation of CDs by starch.The main product is a mixture of α-,β-and γ-CD(with six,seven,and eight glucose units,respectively),which adversely affects the separation and purification of CDs.Therefore,it is important to increase the specificity of the catalytic products for the actual production of a particular type of cyclodextrin,i.e.the product specificity.In recent years,increasing the product specificity of cyclodextrin has been popular.Two main effective strategies are to add organic solvents to the reaction mixture and change the structure of CGTase using sitedirected mutagenesis.Based on β-CGTase gene from Bacillus cereus,a series of studies have been carried out to improve the product specificity of enzymatic synthesis of CDs,and a facile,efficient and highly selective enzymatic synthesis from starch to β-CD using β-cyclodextrin glycosyltransferase method for synthesizing CDs has been established.The relationship between the properties of amino acids at key sites and the catalytic properties of enzymes was explored.Firstly,this thesis studies the catalytic mechanism of β-CGTase enzymatic synthesis of CDs,and optimizes the effect of pretreatment of substrate starch on the yield of CDs,the catalytic conditions of enzymatic synthesis of CDs,and the reaction of alcohol solvent system to synthesize CDs.condition.We found that the substrate starch can greatly increase the yield of CDs after steam treatment or gelatinization.The optimum pH value of β-CGTase for the synthesis of CDs is 8.5,and the optimum catalytic temperature is 55 °C.Alcohol solvents affected the yield and product selectivity of CDs.Among them,tertiary alcohol affected CDs yield(from 54.95% to 68.21%),and secondary alcohol affected product selectivity(β-CD/γ-CD: from 6.25 to 8.05).Secondly,the mechanism of action of alcohol solvent and β-cyclodextrin glycosyltransferase was explored by fluorescence quenching and thermodynamic calculation.Fluorescence quenching analysis showed that the binding constant and entropy of the solvent had an effect on the yield and product specificity of CDs.The thermodynamic parameters revealed that the structure of β-CGTase could be caused by hydrophobic interaction between the alcohol solvent and β-CGTase.The changes,which have an effect on the catalytic properties of β-CGTase,alter the yield and product specificity of enzymatic synthesis of CDs in this system.Finally,sequence alignment of CGTase genes from various sources revealed that the 47 th amino acid of Bacillus cereus-derived CGTase had an important effect on the product specificity of CGTase-synthesized CDs.Using the recombinant plasmid pET22b/CGTase as a template,the alanine was successfully mutated by genetic modification,and 19 mutant strains were successfully constructed.The catalytic performance of the mutant enzyme was detected by CDs synthesis reaction,and the site was analyzed.The relationship between amino acids and CGTase product specificity.It was found that the 47 th amino acid position in the active region is a hydrophobic amino acid,which can increase the yield of CDs(β-CD,γ-CD),and the catalytic product tends to synthesize macrocyclic CDs(γ-CD,etc.).This suggests that increasing the hydrophobic interaction near the key amino acids of β-CGTase may be an effective strategy for the catalytic synthesis of macrocyclic CDs.This study further clarify the catalytic mechanism of β-cyclodextrin glycosyltransferase,which provides a reference for the directed evolution and practical application of the enzyme.