Studies On The Glucosylation Of Phenol Compounds By Branching Sucrase GtfZ-CD2 From Lactobacillus Kunkeei

Glycoside hydrolase 70 family(GH70)glucansucrases,which are widely found in lactic acid bacteria,can catalyze the synthesis of structurally diverse a-glucans using sucrose as a substrate.In addition to polysaccharide and oligosaccharide synthesis,glucansucrases are also capable of catalyzing the glucosylation of organic molecules with hydroxyl groups and are proven to have a wide acceptor substrate promiscuity.Phenolic compounds are a class of compounds containing an aromatic ring with hydroxyl groups.According to the structure,they can be divided into monohydric phenol?dihydric phenol and polyphenols such as flavonoids and resveratrol.Studies have shown that phenolic compounds have antioxidant,anti-tumor and other physiological functions.However,polyphenols generally show low solubility and low bioavailability in their utilizations,which is one of the main constraints that limit the practical application of polyphenols.In the present thesis,we investigated the glucosylation of phenol compounds using a branching sucrase GtfZ-CD2 from Lactobacillus kunkeei DSM 12361.The main findings of the present thesis listed as follows.1.The branching sucrase GtfZ-CD2 from Lactobacillus kunkeei DSM 12361 was shown to catalyze the glucosylation of catechol,resorcinol and hydroquinone to produce their monoglycosylation and diglycosylation products.At a donor substrate concentration of 1 M sucrose,when the concentrations of catechol,resorcinol and hydroquinone are 200 mM,25 mM and 25 mM,respectively,the total glucosylataon efficiency reached 88%,58%and 31%.When considering both glycosylation efficiency and the yield of glycosylation products,200 mM of catechol,resorcinol and hydroquinone and 1 M sucrose were selected as the optimum conditions.Under these conditions,the glucosylation efficiency of catechol,resorcinol and hydroquinone is 88%,36%and 15%,respectively.It is worth noting that the glucosylation efficiency of catechol is significantly higher than resorcinol and hydroquinone.The kinetic analysis showed that the Km of catechol was significantly higher than that of resorcinol and hydroquinone,indicating that its binding affinity was significantly higher than that of resorcinol and hydroquinone.2.Through structural simulation of branching sucrase GtfZ-CD2 and docking analysis with catechol,resorcinol and hydroquinone,the site-specific mutation site was designed and the molecular modification of branching sucrase was completed,and the mutants with high glycosylation efficiency were obtained.According to the structural simulation of GtfZ-CD2 and its docking analysis with hydroquinone,6 amino acid residues at receptor substrate binding sites,including L1559,L1560,L1590,A1635,F1638 and F2065,were selected for semi-rational mutation.Among the 81 mutants constructed,the mutants L1559K and F2065V significantly improved the glycosylation efficiency of resorcinol and hydroquinone,by 24.3%and 15.3%,respectively.The glucosylation efficiency of hydroquinone of mutant F2065V was improved by nearly 3 times.3.Using the mutant enzyme molecule F2065V as a catalyst,the conditions for the production a-arbutin by hydroquinone glucosylation were optimized.Based on the single factor experiments,the effects of branching sucrase enzyme concentration,hydroquinone concentration,sucrose/hydroquinone molar ratio on the production of a-arbutin was investigated by response surface analysis.The optimal conditions for production of ?-arbutin were determined to be:1.5 U/mL branching sucrase GtfZ-CD2,hydroquinone concentration of 206 mM,sucrose/hydroquinone molar ratio of 5.3:1.Under the conditions,the output of theory a-arbutin reached 13.699 mM4.The glucosylation of flavonoids compounds by branching sucrase GtfZ-CD2 were preliminarily investigated.The results showed that GtfZ-CD2 is capable of glucosylating quercetin,morin,naringenin,luteolin,kaempferol,genistein,apigenin,catechins and resveratrol.A variety of polyphenols glucosylation products with different number of glucosyl unit were produced.Among them,GtfZ-CD2showed a higher glucosylation efficiency on apigenin,quercetin,kaempferol,catechins and resveratrol,reaching up to 93%,91%,89%,88%and 86%,respectively.