Expression And Characterization Of Beta-1,6-Galactosidases From Lactobacillus Plantarum

Galactosidase is a kind of glycosidase that hydrolyzes galactosidic bonds.It is not only used in food,pharmaceutical and papermaking widely,but also can degrade galactan and galactooligosaccharides.At the same time,it can also be used in the modification and analysis of polysaccharide structure.Arabinogalactan is an important galactan,which is rich in plant tissue and has various functions.The structure-activity relationship of arabinogalactan is always limited by structure analysis,so it is of great significance to find a specific galactosidase.Lactobacillus plantarum as a probiotic can metabolize galactan,and its genomics research shows that it contains a variety of?-galactosidases.In this thesis,the recombinant expression and enzymatic properties of?-galactosidase from Lactobacillus plantarum were studied in order to find and prepare the enzyme that can be used to analyze arabinogalactan.The main results are as follows:1.lpgal42 and lpgal2,which belong to glycoside hydrolase family 42?GH42?and family2?GH2?,were successfully cloned from Lactobacillus plantarum genome.The target gene was connected to pET-28a?+?vector and transferred to E.coli BL21?DE3?competent cells to obtainrecombinantstrainslpgal42-pET-28a?+?-BL21?DE3?and lpgal2-pET-28a?+?-BL21?DE3?.The recombinant strain was induced at 25? 0.5mM IPTG for 16 h to express the soluble recombinant proteins LpGal42 and LpGal2.After purification by Ni sepharose fast flow affinity chromatography,the target protein with high purity was obtained with molecular weights of 55 kDa and 40 kDa respectively.In order to improve the expression of protein,the recombinant strain was fermented in 5 L fermentor.The expression of LpGal42 and LpGal2 was 10.2 and 7.6 times as much as that of shake flask culture respectively2.The enzymatic properties of the recombinant enzymes LpGal42 and LpGal2 were studied using?-1,6-galactan??-1,6-G?and p-nitrophenyl-?-D-galactoside?pNP?Gal?as substrates.The results showed that the specific enzyme activity of LpGal42 is 0.2 U/mg,the optimal pH is 6.0,and the optimal reaction temperature is 40?.The specific enzyme activity of LpGal2 is 2.2 U/mg,the optimal pH is 7.0,and the optimal reaction temperature is 45?.The results of ion tolerance experiments showd that Mg²⁺and Na⁺can promote the enzymatic activities of the two enzymes.Among them,Mg²⁺increases the enzymatic activities of LpGal42 and LpGal2 to 115%and 126%respectively,while Cu²⁺,Fe³⁺,Ba²⁺,Hg²⁺,Zn²⁺,Li⁺,Ni²⁺and SDS have strong inhibition on these two enzymes.3.The functions of LpGal42 and LpGal2 were characterized by a series of p-nitrophenyl glycosides,?-1,3/1,4/1,6-galactobiose,?-1,3/1,4/1,6-galactan as substrates.LpGal42 can only degrade?-1,6-galactan,and?-1,6-galactobiose is the main product.LpGal2 can degrade pNP?Gal,?-1,6-galactobiose and?-1,6-galactan,releasing galactose.The hydrolysis results of LpGal42 and LpGal2 on other types of polysaccharides showed that LpGal42 and LpGal2could not degrade polysaccharide substrates such as?-1,3/1,4-galactan,glucan,xylan and arabinan.The above results indicated that LpGal42 is a specific endo-?-1,6-galactosidase,while LpGal2 is an exo-?-1,6-galactosidase,which is the first time to find an exo-?-1,6-galactosidase with good specificity.4.The recombinant LpGal42 and LpGal2 were used for enzymatic hydrolysis with type ? arabinogalactan?AG-??as substrate.Due to the complex structure of AG-? and the variety of glycosyl linkages,it was found that two?-1,6-galactosidases could not degrade AG-? either alone or in combination.However,LpGal42 and LpGal2 can play a role in the hydrolysis of AG-? with exo-?-1,3-galactosidase PpGal43 and endo-?-1,3-galactanase FvEn3Gal.This result showd the potential of LpGal42 and LpGal2 in AG-? enzymatic modification and structural analysis.In conclusion,this thesis discovered and prepared two specific?-1,6-galactosidase LpGal42 and LpGal2.The research results not only enriched the types of?-1,6-galactosidase,but also provided new tools for the enzymatic modification and structural analysis of galactose containing polysaccharide compounds such as AG-?.