Recombinant Expression And Characterization Of An Exo-?-1,3-galactanase From Penicillium Oxalicum

?-galactanase is a kind of glycosidase to specifically hydrolyze the glycosidic bond between galactose residues in galactan and arabinogalactan mainly including?-1,3-,?-1,4-and?-1,6-galactanase.?-galactanase is widely used in the analysis of polysaccharide structure,construction of medicinal plant fingerprints,preparation of galactooligosaccharides and food quality improvement,among them,?-galactanase palys an important roles in the structural analysis of polysaccharides which rich in?-galactose such as arabinogalactan.Arabinogalactan is a complex polysaccharide with various physiological activities such as immune regulation,anti-oxidation and neuroprotection,which has the potential to be applied in many fields.Analysis of the fine structure of arabinogalactan could help to studying its relationship between structure and function deeply.In this paper,an exo-?-1,3-galactanase from Penicillium oxalicum was cloned,expressed and characterized,which lays a foundation for the study of the structure of arabinogalactan.The main results are as follows:The recombinant protein PoGal3 was expressed and purified.Recombinant strain of Rosetta-pet32a?+?-pogal3 and GS115-pPICZ?A-pogal3 were successfully constructed.The exo-?-1,3-galactanase PoGal3 was expressed and obtained in Pichia pastoris recombinant strain GS115-pPICZ?A-pogal3.1.2 mg of PoGal3 was successfully purified from 400 mL of culture medium by two steps of ammonium sulfate precipitation and gel filtration chromatography.The induced culture conditions of the recombinant Pichia pastoris strain were optimized,the result of optimization is that the pH of the BMMY induction medium was determined to be 6.0,the OD_{600 nm} of the initial bacterial solution was 1.0,the methanol addition amount was 2.0%?v/v?and the induction time was 2 days.The expression of PoGal3was increased by 3.3 times in optimized conditions.PoGal3 consists of 451 amino acids with a molecular weight of 48.5 KD and an isoelectric point of 6.60,which is a highly stable hydrophilic protein.The enzymatic properties and function of PoGal3 were characterized.Firstly,1,3-?-galactan AG-P-I was prepared by periodate oxidation and Smith degradation,which is high purity and homogeneity with a small amount of side chain structure.Then,the specific enzyme activity of the enzyme was determined as 9.3±0.9 U/mg of crude enzyme and 50±3.7U/mg of pure enzyme with the substrate of AG-P-I.The results of characterizing properties of PoGal3 showed that the optimal reaction temperature of PoGal3 was 40°C,and the optimum pH was 5.0,Zn²⁺could increase the enzyme activity to 128.6%,while Li⁺,Mg²⁺,Cu²⁺,Fe³⁺,Hg²⁺,Mn²⁺,Ni⁺,EDTA and SDS could weaken the enzyme activity.Substrate specificity results indicated that PoGal3 was an?-1,3-galactanase with little activity of?-1,3-glucanase activity,and it was not worked on 1,4/6-?-galactan,1,4/6-?-glucan,xylan and arabinan.PoGal3 was used in enzymatic hydrolysis and analysis with AG-P-I and two classical type II arabinogalactan?LWAG and Acacia?,and the results showed that PoGal3 was an exo-?-1,3-galactanase which could degrade type II arabinogalactan with a small molecular weight and a potential tool for the structural analysis of arabinogalactan.