Structure And Property Analysis Of A Cold-adapted ?-galactosidase From Rahnella Sp. R3 Isolated From Glacier

Cold-adapted enzymes,isolated from psychrotrophic or psychrophilic microorganisms are capable of functioning at low temperatures ranging from 0°C to 30°C,can be potentially used in biotechnology,bio-medicine,and food industry.Cold-adapted?-galactosidases?EC3.2.1.23?possess high catalytic rate at 4°C,thus they have great potential in dairy industry.A cold-adapted?-galactosidase from Rahnella sp.R3?R3-bgal?,which was isolated from a psychrotrophic microorganism discovered from the No.1 Glacier in the Tianshan Mountains in Xinjiang,China,can be functional and stable at 4°C and neutral pH.Although there are several reports about the isolation of new cold-adapted?-galactosidases,it is still lack of deeper information including enzyme characters and three-dimensional.Based on the cold-adapted?-galactosidases originated from Rahnella sp.R3,genetic engineering methods were used to construct engineered strains,and the recombinant enzyme was studied systemically.For further understanding of the enzyme,the three-dimensional structure was solved and studied,and these can provide important theoretical information for the engineering and industrial application of the protein.Major contents and results are as following:?1?The cold-adapted?-galactosidase isolated from Rahnella sp.R3 was cloned through degenerated primers and gene walking,and expressed in different expression systems.The entire R3-bgal gene has an open reading frame of 2064 bp encoding 687 amino acid residues.The nucleotide sequence was analyzed,submitted to Gen Bank and was assigned accession number KM486621.Three expression plasmids were constructed,and expressed in E.coli BL21?DE3pLySs??Bacillus subtilis WB800N?Pichia pastoris SMD1168H,respectively.The enzyme was expressed successfully in these three systems,and the induction conditions were optimized.?2?The best yield was got in E.coli after purification of the recombinant enzymes,secondly in Bacillus Subtilis.Moreover,it could be hardly expressed in Pichia Pastoris.While expressed in E.coli,the purified recombinant enzyme showed a specific activity of 27 U/mg,a single band of approximately 73 kDa on SDS PAGE,and 225 kDa on Native PAGE.The R3-bgal was a homo-trimer belonging to Glycoside hydrolase family 42.The hydrolysis characters toward natural substrate lactose and synthetic substrate were analyzed.The results showed that the optimum temperature of R3-bgal was 25°C,75%of the maximum activity was retained at 4°C,and it was stable under 45°C.R3-bgal was stable between p H 5.5-8.0 with an optimum pH of 7.0,and it was not inhibited by Na⁺and Ca²⁺,thus it has great potential in milk industry.The kinetic parameters indicated that R3-bgal had a higher affinity?K_m=2.2?0.59?for lactose at 4°C,and glucose was not an inhibitor while galactose was a noncompetitive inhibitor.The secondary structural was investigated by circular dichroism spectroscopy?CD?when R3-bgal was incubated under different conditions?temperature,pH,and metal ions?.?3?The initial crystallization screen of R3-bgal was performed by screening 432 conditions using the hanging-drop vapor-diffusion method.Single crystals were obtained under the crystallization condition of 0.2 M sodium acetate,0.1 M Tris-HCl?pH8.0?,and 30%?w/v?PEG1500 after stored at 20°C for 14-21 days.The crystals were immersed in a cryoprotectant solution?50%reservoir solution with 50%w/v PEG 400?and flash-cooled in liquid nitrogen before X-ray diffraction.The crystal showed a hexagon flake structure with a size of 0.1 mm x 0.35 mm.One hundred crystals were screened and the best crystal diffracted to 2.56?.It can provide enough information for protein three-dimensional structure.In the final refined structure,there were two trimers in the crystallographic asymmetric unit.There were 4,099 amino acid residues and a total of32,981 atoms,6 zinc ions,6 acetate ions,and 428 water molecules.The cell parameters were a=146.43?,b=106.83?,c=164,24?;?=90.00°,?=109.0°,?=90.00°,and the spacegroup was P12₁1.In the Ramachandran plot,96%amino acid residues were in the most favorite region and0.5%was outliers.The coordinates and structure factors for R3-bgal were deposited in the Protein Data Bank and the PDB ID is 5E9A.?4?The three-dimensional structure of R3-bgal was analyzed.The shape of the structure of R3-bgal resembles a flowerpot,a larger opening at the top?40??,and a smaller opening at the bottom?10??.An R3-bgal monomer consists of three domains.The catalytic domain is a TIM barrel that contains a?-barrel at the center with 8?-strands that is surrounded by 8?-helices.A pair of catalytic residues,E157 and E314,can be identified as the acid/base catalyst and the nucleophile,respectively.The reaction mechanism for R3-bgal is retaining.Domain B is essential for the stabilization of the structure of R3-bgal.Domain C is a structural element of GH42?-galactosidases.The cold-adaptation mechanism of R3-bgal was proposed through structural based alignment and sequence analysis of GH42?-galactosidases.It was found that intramolecular forces and structural flexibility might be important to the cold-adaptation of GH42?-galactosidases.?5?The cold-adapted?-galactosidase R3-bgal was encapsulated in gellan gum by a mild ionotropic gelation with two different cations(Ca²⁺and Mg²⁺).The hydrogel beads had a spherical shape with smooth and hard surface,and can be reused for many times after encapsulation.Moreover,they had better thermal stability,and shown to be more effective at low temperature?4°C?,can be applied at higher temperature?>45°C?;these hydrogel beads were stable in alkaline pH region,and the optimum retained at 7.0.The surface morphology and cross-sectional image of the freeze-dried gellan gum beads was examined by scanning electron microscope,and the dissimilarity between the two types of hydrogel beads was due to the different cross-linking effects of two cations.GG-Ca hydrogels can be applied in milk to hydrolyze lactose,and 0.8 U/ml GG-Ca hydrogels can hydrolyze over 70%lactose in milk within 7 h.Protein and fat did not have significant effect on the procedure of lactose hydrolysis.