Heterologous Expression And Enzymatic Properties Of α-agarase From Catenovulum Agarivorans

Agarooligosaccharides,functional oligosaccharides composed of 2 to 20 D-galactose linked byα-1,3-glycosidic bonds andβ-1,4-glycosidic bonds,have favorable physiochemical properties such as anti-inflammatory,anti-oxidation,inhibition of tumor cells,improvement of intestinal environment and skin whitening,thus leading to the profound application in food,medicine and chemistry industry.The biological preparation of agarooligosaccharides is to use agarase to hydrolyze agar,which has the advantage of strong product specificity and milder reaction conditions,and is considered to be an important method for the future commercial production of agarooligosaccharides.However,the development process of enzymatic preparation of agarooligosaccharides has been greatly hindered by the lack of enzyme category development,the lack of in-depth research on enzyme properties,and the low expression efficiency and catalytic activity.In order to solve the above problems,we firstly identified anα-agarase from Marine microorganism Catenovulum agarivorans（Ca-AGA）and constructed efficient and stable expression systems in Escherichia coli,Bacillus subtilis and Pichia pastoris.We further analyzed the enzymatic properties of the purified recombinant Ca-AGA,especially its cold adaptation,cold activation and mechanism.Firstly,the basic physical and chemical properties of Ca-AGA were predicted by bioinformatics methods to provide a theoretical basis for the construction of expression.The synthesized target gene was inserted into the vector fragment,and the genetically engineered bacteria E.coli BL21（DE3）（pET-28a（+）/ca-aga）,P.GS115（p PIC9K/ca-aga）,B.Subtilis WB600（p P43NMK/ca-aga）were successfully constructed.It was found that there were significant differences in the expression level and enzyme activity of the genetically engineered bacteria,the highest enzyme activity was E.coli BL21（DE3）（pET-28a（+）/ca-aga）,which was 17.6 U/m L.At the same time,the medium components and fermentation conditions of E.coli BL21（DE3）（pET-28a（+）/ca-aga）were optimized.The highest hydrolytic activity of the recombinant Ca-AGA reached up to 91.7 U/m L when it was incubated in the optimized medium containing 12 g/L yeast extract,24 g/L tryptone,5 g/L sucrose,2.3 g/L KH₂PO₄ and 12.5 g/L K₂HPO₄（p H 9.0）at 25℃for 16 h.0.005 m M IPTG after fermentation for 3 h was the most suitable for expression of the enzyme.Secondly,the Ca-AGA prepared by E.coli BL21（DE3）（pET-28a（+）/ca-aga）was isolated and purified by nickel column affinity chromatography to analyze its enzymatic properties.It was found that the optimum reaction temperature of Ca-AGA was 33℃,but it still had high catalytic activity at 0℃and 25℃,indicating that CA-AGA had strong cold adaptation.In addition,the optimum p H of Ca-AGA was 8.0,and when incubated in p H6.0～11.0 buffer for 6 h,it still maintained more than 80%of the enzyme activity,indicating that the enzyme had good p H stability in neutral and alkaline conditions.The inhibition degree of EDTA at 5 m M was 90%,indicating that the enzyme activity was dependent on metal ions.1 m M Ca²⁺significantly activated the enzyme activity,and the degree of activation was increased by 20.15%.Analysis of Ca-AGA hydrolysates by HPLC revealed that the enzymatic hydrolysis products of Ca-AGA were agarobiose（A2）,agarotetraose（A4）and agarohexaose（A6）.Subsequently,we firstly predicted the structure of Ca-AGA using AlphaFold2 protein modeling software and obtained its structural information.The results showed that Ca-AGA comprised a catalytic domain（V780～H1431）and three carbohydrate-binding modules（CBM）（G631～T779,Q181～H320,and E27～H131）.The catalytic domain and CBMs were linked and adjusted in relative position by flexible Linker.In order to explore the cold adaptation mechanism of Ca-AGA,the structural characteristics of Ca-AGA were analyzed.Firstly,the surface charge distribution of Ca-AGA structure was analyzed.The results showed that there were a large number of acidic amino acid residues on the surface of Linker and CBMs,and the net charge density was as high as-18%,-11.8%,-10.1%and-12%,which was conducive to the solubility and molecular motility of the protein at low temperatures and thus improving enzyme activity.In order to verify the effect of Linker and CBMs on Ca-AGA adapting cold,the Linker and CBMs were truncated to construct mutants:truncation of CBM1（ΔCBM1）,truncation of CBM2（ΔCBM2）,truncation of CBM3（ΔCBM3）and truncation of Linker（ΔLinker）.The relative hydrolytic activity ofΔCBM2 andΔLinker mutants decreased by9.8%and 19.9%at 10℃,respectively.We further constructed the mutant which truncated CBM2 and Linker region（ΔCBM2L）,the optimum reaction temperature ofΔCBM2L was increased to 40℃,and the relative hydrolysis activity ofΔCBM2L decreased by 59.97%at10℃,respectively.The stability ofΔCBM2L at low temperature decreased compared with the wild-type.The wild-type Ca-AGA could maintain more than 95%activity at low temperature for 2 h while theΔCBM2L only had 73.5%initial activity after 2 h incubation.These results suggested that Linker-CBM played an important role in regulating the cold adaptation of Ca-AGA.In addition,in view of the good cold adaptation of Ca-AGA,we explored the use of Ca-AGA for DNA gel recovery and optimized the recovery conditions.The results showed that when Ca-AGA（40 U）was added to the recovery system at ambient temperature for 6 h,the DNA-containing gel could be completely hydrolyzed.Finally,the DNA recovery rate reached 78%.Finally,Ca-AGA could be increased by 5 times after 6 h incubation at low temperature（-20℃～0℃）.In order to investigate this activation,intrinsic fluorescence and circular dichroism（CD）were used to determine the secondary and tertiary structural changes of Ca-AGA before and after 6 h incubation at 0℃.The results of CD spectroscopy showed that theα-helix in the enzyme protein increased from 12.2%to 22.5%after low temperature incubation,and the random coil decreased from 42.3%to 33.4%after low temperature incubation;the results of intrinsic fluorescence spectroscopy showed that the FI_max of the enzyme protein increased sharply from 1329 to 2593,and theλ_max was slightly blue-shifted from 338 to 330.All results indicated that low temperature incubation could make the protein form an effective secondary structure folding,and the hydrophobicity in the protein environment was enhanced,leaded Ca-AGA to fold from loose packing state to compact state.