A Multi-enzyme Self-assembly Study For The Production Of Glucaric Acid From Hemicellulose

Glucaric acid is a high value-added product that has been hailed as one of the "Top value added chemicals from biomass" and widely used in many fields.It has great potential economic value for emerging biomass energy.The traditional synthesis method of glucaric acid is chemical method,but it is limited by a large amount of reagent consumption and serious pollution.The microbial fermentation method is a common method to realize organic acid production.However,this method has some disadvantages.Therefore,the enzymatic which has efficient,safe and environmentally friendly become the first choice.UDH is an enzyme that is indispensable for the enzymatic production of glucaric acid.The high temperature can improve the substrate solubility,reduce industrial costs and prevent microbial contamination.Therefore,The dehydrogenase derived from Thermobispora bispora was selected.The combination of XynB and AguA and other enzymes is also required for the synthesis of high value-added glucaric acid from low-cost xylan.In order to enhance the synergism and catalytic efficiency of multiple enzymes,the multi-enzyme self-assembly was constructed based on the specific recognition mechanism of PDZ domain,which made the enzyme and enzyme spatially approached to form a substrate channel,which effectively improved the conversion efficiency.The sequence of GVKESLV was chosen as PDZIig,and it was fused to the C-terminal of glucuronidase gene,and the recombinant plasmid pHsh-aguA-PDZlig was constructed and induced in E.coli.The enzyme activity(0.7 U/mL and 0.3 U/mg)of AguA-PDZlig were significantly increased 10.7 and 11.5 fold compared to the AguA(0.06 U/mL and 0.024 U/mg).The optimum pH of AguA and AguA-PDZlig was found to be 7.5 and 8.5.respectively,and the optimum temperature was 85? and 75? respectively.The temperature stability of AguA-PDZlig decreased.The kinetic parameters of AguA and AguA-PDZlig were analyzed.The Km were 0.148±0.0062 and 0.166±0.0051 g/mL,respectively,and the kcat were 70.67±0.929,322.56±11.26 s-1,kcat/Km were 477.48 ±2.625,2209.3±26.34 s-1.(g.mL-1)-1,the kcat/Km of AguA-PDZlig increased by 3.6 fold.The dehydrogenase gene from Thermobispora bispora was selected to construct the recombinant plasmid pET-20b-Tb-UDH.The PDZ(PSD95/Dlgl/zo-1)domain was selected and the PDZ was fused to the C-terminal of the aldehyde dehydrogenase gene to construct the fusion proteins Tb-UDH-LR-PDZ?Tb-UDH-LF3-PDZ?Tb-UDH-LF4-PDZ?Tb-UDH-LF5-PDZ?Tb-UDH-LF6-PDZ.It was found that only Tb-UDH-LR-PDZ can be purified by Ni column.The enzymatic activities and specific activities of Tb-UDH-LR-PDZ(2.54 U/mL and 3.35 U/mg)were significantly reduced by 89%and 84.8%compared to Tb-UDH(23.98 U/mL,22 U/mg).The optimum pH of both Tb-UDH and Tb-UDH-LR-PDZ were 7.0,and the optimal temperature was 60?.The kinetic parameters of the Tb-UDH and Tb-UDH-LR-PDZ were analyzed.When glucuronic acid was used as substrate,Km of Tb-UDH and Tb-UDH-LR-PDZ were all 0.165 mM,kcat was 65.882 and 9.14 s-1,and kcat/Km was 400.014 and 55.55 s-1.mM-1,respectively.For galacturonic acid,Km of Tb-UDH and Tb-UDH-LR-PDZ were 0.115 and 0.116 mM,kcat was 58.333 and 4.34 s-1,and kcat/Km was 509.018 and 37.59 s-1.mM-1 respectively.For NAD,Km of Tb-UDH and Tb-UDH-LR-PDZ were 0.120 and 0.139 mM,kcat was 74.666 and 17.75 s-1,kcat/Km was 622.220 and 127.8 s-1.mM-1 respectively.In order to improve the enzymatic hydrolysis efficiency of AguA and UDH,the optimal hydrolysis conditions were optimized.The optimum conditions of AguA were 4%substrate concentration,5 U/g enzyme,pH8.5,temperature 75?,reaction time 4 h,the glucuronic acid release reached the maximum of 2.6 mg/mL under these optimal conditions.The optimal conditions of UDH were substrate concentration 1 mM,enzyme dosage 60 U/g,pH 7.0,temperature 60?,reaction time 30 min,the release of glucosaccharic acid reached up to 170 mg/L under these optimal conditions.The results showed that the molar ratio of Tb-UDH-LR-PDZ to AguA-PDZlig was 5:1 and the release of glucaric acid reached 38 mg/L when assembled for 4 h.After self-assembly,the activity of AguA-PDZlig decreased compared with that before assembly.Tb-UDH-LR-PDZ activity increased.The formation of self-assembly was verified by live gel and electron microscopy.No significant changes were found in the optimal pH,temperature and their stability of the enzyme solution before and after self-assembly.In the 4%beech xylan hydrolyzate as a substrate,adding 10 U/g substrates before and after the enzyme assembly,in 65?,pH8.0 imidazole buffer for 30 min,before self-assembly solution released 600 mg/L of glucaric acid,and the after self-assembled enzyme solution released 700 mg/L of glucaric acid.This suggests that the double enzyme assembly may form a substrate transport channel between the two enzymes,thus increasing the release of the target product.