Expression And Activity Analysis Of Xylanase Gene In Synechocystis Sp. PCC6803

Xylanase is a complex enzyme system that degrades xylan into xylo-oligosaccharides and xylo-monosaccharides throughβ-1,4-D-endoxylanase andβ-D-xylanase.With the development of science and technology,xylanase has been widely used in papermaking,fruit and vegetable,feed,energy and food.Compared with heterotrophic microorganisms,Synechocystis sp.PCC6803 can realize its own growth and foreign gene expression by using CO₂,light energy and some simple inorganic salts.In this study,xylanase gene sxyl containing signal peptide and xyl without signal peptide from Bacillus subtilis were successfully integrated into the genome of Synechocystis sp.PCC6803.The xylanase photosynthetic organism expression engineering strain was constructed.Compared with E.coli and S.cerevisiaet expression systems,the engineered strain can use light energy,simple inorganic salts and carbon dioxide in the air to synthesize the target product,greatly reducing production costs.At the same time,the culture condition is simple,no toxin,and the downstream purification cost is reduced.The pBluescript SK was used as the starter plasmid to connect with the upstream arm and photosensitive promoter Pups,Sdw,terminator Eter and Kana resistant KanR gene fragments through DNA recombination technology to construct the integrated vector pBluesys used in this study.Xylanase genes sxyl and xyl were linked with linearized pBluesys plasmids by recombinant enzyme,and homologous recombinant expression plasmids pBluesys-sxyl and pBluesys-xyl were constructed,and then transferred into Syzymosis PCC6803 by natural transformation method for further screening and verification.The xylanase-producing engineered algae strains PSNC9 and PSXC9 were obtained.The enzyme activities of PSNC9 and PSXC9 were 1.3801U/mL and 1.4161U/mL respectively,which were about 3.5 times higher than those of wild-type algae.However,no exocrine secretion was detected in PSNC9 containing signal peptide.SDS-PAGE was used to verify the recombinant enzyme protein size of PSXC9.The enzymatic properties of recombinant xylanase were investigated by different pH,temperature,metal ions and EDTA.The results showed that the optimal catalytic pH value was 6,the optimal catalytic temperature value was 60℃,Cu²⁺,Ca²⁺and EDTA had a certain inhibitory effect on the enzymatic reaction,and Na⁺and Fe³⁺had a certain promoting effect on the enzymatic reaction.Among them,Fe³⁺had the most significant promoting effect,and the enzyme activity increased by about 11%,while Mg²⁺and K⁺had little effect on the enzymatic reaction.By plotting the growth curves of the wild-type algae strain and the engineered algae strain PSXC9,and comparing the contents of chlorophyll-a,total carotenoids and cyanobacterin in the two different algae strains,it was found that the engineered algae strain PSXC9 had the performance of growth lag.From three aspects of culture conditions:temperature,pH and light intensity,the optimal conditions of engineering algae strain PSXC9 were explored,and it was found that the growth condition of engineering algae strain PSXC9 was the best under pH 9,temperature 28℃and light intensity 1400L Lux.