Immobilized Bacteria With PH-response Hydrogel For Self-healing Of Concrete And Molecular Mechanism Study On Steroselectivity Of α Or β Hydroxysteroid Dehydrogenases

Immobilized bacteria with pH-response hydrogel for self-healing of concrete:Concrete is the most significant material in construction industry.However,there are still some defects which is hard to avoid,such as its brittleness.The appearance of cracks will not only damage the integrity and carrying capacity of concrete,which affects the normal use of concrete,but also shorten the using life of concrete.In recent years,a self-healing technology for concrete cracks based on microbial living activities has shown great advantages in a series of self-healing strategies.The research in this paper includes the cultivation of Bacillus pseudofirmus,which work for the healing of concrete;the preparation of encapsulating material hydrogel and their swelling properties in different pH conditions;the preparation of concretes and their mechanical performance assessment;the self-healing of concrete.The results are summarized as follows:(1)The swelling properties of calcium alginate was improved by adding chitosan.The results show that the swelling ratio of hydrogel beads decreased as the increasing of pH when the content of chitosan in the chloride-chitosan mixed solution was 1.0%and 1.5%,respectively.The calcium alginate-chitosan hydrogel beads,with a chitosan content of 1.5%,exhibit stronger swelling property in acidic buffer,whose swelling ratio is 67.59%in pH 6 while 51.72%in pH 12,opposite to calcium alginate.(2)The mechanical properties of concretes were improved with the addition of hydrogel beads.After adding 1.2～1.5%calcium alginate-chitosan(1.0%)hydrogel in concrete and 28 days’ curing,the compressive strength and flexural strength of concrete were increased by 9.57%and 12.0%,respectively.And the ratio of the flexural strength to the compressive strength is 17.14%.(3)The properties of self-healing was revealed when adding bacterial spores encapsulated by calcium alginate and calcium alginate-chitosan(1.0%)hydrogel,respectively.We got two healing crack of 4 cm length and 1 mm width,which was encapsulated by calcium alginate,as well as healing crack of 3 cm length and 0.8 mm width,which was encapsulated by alginate-chitosan(1.0%)hydrogel,with the amount of bacterial spores of 2.54～3.07×105 every cubic centimeter concrete.Molecular mechanism study on stereoselectivity of α or βhydroxysteroid dehydrogenases:Hydroxysteroid dehydrogenases(HSDHs)are a type of NAD(H)/NADP(H)-dependent oxidative reductase that can specifically catalyze the reduction of carbonyl to hydroxy which share steroidskeleton structures,as well as their reversible reactions.All HSDHs exhibit strong stereospecificity during catalytic reactions,resulting in a pair of stereo heterogeneous specific products in reduced reactions,which show big differences in properties,functions,etc.In this article,we compared and analyzed the binding model of enzymes and coenzymes which from short-chain dehydrogenase(SDR)superfamily and aldo-keto reductase(AKR)superfamily firstly.And then the crystal structures of 7 β-HSDH(PDB:5GT9),17β-HSDH(PDB:3QWH)from SDR superfamily,and 3(17)α-HSDH(PDB:2P5N)from the AKR superfamily were selected for the binding model analysis between enzymes and coenzymes as well as the binary complex and substrates/products.A pair of enzymes,7α-HSDH and 7β-HSDH,which can specifically catalyze C7 site oxidative reduction,were mainly used for the study of HSDHs’stereospecificity.A binary crystal structure of 7β-HSDH-NADP+ and its substrate 7-ketolithocholic acid(7-KLA)were used for molecular docking via YASARA to explore the substrate binding model,and a possible binding model of 7β-HSDH and 7-KLA was obtained.The α side of 7-KLA towards NADP+in 7β-HSDH,while the β side of 7-KLA towards NAD+in 7α-HSDH.This may be the key factor which made the orientations of C7-OH different in the reduced products of ursodeoxycholic acid and chenodeoxycholic acid.The interaction existed in pyrophosphate of NAD(P)+[Ser193-OG…3.11(?)…O1N-PN]and Ser193 of enzyme caused the upturning of PN-phosphate group,which formed a barrier with the side chain of His95 to make 7-KLA only able to bind to 7β-HSDH with α side towards nicotinamide of NADP+.A possible interaction of Tyr253 and C24 of 7-KLA may help to the orientation maintaining in 7β-HSDH.Several key amino acid residues of His95,Ser193,and Tyr253 are conserved in 7β-HSDHs,which show a significant difference to 7 α-HSDHs.The conclusion above was furtherly clarified by the molecular docking results of other two enzymes,17β-HSDH and 3(17)α-HSDH.