Molecular Design And Application Research On The Metabolism Of Thermoanaerobacterium And The Catalysis Of Nitrilase

To address the increasing serious environmental and fossil energy crisis, much attention hasbeen paid to bioeconomy, which based on the bioscience and biotechnology but not fossilfuels. Engineering the cells, subcellular fractions or biological macromolecules to bebioenergy and chemicals industrial producers is one of the key policies. In this work, aThermoanaerobacterium strain was metabolic engineered to improve lactic acid productionfrom various lignocellulosic derived sugars, and a nitrilase was fused with self-assemblypeptides to improve the operation stability in biocatalysis.On one hand, by the rational design, suitable microbial should be engineered to be microbialcell factories for bioenergy and chemicals production. Herein, Thermoanaerobacteriumaotearoense SCUT27, a cellulose and hemicellulose degradable strain, was used as chassisand metabolic engineered. A knockout vector pPuKAd, which contained a thermostablethermostable kanamycin resistant cassette, was constructed and transformed into SCUT27toblock the acetate pathway (pta-ack) by homogenous recombination. The mutant T.aotearoense LA1002is genetically stable, and the carbon flux was redirected to significantlyimprove the concentration and yield in L-lactic acid production by using simultaneous pentoseand hexose as substrate. The optical purity of L-lactic acid is more than99%in allfermentation.The genetic manipulation resulted in1.8and2.1fold increase of the lactic acid yield using10g/L of glucose or10g/L of xylose as substrate, respectively. In particular, LA1002canproduce lactic acid from cellobiose, mannose, dextran, or xylan as the single substrate withoutany pretreatment or enzyme addition. In the5-L fermenter, the maximum L-lactic acid yield of0.93g/g glucose was obtained with47.17g/L lactic acid by LA1002from50g/L of substrate,which is very close to the theoretical value (1.0g/g of glucose). LA1002can also producedupto39.72g/L,43.56g/L L-lactic acid at the yield of0.79g/g,0.86g/g sugar with by using50g/L of xylose or mixed sugar (glucose:xylose=1:1, w/w) as substrate, respectively. Thenon-sterilized fermentative production of L-lactic acid was also carried out, achieving valuesof44.89g/L and0.89g/g mixed sugar for lactic acid concentration and yield, respectively.Combined with the fermentation merits, T. aotearoense LA1002is encouraging andpotentially well-suited for optically pure L-lactic acid production from lignocellulosic biomassin an economic feasible way.On the other hand, this study also focuses on improving the operation stability of biocatalyst.For the special self-assembly properties, two self-assembling amphiphilic peptides (18A and R18A were fused at the C-terminus of an oligomeric nitrilase from A. faecalis JM3through molecular cloning, respectively. The fusion enzyme, designated as Nit-SEA, canspontaneously assembled into active aggregates in Escherichia coli with>95%nitrilase. Nit-SEA can be obtained by simple cell disruption and washing with high enzymeactivity retention. The special activity of purified Nit-18A and Nit-R18A were77.4%and107.3%of native nitrilase (Nit), respectively. The half-life time of Nit-SEA was about3.6-to6.7-fold longer than the native nitrilase at45°C and50°C. By fusedself-assembling peptide (18A or R18A), active self-assemble enzyme aggregates of targetenzyme can be obtained to improve its thermostability.Furthermore, the enzyme aggregates were purified through cell lysis and centrifugationled to the facile preparation of immobilized particles (Nit-iSEA). Approximately86%of the initial nitrilase activity was incorporated into the Ca-alginate entrapment beads(final1.5%(w/v) Na-alginate,0.5mol/L CaCl2, immobilized for90min). Thethermostability of Nit-iSEA was about9.9-and5.5-fold more stable than the nativenitrilase at45°C and50°C, respectively. The nitrile tolerance was also dramaticallyimproved, no apparent substrate inhibition was observed for Nit-iSEA upto120mMmandelonitrile. Additionally, the Nit-iSEA could be recycled20times with~5%loss inactivity. Nit-SEA, which can further improving the thermal stability, substrate resistance andstability of reusing operation by simple entrapment, is alternative in industrial biocatalysis.In deeply, structural characteristics of Nit-SEA were studied. TEM and Cryo-TEM showedNit-SEA formed an ordered fiber-like structure with a diameter of about10nm. FTIR andsecond derivative analysis showed an increase-helix proportion in Nit-SEA. Meanwhile,Nit-SEA did not bind thioflavin T and have a9.5reflection, which represents thecoiled-coil structure by-helices, was obtained through X-ray diffraction. The preliminarystudy of Nit-SEA structure suggests Nit-SEA form helix fibrils by coiled-coil structure of18Aor R18A.