Molecular Engineering Of Streptomyces Griseus Trypsin

Bacterial Streptomyces trypsin （SGT, EC3.4.21.4） is one of the serine proteinases inStreptomyces griseus and acts as a key mediator during microorganism growth and cellulardifferentiation. It is also a sort of serine protease which has potential applications in leatherbating, food processing, pharmacy, clinical diagnoses and biochemical tests. S. griseus trypsinis highly identical to bovine trypsin with respect to the structure and function. Although theproduction process was easily controlled and natural SGT was immunogenicity to humanbeing, commercial trypsin is mainly from mammal production. For S. griseus fermentationperiod was long and its production level was low. As a result, heterologous production ofStreptomyces trypsin was an attractive alternative for its protein engineering and application.This study was involved in heterologous expression of Streptomyces trypsin, especiallyfocused on rational design of its propeptide, analysis and optimization of its enzymecharacters and production by site-directed mutagenesis of the R145and construction ofauto-activated Streptomyces trypsin. Major results were listed below:（1） After comparatively investigated different heterologous expression systems pGAP，pAOX1and pFLD in different hosts P. pastoris GS115, P. pastoris SMD1168, P. pastoris X33and P. pastoris KM71). pAOX1and P. pastoris GS115were identified as the bestcombination. The highest recombinant Streptomyces trypsin production9.6U·mL^–1wasinduced with20g·L^–1methanol for5days at30°C co-feeded with4g·L^–1glycerol in shakeflask fermentation. The recombinant P. pastoris GS115/pAOX1/mt had the highest trypsinexpression level of15.6U·mL^–1under the optimized condition with the high cell densityfermentation in3L fermentor.（2） Through the heterologous expression of Streptomyces trypsinogen and its propeptidedeteletion mutants, the residue at P1site next to the N terminus of the mature trypsin wasproved to be the most necessary amino acid for the inhibition effect of the propeptide.Moreover, the major features of native trypsin were characterized. First, three disulfide bondsbetween residues C168-C182, C191-C220, and C42-C58held the substrate binding pocketrigid and the correct fold was observed. Second, three Hydrogen （H） bonds among thecatalytic triad （H57, D102and S195） maintained the accurate conformation of the catalyticcenter. Third, one H-bond had formed between V16and loopD189-D194and this interactionstabilized the structure of trypsin. Novel Streptomyces trypsin mutant Exmt （YVEFmt） andIVEFmt were obtained by the rational design of the propeptide. The specific activity towardsamidase substrate BAPNA of Exmt （YVEFmt） is852U·mg^–1, yet the specific activitytowards amidase substrate BAPNA of IVEFmt decreased to242U·mg^–1. Finally, through therational design of propeptide, the optimized recombinant Exmt was obtained. （3） Compared the enzyme characteristics of recombinant Streptomyces trypsin （Exmt）with wild Streptomyces trypsin （wSGT） and Bovine trypsin （BT）. The optimized catalytic pHof Exmt, wSGT and BT were8.0,8.0and10.0respectively. The optimized catalytictemperature of Exmt, wSGT and BT were50°C,50°C and60°C respectively. The Nterminal propeptide YVEF of Exmt improved its pH tolerance and thermostability. The Exmtshowed significant increase of the thermostability, which values of t1/2were1.8-fold,2.5-foldand31.2-fold of that of the SGT at40°C,50°C,60°C respectively. Furtherly, the N terminalpropeptide YVEF of Exmt increase its resitance to metal ions. Its tolerance to organic solventswere decreased, but its esterase activity was increased by the organic solvents. Moreover, thecatalytic efficiency （representing as specificity constant, kcat/Km） of Exmt was also improvedby3.1-fold and5.5-fold towards BAPNA and BAEE respectively, because of the increase ofthe kcatand decrease of the Km. In summary, these improvements were mainly because of theincrease of protein structure flexibility and inner molecular hydrogen bonds.（4） Through the site-directed mutagenesis of the R145of Exmt, all mutants showedincrease of recombinant trypsin expression level in shake flask fermentation. Furtherly,Exmt（R145I） was obtained of the specific activity1242.85±99.15U·mg^–1towards BPANA.Compared with Exmt and wSGT, it increased1.46-fold and3.61-fold. Moreover, And it hasthe specific activity101491.58±1225.56U·mg^–1towards BAEE. Compared to Exmt andwSGT, it has increased0.3-fold and2.9-fold. What’s more, the catalytic efficiency（representing as specificity constant, kcat/Km） of Exmt（R145I） was also improved by0.2-foldand0.3-fold towards BAPNA and BAEE respectively. The resitance to the auto hydrolysis ofExmt（R145I） was also improved. Finally, the recombinant P. pastorisGS115/pAOX1/Exmt（R145I） have the highest trypsin production of18.7U·mL^–1under theoptimized condition with the high cell density fermentation in3L fermentor.（5） The non-autoactivated hybrid Streptomyces trypsinogen VD4Kmt was activated byenterokinase. It was mainly because of the propeptide VD4K buried into the inner space ofthe trypsin structure. The autoactivated fusion mutant TLmt（D4K） can be acitivated by itselfand obtained the recombinant trypsin production of5.8U·mL^–1in shake flask. Furtherly, wehave proved that the autoactivation process of TLmt（D4K）. Firstly, the fusion part TrxAdoesn’t inhibit the trypsin activity during expression; Secondly, the active trypsin part cleavedat K site in the linker part to remove the fusion part TL（D4K）. Finally, the recombinant P.pastoris GS115/pAOX1/TLmt（D4K） had the highest trypsin expression level of19.85U·mL^–1with the optimized condition by the high cell density fermentation method in3L fermentor.