Purification And Characterization Of Poly (L-lactic Acid)-degrading Enzymes

As a backbone in material industry,petroleum-based plastic has played an important role in everyday life.However,due to the ever-increasing pollution caused by plastic and lack of petroleum resources,green polymer materials gradually is becoming more and more popular than petroleum-based plastic.The green polymer materials mainly include polyesters,which have the advantage of biodegradability and renewability.The mechanical properties of natural or modified polyesters are equal to or even better than those of the conventional plastic.Among all polyesters,Poly (L-Lactic acid)(PLA)is especially promising with its excellent physicochemical properties and the potential price benefit.PLA has been successfully applied to medical,textile and packaging industries,which is considered as a novel kind of ecological material to substitute the existing plastic.Although PLA has distinct advantages in properties and scale-up production over other polyesters,the study of its biodegradation is relatively lagging behind its synthesis.PLA is degraded slowly in nature,moreover,microorganisms capable of degrading PLA and involved enzymes have been found to be very special in some aspects,which have limited the development of an efficient recycling system for PLA. In order to realize the actual application of PLA environmental friendly,mechanisms of its fast and complete biodegradation must be studied systematically.In this thesis, we studied the properties and the catalytic characteristics of PLA degrading enzymes isolate from an efficient PLA-degrading bacterium.The mechanisms involved were also discussed from the point of enzymatic catalysis and evolution of enzymes.The major results of the thesis are as follows:1.Poly(L-lactic acid)(PLA)-degrading strain,Amycolatopsis orientalis,was isolated by screening soil samples and culture collections as the most effective PLA-degrading microbe.It seems that microorganisms capable of degrading high-molecular-weight PLA are not distributed widely in the environment because previously isolated PLA-degraidng microbes were often found to phyiogeneticaily belong to Pseudonocardiaceae family and related genera.In this work,some PLA-degrading microbes were isolated by screening soil samples and culture collections for the ability to form clear zones on the PLA-emulsified mineral agar plates.Amycolatopsis orientalis was identified to be the most effective strain in PLA degradation.The isolated Amycolatopsis orientalis grew well with PLA as the sole carbon source with 80%of PLA film being degraded and assimilated within 8 days under aerobic conditions.The pH of the culture supernatant dropped slightly with the culture probably due to the production of some oligomers of lactic acid.On the other hand,PLA-degrading activities of Amycolatopsis orientalis proved to be inducible by some proteins as well as PLA, implying that the secreted PLA degradig enzymes were protease-related enzymes. Gelatin was found to the best inducer for Amycolatopsis orientalis to produce the PLA-degrading enzymes.2.Three novel PLA-degrading enzymes were purified to homogeneity from the culture supernatant of Amycolatopsis orientalis.Up to date,only a few PLA-degrading enzymes have been isolated.In this work, three novel PLA-degrading enzymes,named PLAaseⅠ,ⅡandⅢ,were purified to homogeneity from the culture supernatant of Amycolatopsis orientalis simultaneously by a series of chromatographic steps.The molecular masses of these three PLAases as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis were 24.0, 19.5 and 18.0 kDa with the pH optima being 9.5,10.5 and 9.5,respectively.The optimal temperatures for the enzyme activities were 50-60℃.All the purified enzymes could degrade high-molecular-weight PLA film as well as casein,and the PLA-degrading activities were strongly inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride and aprotinin,but were not susceptive to chymostatin and pepstatin.Taken together,these data demonstrated that Amycolatopsis orientalis produces multiple serine-like proteases to utilize extracellular polylactide as a sole carbon source.Despite clues pointing to their potential relationship to serine proteases, efforts to identify,PLAaseⅠ,PLAaseⅡand PLAaseⅢby N-terminal amino acids and mass spectrum failed to find the exact match in the available database,suggesting that these enzymes may be novel proteins which have not been registered so far.3.The plaase2 and plaase3 genes encoding PLAaseⅡand PLAaseⅢwere cloned and their heterologous expression was primarily achieved.There have been only two reports on the isolation of genes encoding PLA degradig enzymes.In this report,genes encoding PLAaseⅡand PLAaseⅢwere cloned by an efficient chromosome walking--Self-formed adaptor PCR by use of degenerate primers designed on the base of the N-terminal amino acid sequences and the conserved serine active sites conjectured according to sequence analysis of the PLAases.The isolate genes were further tried for the heterologous expression in E.coli and B.subtilis,respectively.Results of SDS-PAGE and activity assay confirmed that the recombinant enzymes could be expressed in their active forms.4.Specific structural features of the isolated PLAases were revealed by sequence and structure analyses.The sequence and structure of the PLAases were analyzed by various bioinformatics softwares.Results showed that the PLAases belong to the S1A (chymotrypsin)family of serine proteases with the absolute conservation of the serine protease catalytic triad in PLAases.Like other chymotrypsin family members, PLAases consist of twoβ-barrels with the putative active site located in the cleft between twoβ-barrel domains.Residue S214 supposed to contribute significantly to the polar environment,as well as residue G193 forming an oxyanion hole were also conserved in PLAase2 as S215,G188,and in PLAase3 as S150,G133,respectively. However,dissimilarities at some key sites were also evident between the PLAase and elastase.Firstly,residue V216 in the S1 site affecting the substrate specificity for nonbulky residues or bulky residues in S1 were replaced in PLAases.Secondly, residues C191 and C220(chymotrypsin numbering)known to form disulfide linkage bonds for the structure of the P1 pocket were also missing in PLAases.This disulfide bridges the two walls of the S1 site and likely provides a degree of structural rigidity to the substarte bindig cavity.Thirdly,residues E70 and E80 of porcine pancreatic elastase I hae been shown to be involed in calcium binding.Again,these residues are absent in PLAase.Moreoer,results of homology modeling indicated that although there are no significant differences regarding the struactural backbone between PLAases and the template enzyme,some apparent changes indeed exist in some surface loops which surround the extended substrate binding site.Thus,despite the conservation of the active site and relatively high degree of similarity of the overall structure between serine protease and PLAases,the substarte binding site of the PLAases might be somewhat divergent from that of elastase or other serine proteases. On the other hand,phylogenetic affiliations showed that the PLAases have relatively high degree of identity with the serine proteases from Gram-positive strain with high GC content,but with little homology with other serine proteases,suggesting that the PLAases were evolutionarily unique with primitive enzymatic scaffolds but plastic active pockets.5.Mechanism of enzyme-catalyzed degradation of PLA was supposed.Based on the above results and analyses,mechanism of enzyme-catalyzed degradation of PLA was discussed.The comparability between PLA and peptide with regard to the similarity in both the construction unit(Ala vs Lactic acid)and the linking bond(vs)constitutes the base of the degradation,while enzymes with adaptable substrate specificities make the degradation doable.We suppose that not only the conserved active site of serine proteases,but also the unique substarte binding pocket as well as the surface loops surrounding the binding pocket suffice the PLAase-mediated degradation of PLA.From this point,it seems that the PLAases are kinds of promiscuous enzymes with ability to catalyze reactions other than the ones they evolved for.So,compared with enzymes with high specificity,these primitive enzymes with higher degree of catalytic plasticity may be excellent starting materials for engineering to generate novel enzymes possessing a range of useful properties.