Study On The New Lipase Resources From Extreme Environmental Bacteria And Their Application Exploration In The Field Of Biopharmaceutical

Lipases,as a group of important industrial enzymes,can catalyze a variety of types of reactions including hydrolysis,esterification and transesterification reactions on a wide range of substrates.They are playing increasingly important role in biopharmaceutical and the processing of healthcare products.However,the lipases involved in the resolution of chiral drugs or medicine intermediates and the enrichment of polyunsaturated fatty acids these reactions have some disadvantages,including limited species diversity,poor enantioselectivity or fatty acid-specificity,low initial enzyme activity,easily inactivation,weak tolerance to high temperature and/or acid environment,and/or salinity,and/or organic solvents,hard to recycle,instability and poor reusability.The reported lipases and commercial lipases are far from meeting the demands of rapid development of modern industries,especially for biopharmaceutical industry.Therefore,customized lipases with specific properties are required for the purpose of the resolution of chiral compounds and enrichment of polyunsaturated fatty acids from vegetable and animal oil sources.Among various lipase resources,bacterial lipases generally possess better catalytic ability in the aqueous or organic phase due to higher activity,better stability and higher resistance.Moreover,lipases produced by bacteria from such particular environments may also accordingly have some special properties due to adaptation to extreme environments.So,it is of great importance to discover lipases from extremophile bacteria characterize their enzymatic properties and further explore their preliminary industrial applications.In this study,various soil samples from oil contaminated or extremophile environments were collected from the South China Sea,Ny-?lesund of the Arctic,Xinjiang Autonomous Region,and Ma'anshan Forest Park of Wuhan City.,Based on combining lipase resource mining techniques such as olive oil-plates screening,touchdown PCR and genome walking,some novel lipases were discovered.Protein purification and covalent immobilization were further employed to obtain lipase biocatalysts with satisfactory features for potential use in biological medicine,food and health care,etc..The main achievements are summarized below.1.By using three simple kinds of active plates with olive oil as the substrate,eight lipase-producing strains were screened.Among them,four strains TY1-DH,TY4-BXN,TY4-HX and TY5-SH were from the oil contaminated soil samples of oil mills of Jiangxi Province,strain 11E105 isolated from the mud samples of the seabed of the South China Sea,strain R0-14 screened from soil samples of the bare mining area of the Ny-?lesund of the Arctic after the recession of Midre Lovénbreen glacier,strain M9 came from soil samples of arid area of Xinjiang Autonomous Region,and strain SL-4 was cultured from soil samples contaminated by cooking oil and salt of the barbecue area of Ma'anshan Forest Park of Wuhan City.Based on sequence analysis of their 16 S rRNA,they belong to genera Pseudomonas,Acinetobacter,Sphingomonas,Burkholderia and Psychrobacter,respectively,and named as P.mendocina TY1-DH,A.lwoffii TY4-BXN,S.paucimobilis TY4-HX,B.cepacia TY5-SH,P.pacificensis 11E105,Pseudomonas sp.R0-14,P.moraviensis M9 and B.ubonensis SL-4,accordingly.These strains enrich lipaseproducing sample library.Among them,strain R0-14 from the Arctic has certain particularity,and no lipase is reported from P.moraviensis and B.ubonensis.Thus,some lipases from the two strains have bigger research value than others in the field of biopharmaceutical.2.A new lipase SL-4 from B.ubonensis SL-4 was purified by ammonium sulphate precipitation,anion exchange chromatography and gel filtration chromatography.Designing primers according to the protein sequence,the lipase gene SL-4 was successfully cloned.Amino acid sequence alignment revealed that lipase SL-4 has 88% identity to lipase YCJ01 from B.ambifaria YCJ01,lipase S31 from B.ceapcia S31 and lipase BCL from B.cepacia,86% identity to lipase LipA from B.cepacia ATCC 25416,and 81% identity to lipase Lip A from Burkholderia sp.HY-10.Based on the phylogenetic analysis,lipase SL-4 belongs to the bacterial lipase subfamily I.2.Enzymatic characterization indicated that lipase SL-4 exhibited optimum activity toward p-NP myristate(C14)at pH 8.5 and 65 °C with a Km of 0.72 mM,a kcat of 391.63 s-1 and a kcat/Km of 543.93 s-1 mM-1.Interestingly,lipase SL-4 could remain so more than 60% activity after incubation at 65 °C for 12 h and could keep over 90% of its original activity after incubation at 50 °C for 24 h that it had high thermostability.Moreover,after being incubated under 4 M Na Cl at 50 °C,pH 8.5 for 24 h,lipase SL-4 still retained over 50% activity of its original activity,and showed high tolerance to salt.Additionally,lipase SL-4 also possessed non-ionic detergent stability and organic solvent stability.Lipase SL-4 was successfully immobilized on the Fe3O4 magnetic nanoparticles by covalent binding.Under the optimal condition,the immobilization efficiency was 94.45%,and the enzyme activity of transesterification were and 385,066.8 U/min/g protein.When preliminarily employed to chiral resolution of(R,S)-1-phenylethanol,under the optimal condition,the immobilized lipase SL-4 could attain a substrate conversion rate of 49.54% and an enantiomeric excess value of 99.90%.After being continuously used for 10 cycles,it yet retained the substrate conversion rate of 46.11% and the enantiomeric excess value of 84.93%,which stated that this immobilized lipase SL-4 possess good reusable stability.These results demonstrated that the new thermo-solvent-stable lipase SL-4 possesses an attractive potential for biotechnological applications as biocatalyst,especially in bio-pharmaceutical and organic synthesis.3.A gene lipR was cloned from Pseudomonas sp.R0-14 by touchdown PCR and genome walking.The gene encodes a 562-amino acid polypeptide showing a typical ?/? hydrolase structure with a catalytic triad consisting of Ser153-Asp202-His260 and one ?-helical lid consist of 103-113 residues.Based on the phylogenetic analysis,lipase LipR belongs to the bacterial lipase subfamily I.2.Then,LipR was successfully expressed in E.coli,unfolded by urea,refolded by dialysis and purified by Ni-NTA column from inclusion bodies,and then biochemically characterized.By enzymatic characterization,the recombinant LipR preferred to p-nitrophenyl butyrate at pH 8.5 and 60 °C with a Km of 0.37 mM and a kcat of 6.42 s-1.Moreover,LipM can exhibit certain activity within the temperature range of 0-100 °C,thus is a eurythermic lipase.Thermal stability experiments demonstrated that LipR retained over 90% of its original activity after incubation at 50 °C for 12 h,and showed high thermostability.Moreover,LipR showed good tolerance to organic solvents acetonitrile,isopropanol,acetone,methanol,and tert-butanol.Additionally,LipR possessed strong ability of enrichment of polyunsaturated fatty acids.It could improve the relative content of EPA in algal oil from 0.74% added to 1.22%,DPA from 10.41% to 21.24%,DHA from 27.79% to 36.98%,and their relative total content from 38.94% increased to 59.44%.This unique character of LipR render it has great prospect in industrial applications,especially in health care,biomedicine products,food,for such these research fields involve selective hydrolysis.4.The lipM gene was first cloned from P.moraviensis M9 via touchdown PCR and genome walking.Amino acid sequence alignment reveals that the predicted amino acid sequences of LipM has 82% identity to lipase LipT from P.mandelii JR-1 and lipase LipR from Pseudomonas sp.R0-14,and 54% identity to lipase PML from Pseudomonas sp.MIS38 and lipase Lip I.3 from Pseudomonas sp.CR-611.Based on the phylogenetic analysis,lipase LipM also belonged to the bacterial lipase subfamily I.3.The lipase LipM was expressed in E.coli,unfolded by guanidine hydrochloride,refolded by dialysis and purified by Ni-NTA column from inclusion bodies,and then further biochemically characterized.Enzymatic characterization showed that the recombinant LipM preferred to hydrolyze p-nitrophenyl octoate(Km: 0.36 mM;kcat: 35.7 s-1;kcat/Km: 99.17 s-1 mM-1),and exhibited optimum activity at pH 8.0 and 65 °C.Biochemical characterization of Lip M suggested that it was a eurythermic lipase,which could show certain activity between 10 °C and 95 °C.Thermal stability study indicated that LipM possessed high thermostability with more than 90% of its original activity remaining after incubation at 65 °C for 12 h.In addition,LipM had medium tolerance to methanol,ethanol,and isopropanol.LipM also showed strong ability of enrichment of polyunsaturated fatty acids,it could enhance the relative content of EPA in algal oil from 0.74% added to 1.25%,DPA from 10.41% to 17.61%,DHA from 27.79% to 47.02%,and their relative total content from 38.94% to 65.88%.At the same time,the relative content of diacylglycerols(DAGs)could attain 30.73%.The desirable characteristics infer that LipM is one of the few eurythermic lipases,and similar to LipR,LipM can be potentially applied in health care,biomedicine products,food and other industries.