Screening Of Lipase Producing Strains For Aquatic Animals And Clonging Of Lipase Genes And Its Enzyme Charactritics

In recent years, as prices climb quickly in liquid source, a cheap poor lipid are widely used incultivation industry which can cause negative effect, such as the oil peroxide, fatty liver in fish,metabolic disorders, low fish quality and aquatic pollution. Bes ides, because of its special livingcondition for fish which could also limited endogenous lipase activity. Therefore, to dissolve theseproblems by bioengineering technology is turned persistent and feasible. Lipases from microorganismshave been utilized widely in industrial fields due to substrate specificity, enantiomeric selectivity,thermostability, alkaline stability and higher catalytic efficiency.The purpose of this study was to build a specific and effective method to screen lipase strain frompond sediment. The isolated process are included using low and high temperature culture strategy withtwo step screening process and detection of lipase activity from induced fermentation solution ofisolated strains. The certified strains were cloned lipase gene and heterogeneous expression, purifiedand detected enzymatic properties.Five low temperature lipase strains and four high temperature lipase strains were screened frompond sediment. The low temperature lipases strains were identified as Acinetobacter and Pseudomonas,the high temperature lipases strains were mainly identified as Geobacillus. A low temperature lipasestrain Acinetobacter sp. G1and high temperature lipase strain Geobacillus sp. B2were detected higherlipase activity by spectrophotometer, and used as the next study.Depending on the amino acid sequence of low temperature aklaine lipases in Acinetobacterobtained from the NCBI, two conserved motifs A-P-D-Y-E-G-L-G-（T/E） andT-K-G-[T/A]-[I/V]-A-[V/L]-A-P-A-S were found. A set of degenerate primers （Acinet alkaline F andAcinet alkaline R） was designed based on these conserved motifs. The full-length genes of G1fromAcinetobacter sp. G1were obtained using TAIL-PCR technology. The G1gene is1227bp whichencoded a protein of406aa and a stop codon. Its deduced amino acid sequence showed66%identity toAcinetobacter lwoffii SH145. Degenerate primers based on the two conserved motifs of the oxyanionhole and the active sites were used to clone Geobacillus sp. B2lipase gene. The sequence resultsshowed that B2gene indentified the same to the whole genome sequencing of Geobacillus sp. WCH70.The B2gene is795bp, comparising one open reading frame encoding a polypeptide of264aa, whichbelonged to GDSL family lipase.G1and B2were expressed in Escerichia coli BL21（DE3） respectively. The lipase activity wasdetected after induction by IPTG, the unique bands were excised from SDS-PAGE through massspectrometry. The recombinant proteins G1and B2were purified by Ni-Column and characterized.The optimum temperature of lipase G1activity was40℃. After kept at20-40℃for2h, the retainedactivity were left more than50%. While50℃kept for1h, there was retained little activity, whichshowed a typical cold-adapted lipase. The optimum pH was8.0, G1showed good stability over the pHrange of4.0¹2.0. The lipase G1was resistant to great majority metal ions, organic solvent，protease. The study of substrate specificity result showed G1was suitable to degrade carbon chain between C8and C12fatty acid. The substrate specificity for C8was8908U/mgB2was showed the highest activity at65℃and pH7.5, had strong stability at higher temperatureand wide range pH4.0¹2.0. The lipase activity was stimulated by Zn²⁺, K⁺, Li⁺, Na⁺andβ-mercaptoethanol, while inhibited by Ca²⁺, Co²⁺, Ni²⁺, Cu²⁺, Mn²⁺and Pb²⁺. The lipase B2was resistantto great majority organic solvent, detergent and protease. The Km value was0.26mmol/L with a Vmaxof149.25μmol min–1mg–1for substrate C8, and the Km value was0.41mmol/L with a Vmax of71.1μmol min–1mg–1for substrate C10.Our study demonstrates that a quick specificity screening was built by two-step isolated process andwith measuring lipase activity from induced ferment solution. The enzymatic properties of G1and B2were showed that both of them could a potential app lication in aquatic animal.