Research On Urease And Lipase Of Microorganisms Associated With Marine Sponges

Urea represents one of the dominant organic nitrogenous compounds in theoligotrophic oceans. Compared to the knowledge of nitrogen transformation ofnitrogen fixation ammonia oxidization, nitrate and nitrite reduction, the ureautilization in sponges remains nearly unknown. Phylogenetically diverse bacterialureC genes were detected in the total genomic DNA of sponge microbiota, i.e.,19operative taxonomic units (OTUs) in ureC library. Blast and phylogenetic analysesshowed that most of the ureC sequences were similar with the urease alpha subunit ofProteobacteria, which are the predominant components in sponge X. testudinaria, andthe remaining ureC sequences were related to Magnetococcus, Cyanobacteria, andActinobacteria. In addition, we screened a strain producing urease from spongeHalichondria regosa, which was identified as Bacillus licheniformis through16SrRNA sequence. Urease activity of the crude enzyme solution were measured, andfound Pb2+and Cu2+could largely inhibit urease activity, which could offer aninstruction on biosensor in the detection of heavy metal ions. This study highlightedthe role of sponge bacterial symbionts in the urea utilization, extending ourunderstanding of nitrogen cycling mediated by sponge microbiota.In recent years, with the development of metagenomic technology, it has gonedeep into the research areas of the oceans, soils. However, there is little reasearch onapplying metagenomic library to screen novel enzymes from the sponge tissue. In thisarticle, using pUC18as vector, metagenomic library of sponge was constructed, andobtained6568clones. A clone containing lipase gene was screened using substratesselective medium. NCBI-blastx analysis demonstrated that it had a maxmiumsimilarity to the lipase gene from Aeromonas media WS and the similarity was61%.Molecular weight was expected to be30.64kDa. Expression vector of the lipase geneand pET28a(+) was constructed, and transferred to competent cells BL21(DE3). Thelipase gene product was present in the form of inclusion bodies according to SDS-PAGE. Then denaturied, purificatied, renaturatied and determined of lipaseactivity. The optimum temperature of the lipase was40℃. Thermalstability testshowed that the lipase was relatively stable at4℃, Activity of the enzyme maintainedmore than90%after incubated in4℃for90min; The enzyme had poor stability inhigh-temperature environments, Activity maintained42.39%after incubated in40℃for120min; maintained30.54%after incubated in50℃for120min. Activity also hada significant decline in60℃, less than10%of enzyme activity after incubated in60℃for120min. From the results, we could conclude that the enzyme was sensitive totemperature changes. The optimum pH was9.0, the enzyme showed a relatively highenzymatic activity between pH8.0-10.0, so it belonged to the range of alkaline lipase.We also found that the lipase was more stable in an alkaline environment after pHstability testing. Na+, Ca2+, Mg2+could greatly promote the enzyme activity. On thecontrary, EDTA, K+, Cu2+, Co2+could inhibit the enzyme activity, especially Cu2+,Co2+. Methanol, Acetone could greatly promote the enzyme activity, with the increaseof the concentration, the effect was more obvious. Low concentrations of isopropanolcould promote enzyme activity, while high concentration of isopropanol could inhibitenzyme activity. In addition, ethanol and DMSO inhibited enzyme activity, especiallyethanol. Determination of lipase kinetic parameters found that lipase35F4had betterdecomposition of long chain fatty acids, For p-Nitrophenyl myristate, the activity was17.59±1.83U/mg, Km was0.42±0.012, Vmax0.0133±0.00279mg.product/min mgprotein. For p-Nitrophenyl caprylate, the activity was5.07±0.79U/mg, Km was1.35±0.026, Vmax0.00068±0.000142mg.product./min mg protein. In this study, weacquired a novel lipase gene from metagenomic library of sponge associatedmicroorganisms, it has a guiding role for the development of genetic resources ofsponge.