Strategies for isolation of novel enzymes by using metagenomics approach

Microorganisms have been used for many biotechnological applications. Due to their both scientific and industrial importance, they have attracted many researchers. In traditional microbiological methods, microorganisms are isolated from their environment and grown in cultures. Until 2000s, it was believed that the microorganisms could not be characterized without culturing. However, scientific observations revealed the fact that only tiny amount of microorganisms could be grown in pure cultures. At the beginning of this century, a new field called metagenomics was entered to the literature. It is defined as the culture independent study of microorganisms that live in any environment. Metagenomic studies can be accomplished by function or sequence based approaches. In function based approach, a metagenomic library, as similar to genome library, is constructed and screened for desired phenotype. In sequence based metagenomics, either the metagenome is sequenced or already known sequence information is used to amplify specific regions from the metagenome. Extremophiles, as the most interesting group of microorganisms have great potential in industrial biotechnology, due to their macromolecules that conserve their stability in extreme conditions. Among these macromolecules, extremozymes are usually stable and active in harsh conditions, which is desired for many industrial enzyme catalyzed reactions. However, due to their extreme characteristics, it is not always possible to isolate an extremophile from its natural habitat and grow in laboratory conditions. At this point, metagenomics become an efficient approach to isolate novel enzymes from extremophiles. In this study, it is aimed to isolate lipolytic enzymes from extreme environments by metagenomics approach. Both function based and sequence based approaches were applied. Environmental samples were collected from acid mine drainage site (Balıkesir, Turkey; acidic environment) and a hypersaline lake (Acıgöl, Turkey; high-saline environment). In previous studies, a metagenomic fosmid library was constructed from acid mine drainage site and two potential clones (named F1 and F3) were detected. Within the scope of this thesis, strategies that can be applied in function based metagenomics approach are presented to identify the potential lipolytic enzymes. Firstly, subcloning library of F3 fosmid was constructed. F3 fosmid was partially digested with HindIII and EcoRI and ligated to pUC19 plasmid. Resulting constructs were transformed to E. coli DH5α cells and each clone was screened for lipolytic activity. One of the transformants gave positive result but the gene could not be identified by sequence analysis. As a second strategy, preliminary functional analysis was perfomed. Crude lysates of F1 and F3 clones were prepared and tested for lipolytic activity on agar plates. Once the positive results were obtained, crude lysates of F1 and F3 were partially purified by ammnonium sulfate precipitation. Each fraction was tested for lipolytic activity using para-nitrophenol palmitate as substrate. No activity was detected for F3 but 45%-70% fraction of F1 has 1.45 fold more activity compared to control. To further analyze the proteins in cleared lysates of F1, they were fractioned according to their molecular weight. Fractions between 30-50 and 50-100 kDa were analyzed by two dimensional gel electrophoresis. Gel results for F1 and FC were compared and spots that differ between gels were cut for analysis with mass spectrometry. Although the activity was verified with the preliminary functional studies, the targeted protein could not be identified with proteomic approach. As a third alternative, fosmid sample from F3 clone was sequenced. The partial Sanger sequencing revealed that the cloned insert match with nucleotide sequences from Acidithiobacillus species, verifying the acidophilic origin of the genome library. To identify the whole fosmid sequence, sample was sent for next generation sequencing. Partial sequences belonging to acidophilic microorganisms were obtained. A potential sequence similar to alpha/beta hydrolase fold enzymes was detected. Detailed analysis is going to be conducted with further studies. In the second part of the study, sequence based metagenomics approach was applied by using environmental samples collected from hypersaline lake Acıgöl. In previous studies, the microbial diversity of the samples were identified. Within the scope of this study, nucletide sequences of the lipolytic enzymes of these microorganisms were retrieved from databases. They were grouped according to their similarities. Each group was used for degenerate primer design, targeting conserved regions among lipolytic enzymes. Primers were designed by primer4clades online tool, using CODEHOP strategies. A total of nine degenerate primer pairs could be designed for 3 groups, including lipases and lipolytic enzymes, α/β hydrolase fold enzymes and carboxylesterases. While PCR was set for all primer pairs, only one positive result was obtained with the set of primers targeting α/β hydrolase fold enzymes. The obtained amplicon was cloned to pCRTM-4TOPO® vector and transformed to E. coli TOP10 cells. Sequence analysis revealed an insert of 480 bp as expected. When compared with BLAST, the sequence matched with Halolamina sediminis strain with an identity of 75%. To identify the unknown 5' and 3' ends, genome walking strategy was applied. For each end, two sets of gene specific primers were designed according to the partial sequence obtained by degenerate PCR. Metagenomic DNA was digested with restriction enzymes and adaptors were ligated to ends of digested DNA fragments. Using gene specific and adaptor specific primers, unknown 5' and 3' ends were amplified. After sequence analysis, fragments were assembled to yield a full length gene. The gene was composed of 783 bp, which corresponds to 260 amino acids. The gene starts with ATG start codon and ends with UGA stop codon. The gene sequence best matched with Halolamina sediminis with an identity of 75%. Deduced amino acid sequence best matched with the esterase from Halomonas gudaonensis with an identity of 91%. This result showed that a novel enzyme was successfully isolated from an environmental sample. The molecular weight and pI of the protein was calculated as 28368 Da and 5.27, respectively, using the ExPASy tool. To express the novel enzyme, primers targeting 5' and 3' of the novel gene with EcoRI and HindIII restriction sites were designed. The gene was amplified using metagenomic DNA as template and cloned into pET-28a(+) expression vector. The construct was transformed to E. coli C43 (DE3) chemically competent cells. To optimize the expression, 0.1, 0.5 and 1 mM IPTG concentrations were tried. In addition, induction at 25ºC for 16 hours, 30ºC for 6 hours and 37ºC for 3 hours were tried. The optimum expression was observed with 0.1 mM IPTG at 30ºC for 6 hours. After the gene was successfully expressed, purification studies were conducted. The protein was purified using His-tag method. Analysis with SDS-PAGE showed that the purification was successful. The apparent molecular weight of the protein (28.2 kDa) was consistent with the theoritical molecular weight, which was calculated as 28.4 kDa. The protein was found to be more stable in the presence of 10% NaCl, consistent with the halophilic nature of the protein. The activity of the protein was determined with kinetic assay, using para-nitrophenyl esters as substrate. The enzyme hydrolyzes the ester bond and the increasing absorbance of released pNP is measured at 410 nm. The path length for spectrophotometric measurements and the extinction coefficient of pNP at each reaction condition was calculated before activity calculations. To determine the substrate specificity, activity towards pNP-butyrate, pNP-hexanoate, pNP-octanoate, pNP-decanoate, pNP-dodecanoate and pNP-hexadecanoate were calculated. The enzyme showed a maximum activity towards pNP-hexanoate with a kcat value of 12.30 s-1. Also, the activity towards pNP-butyrate (6.03 s-1) and pNP-octanoate (6.26 s-1) was high. Its preference for short carbon chain length substrates indicates that it is an esterase. The optimum pH of the enzyme was found to be 9 and the enzyme was stable after storage at pH 8 to 10 for 24 hours. The enzyme completely losts its activity after storage at pH values below 6. The optimum temperature of the enzyme was found to be 30ºC but it is retains more than 50% of its maximum activity between 5ºC and 40ºC. The enzyme retains 96% of its maximum activity after incubation at 55ºC but the activity was dropped to 27% of its maximum after incubation at 60ºC. As the NaCl concentration increases, the activity of the enzyme drops but storage with increasing NaCl concentrations (10% to 25%) did not affect the stability of the enzyme. The effect of organic solvents (10%) on the activity of the enzyme was also investigated. While the presence DMSO did not affect the activity of the enzyme, glycerol slightly increased the activity. Activity in the presence of methanol was 80% of its maximum and other solvents showed inhibitory effect on the enzyme. The enzyme was found to be organic solvent tolerant that when stored with different organic solvents at 15% and 30% concentrations for 24 hours, except for 30% isopropanol and 30% 1-propanol, it retains more than 80% of its maximum activity. The effect of various metal ions, detergents and inhibitors were also investigated. The esterase was found to highly tolerant to metal ions as compared with the similar enzymes in the literature. The enzyme was inhibited by PMSF, confirming that it is a serine hydrolase. The enzyme is stable in storage conditions, that it retains its activity in storage buffer until 14th day at 4ºC.