ABSTRACT
Sequence-based metagenomic study for the assessment of microbial diversity followed by functional approach for the production of lipase/esterase enzyme system and genomic study of lipase-producing culturable bacteria lead to following conclusions:
A simple and suitable method is developed for the isolation of soil metagenomic DNA (mgDNA). NaCl, Sodium dodecyl sulphate (SDS) and heating at 72°C temperature to lyse the bacterial cells followed by isopropanol precipitation without using phenol cleanup. The yield of mgDNA is 3.8 pg/g soil with the purity of 1.6 and 0.8 with respect to protein and humic acid, respectively. Further purification of the extracted mgDNA using Sephadex G-50 column (MP1 method) has yielded higher amount of DNA suitable for further molecular analyses. Therefore, DNA extracted using the method M5 (reduction in humic acid), followed by purification (MP1) is successfully used for the construction of mgDNA library.
The metagenomic study of microbial diversity present in a bakery soil sample has revealed three 16S rRNA gene clones which are named as KBSR1, KBSR2 and KBSR3 with sequence-based analysis. Subsequently, the 16S rDNA gene sequences are deposited in GenBank of NCBI with accession numbers KJ685805, KJ700876 and KJ700877 respectively. 16S rRNA gene clones with accession numbers KJ685805 (KBSR1), entries KJ700876 (KBSR2) and KJ700877 (KBSR3) have represented 97-99% sequence homology with those of unculturable bacteria. The present study has confirmed that 99% microorganisms in a particular soil sample are unculturable.
The metagenomic library is constructed into pET-32a vector with the insert size of 0.5-3.0 kb from the soil metagenome. The functional metagenomic approach for the lipase enzyme coding gene from soil metagenome has revealed a positive clone KBS-plip1, 202showing lipolytic activity after screening in tributyrin (1%) agar medium and the size of the lipase coding gene is 891 bp. Subsequently, the nucleotide sequence of lipase coding gene is deposited in GenBank of NCBI data library and obtained the accession number KF743145 for the uncultured bacterium KBS-plip1. The sequence homology of KBS-plipl has represented 84-99% identity with that of the uncultured bacterium.
The expression study of KBS-plipl has represented the overexpression of the cloned gene (89l bp) in the presence of 0.5 mM isopropyl P-D-1-thiogalactopyranoside (IPTG) for 5 h at 30°C. The purification of the expressed protein using Ni(II)-nitrilo- triacetic acid (Ni-NTA) affinity chromatography has revealed a single band of protein approximately 40 kDa in size which is quite higher than that of the expected molecular weight of KBS-plipl that is 32,560 kDa. However, the increased molecular weight of the protein could be due to the fusion of His-tag (l2.7 kDa) with the expressed protein.
The nucleotide sequence of KBS-plipl on translation to the protein sequence (ExPASy translation tool) has exhibited the presence of 296 amino acids. The BLAST result of the amino acid sequence of KBS-plipl has revealed a maximum of 55% sequence homology with the lipase enzyme (3V9A) produced by the uncultured bacterium.
The multiple sequence alignment of KBS-plipl has showed the presence of conserved HGGG motif (amino acids 23-26) upstream of the active site, catalytic consensus pentapeptide GDSAG, catalytic triad DPM (amino acids l45-l47) and HVF (amino acids 228-230) referring to the conserved sequences of hormone-sensitive lipase (HSL) family. This confirms that KBS-plipl belongs to the HSL family. KBS-plipl contains GDAAG in place of GDSAG and it could be the novel functional characteristic for the lipase enzyme.
Homology models and validation for protein structure prediction for KBS-plipl have revealed 72% structural similarity with that of ABZ9 template and showed a generated ribbon model in a dimmer fold, consisting of nine P-strands attached with seven a-helices. The Ramachandran plot for modelled enzyme has consisted of favoured (95.6%), allowed (4%) and outlier (0.4%). The result 203is significant as a high percentage of residues is in the favoured region (> 95%). This indicates that the built in model is of good quality.
Biochemical characterization of KBS-plip1 for dose-dependent study has showed the required concentration of 1.0 qg-mi 1 for maximum enzyme activity in the presence of 1% tributyrin. The enzyme kinetics for KBS-plip1 has revealed V max and K m values for the enzyme catalysed reaction to be 227 U/mg and 0.0806 mg/ ml, respectively.
The optimum pH and temperature for the maximum enzyme activity of KBS-plip1 is found to be 7.5 and 37°C, respectively. It has showed maximum lipase activity in the presence of 0.5% CTAB, 0.5% gum arabic and 1 mM EDTA. Metal ions Ca2+, Mn2+, Fe2+ and Zn2+ in the final concentration of 1 mM and salt NaCl at 1.5 M concentration have enhanced the enzyme activity of KBSplip1 by two fold. KBS-plip1 has exhibited solvent tolerance activity and revealed maximum activity in the presence of 30% of 2-propanol. Solvents like ethanol, 1-propanol, glycerol, acetone, acetonitrile and DMSO have also enhanced the enzyme activity.
The KBS-plip1 derived lipase enzyme has revealed its stability at 37°C for 100 min and the same decreased drastically between 45 and 55°C with half-life of 60 and 20 min, respectively.
Five different lipase producing culturable bacterial isolates KBS-101, KB2F, KBS-103, KBS-105 and KBS-107 are isolated from a bakery waste deposited soil, Tezpur, Assam after screening in tributyrin agar plate medium. All five bacterial strains have exhibited zone of hydrolysis 28-25 mm. Morphological and biochemical screening of the lipase producing bacterial strains have showed the strains KBS-101, KB2F and KBS-105 to be rod shaped gram positive Bacillus sp.; KBS-103 and KBS-107 rod shaped Gram-negative Enterobacter sp.
The culturable bacterial strains KBS-101, KB2F, KBS-103, KBS-105 and KBS-107 have yielded genomic DNA 73.4, 58.8, 44.1, 33.9 and 72.2 qg/ml, respectively with the purity of 1.85-1.94.
The lipase producing culturable bacterial strain KBS-101 has showed maximum growth at 37°C during 16 h, KBS-102 at 45°C 204during 24 h, KBS-103 at 37°C during 16 h, KBS-105 at 37°C during 24 h and KBS-107 at 37°C during 24 h of incubation.
The crude bacterial lipases extracted from the bacterial strains have exhibited high degree of thermal stability, and retained 50% of their catalytic activity at 60°C after 80 min. Compatibility study of 205the culture supernatant containing crude lipases has exhibited higher activity with the commercial detergents. The culture supernatant containing crude lipases have retained their 50% activity even after 30 days of storage at 4°C.
An inoculum size of 2.0% gives maximum lipase production. Glucose for the strains KBS-101 and KBS-105; lactose for KB2F; and galactose for KBS-103 and KBS-107 are the effective carbon source for maximum lipase production. Beef extract for KBS-101 and KB2F, yeast extract for KBS-103 and KBS-107 and peptone for the strain KBS-105 are good nitrogen sources for maximum lipase production.
KBS-101and KBS-103 have showed maximum lipase production after 24 h with maximum cell dry biomass; but strains KB2F, KBS-105 and KBS-107 showed maximum lipase production after 48 h with the highest cell dry biomass.
The optimum pH for maximum lipase production by KBS-101 and KBS-105 is 8.5, KB2F and KBS-103 at 7.5, whereas KBS-107 at 9.0 pH. The optimum temperature for maximum lipase production by strains KBS-101, KBS-103, KBS-105 and KBS-107 is 37°C and by KB2F 45°C. The maximum lipase production from KBS-101, KBS-103, KBS-105 and KBS-107 is achieved after 24 h of culture at 37°C while KB2F after 48 h of culture at 45°C. The optimum agitation rate for maximum lipase production from all five bacterial strains is 200 rpm.
Olive oil is found to be a good substrate for maximum lipase production by the bacterial strains KBS-101, KBS-103 and KBS-105, and tributyrin for the highest lipase production by KB2F and KBS-107.
The divalent metal ion Ca2+ enhanced the activity of the culture supernatant containing crude lipase enzyme extracted from KBS-101 and KBS-103; Fe2+ from KB2F and KBS-107, and Co2+ from KBS-105.
The culture supernatant containing crude lipase from the bacterial strain KBS-101 has showed the highest antibacterial activity against Gram-positive and Gram-negative bacteria; and antifungal activity against Fusarium oxysporum (MTCC 284).
