# Biochemical Characterization and 16S rRNA Sequencing of Few Lipase-Producing Thermophilic Bacteria from Taptapani Hot Water Spring, Orissa, India

Authors: Satpal S. Bisht; Amrita K. Panda
Journal: Biotechnology Research International
 (2011)

---

## Abstract

Three lipase-producing thermophilic bacteria (AK-P1, AK-P2, and AK-P3) were isolated from the Taptapani hot water spring in Orissa, India. The crude extra cellular lipases from cell-free culture supernatant were reacted in an olive oil mixture, and their lipolytic activities were compared. Identification of the bacteria was carried out using biochemical tests, 16SrRNA sequencing and sequences submitted to NCBI GenBank. Strain AK-P3, exhibited the highest lipolytic activity of 5.5 U/mL was identified asPorphyrobacter sp. The lipolytic activities of strains AK-P1 and AK-P 2 were 4.5 U/mL and 3.5 U/mL, respectively. Strains AK-P1 and AK-P2 were identified as Acinetobacter sp. and Brevibacillus spp. The GenBank accession numbers of the 16S rRNA gene sequences determined in this study for the strains AK-P1, AK-P2, and AK-P3 are HM359120, HM359119, and HM359118, respectively.


---

## Body

## 1. Introduction

Thermophiles are a group of microbes adapted to thrive in ecological niches such as deep-sea hydrothermal vents, hot springs, and solfataric fields. Thermophilic microorganisms are found to be potential and good alternative source of thermostable enzymes [1, 2]. The investigation and application of thermostable enzymes from thermophilic microorganisms have received a lot of attention in industries ranging from food industry to waste management industries.Thermophiles are of interest to enhance the thermostability of industrial enzymes. The modern concept of the nature of the thermostability of the enzymes from the thermophilic microorganisms can be reduced to two basic ideas. Firstly, the enhanced thermal stability of enzymes in thermophilic cells could be due to their molecular structure being different from that of their counter part mesophilic proteins. Secondly, the increased stability of the enzymes could be due to their supramolecular association with other components of the cell (lipids, polysaccharides, or proteins). In order to survive at high temperatures, thermophilic prokaryotes (Archaea and Eubacteria) adopt different strategies like GC-rich codons, the ratio of charged amino acids compared to uncharged amino acids, ionic interactions, amino acid preferences and their distribution, posttranslational modifications, and solute accumulation [3]. Most of the industrial microbial lipases derived from fungi and mesophilic bacteria [4]. Bacteria produce different classes of lipolytic enzyme, including carboxylesterases (EC 3.1.1.1), which hydrolyse small ester containing molecules at least partly soluble in water, true lipases (EC 3.1.1.3), which display maximal activity towards water insoluble long-chain triglycerides, and various types of phospholipase [5].More than 50 lipases have been identified, purified, and characterized to date which originate from natural sources such as animals, plants, and microorganisms (native or genetically engineered). A large number of beneficial thermophiles which produced lipases with good thermal stabilities have been found in diverse habitats [6, 7]. Their thermal stabilities particularly in the absence of sufficient amounts of water increase their popularity [8].The enormous potential of microbial lipases arises from the facts that they are (1) quite stable and active in organic solvents, (2) do not require cofactors, (3) exhibit a high degree of enhantio- and regioselectivity, and (4) possess a wide range of substrate specificity for the conversion of various unnatural substrates [9, 10]. Therefore screening and identification of thermostable lipolytic bacteria will act as a meaningful addition to the database on bacterial lipase research.


## 2. Material and Methods

### 2.1. Conventional Identification Tests

All isolates were initially evaluated by conventional tests, that is, Gram stain, growth, and morphometric characteristics on thermus agar ATCC 697 medium, growth at 42°C, catalase, oxidase, motility, indole production, gelatin liquefaction, oxidative fermentative carbohydrate utilization, decarboxylation of lysine, and  urease activity. Additional tests included phenylalanine deamination, nitrate reduction, citrate utilization, H2S production, hydrolysis of starch, and growth in the presence of 6.5% sodium chloride.


### 2.2. Culture Maintenance

The cultures AK-P1, AK-P2, and AK-P3 isolated from Taptapani hot water spring (19° 30′ 0N lat and 84° 24′ 0E long) of Ganjam District, Orissa, India were maintained on thermus agar and stored at 4°C.


### 2.3. Screening for Lipase-Producing Bacteria

Screening of lipase producers was carried out using tributyrin agar plate according to Lawrence 1967. Each culture was streaked onto the tributyrin agar plate and incubated at 48°C for 2 days. The lipase producing bacteria were identified by the presence of surrounding clear hydrolytic zones.


### 2.4. Lipolytic Assay of Lipase Producers

The identified bacterial lipase producers were cultivated in production medium (50 mL) in triplicate (one control and two replicates/sample). After incubation at 48°C for 36 h the cultures were centrifuged at 8000 rpm at 4°C for 15 min. The crude lipase solution was obtained by filtering through a millipore 0.22μm filter membrane and lipolytic activity was measured by universal titrimetric method [11, 12]. Each reading was taken in triplicate and the activity was calculated as amount of enzyme required liberating one micromole equivalent fatty acid per mL/min.


### 2.5. DNA Preparation and PCR Amplification

Genomic DNA was extracted from the isolates using Chromous Genomic DNA isolation kit (RKT09). Each genomic DNA used as template was amplified by PCR with the aid of 16SrDNA primers (16S Forward Primer: 5′-AGAGTRTGATCMTYGCTWAC-3′ 16S Reverse Primer: 5′-CGYTAMCTTWTTACGRCT-3′) with the programme consisted of denaturation at 94°C for 5 min and subsequent 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 2 min followed by final extension at 72°C for 5 min. The presence of PCR products was determined by electrophoresis of 10μLof the reaction product in a 1% agarose gel.


### 2.6.  16S rRNA Sequencing and Data Analysis

Sequencing analysis was performed on a 1500 bp PCR product. The sequence analysis was performed using the ABI 3130 genetic analyzer and Big Dye Terminator version 3.1 cycle sequencing kit. The three 16SrRNA sequences were aligned and compared with other 16SrRNA genes in the GenBank by using the NCBI Basic Local alignment search tools BLAST n program (http://www.ncbi.nlm.nih.gov/BLAST). A distance matrix was generated using the Jukes-cantor corrected distance model. The phylogenetic trees created using Weighbor (Weighted Neighbor Joining: A Likelihood-Based Approach to Distance-Based Phylogeny Reconstruction) with alphabet size 4 and length size 1000. The 16SrRNA gene sequences have been deposited to Genbank using BankIt submission tool and has been assigned with NCBI accession numbers.


## 2.1. Conventional Identification Tests

All isolates were initially evaluated by conventional tests, that is, Gram stain, growth, and morphometric characteristics on thermus agar ATCC 697 medium, growth at 42°C, catalase, oxidase, motility, indole production, gelatin liquefaction, oxidative fermentative carbohydrate utilization, decarboxylation of lysine, and  urease activity. Additional tests included phenylalanine deamination, nitrate reduction, citrate utilization, H2S production, hydrolysis of starch, and growth in the presence of 6.5% sodium chloride.


## 2.2. Culture Maintenance

The cultures AK-P1, AK-P2, and AK-P3 isolated from Taptapani hot water spring (19° 30′ 0N lat and 84° 24′ 0E long) of Ganjam District, Orissa, India were maintained on thermus agar and stored at 4°C.


## 2.3. Screening for Lipase-Producing Bacteria

Screening of lipase producers was carried out using tributyrin agar plate according to Lawrence 1967. Each culture was streaked onto the tributyrin agar plate and incubated at 48°C for 2 days. The lipase producing bacteria were identified by the presence of surrounding clear hydrolytic zones.


## 2.4. Lipolytic Assay of Lipase Producers

The identified bacterial lipase producers were cultivated in production medium (50 mL) in triplicate (one control and two replicates/sample). After incubation at 48°C for 36 h the cultures were centrifuged at 8000 rpm at 4°C for 15 min. The crude lipase solution was obtained by filtering through a millipore 0.22μm filter membrane and lipolytic activity was measured by universal titrimetric method [11, 12]. Each reading was taken in triplicate and the activity was calculated as amount of enzyme required liberating one micromole equivalent fatty acid per mL/min.


## 2.5. DNA Preparation and PCR Amplification

Genomic DNA was extracted from the isolates using Chromous Genomic DNA isolation kit (RKT09). Each genomic DNA used as template was amplified by PCR with the aid of 16SrDNA primers (16S Forward Primer: 5′-AGAGTRTGATCMTYGCTWAC-3′ 16S Reverse Primer: 5′-CGYTAMCTTWTTACGRCT-3′) with the programme consisted of denaturation at 94°C for 5 min and subsequent 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 2 min followed by final extension at 72°C for 5 min. The presence of PCR products was determined by electrophoresis of 10μLof the reaction product in a 1% agarose gel.


## 2.6.  16S rRNA Sequencing and Data Analysis

Sequencing analysis was performed on a 1500 bp PCR product. The sequence analysis was performed using the ABI 3130 genetic analyzer and Big Dye Terminator version 3.1 cycle sequencing kit. The three 16SrRNA sequences were aligned and compared with other 16SrRNA genes in the GenBank by using the NCBI Basic Local alignment search tools BLAST n program (http://www.ncbi.nlm.nih.gov/BLAST). A distance matrix was generated using the Jukes-cantor corrected distance model. The phylogenetic trees created using Weighbor (Weighted Neighbor Joining: A Likelihood-Based Approach to Distance-Based Phylogeny Reconstruction) with alphabet size 4 and length size 1000. The 16SrRNA gene sequences have been deposited to Genbank using BankIt submission tool and has been assigned with NCBI accession numbers.


## 3. Results and Discussion

### 3.1. Comparative Lipolytic Activity of Lipase Producers

Lipolytic activities of three lipase-producing bacteria are shown in Figure1. All the three cultures showed good lipolytic activities. Strain AK-P3 exhibited the highest lipolytic activity of 5.5 U/mL followed by strains AK-P1 and AK-P2 with an activity of 4.5 U/mL and 3.5 U/mL, respectively,  (Figure 1).Figure 1
Lipolytic activities of lipase-producing bacteria isolated from Taptapani hot water spring.


### 3.2. Thermostability of Crude Lipase of AK-P3

The enzyme has shown very good stability up to 65°C for 20 min. Further it decreased considerably at 70°C followed by a sharp decline in enzyme activity after 70°C. The exposure of the enzyme at 75°C for 20 min decreases the enzyme activity to 26% of the total activity (Figure2).Figure 2
Thermal stability of crude lipase produced by AK-P3.


### 3.3. Colony Morphology, Gram Reaction

See Tables1 and 2.Table 1
Colony morphology of isolates.
StrainColony size (mm)Colony morphologyGram reactionCellular morphologyAK-P11Circular, entire edge, Smooth surface−veCoccoidAK-P22Irregular and spreading Raised margin+veRodsAK-P31Circular, entire edge, Smooth surface, Orange pigmented−veOvoid to short rodsTable 2
Results of biochemical tests.
Biochemical testsResultsAK-P1AK-P2AK-P3Carbohydrate fermentationGlucose+ve−ve−veAdonitol−ve−ve+ve, weakLactose−ve−ve−veArabinose−ve−ve+ve, weakSorbitol−ve−ve−veCitrate utilization−ve−ve−veIndole−ve−ve+veMotility−ve−ve+veNitrate reduction+ve+ve−veLysine utilization−ve−ve−veOrnithine utilization−ve−ve−vePhenylalanine deamination−ve−ve−veUrease production−ve−ve−veH2S production−ve−ve+veCatalase+ve+ve+ve, weakOxidase−ve−ve−veMethyl Red−ve−ve−veVoges-Proskauer−ve+ve−veStarch hydrolysis−ve+ve+veGelatin liquefaction−ve−ve−ve+ Positive result; − Negative result.


### 3.4.  16S rRNA Sequence Analysis

More than 1400 bp of the 16S rRNA genes of strains AK-P1, AK-P2, and AK-P3 were sequenced. Analysis of the 16S rRNA sequences confirmed the strain AK-P1 was most similar to Acinetobacter sp. 01B0 (Figure3). The strain AK-P2 was found to be most similar to Brevibacillus sp. B2 (Figure 4) and the strain AK-P3 shares closest homology with Porphyrobacter cryptus (T) ALC-2 (Figure 5).Figure 3
Phylogenetic position based on 16S rRNA gene sequence analysis of strain AK-P1.Figure 4
Phylogenetic position based on 16S rRNA gene sequence analysis of strain AK-P2.Figure 5
Phylogenetic position based on 16S rRNA gene sequence analysis of strain AK-P3.


## 3.1. Comparative Lipolytic Activity of Lipase Producers

Lipolytic activities of three lipase-producing bacteria are shown in Figure1. All the three cultures showed good lipolytic activities. Strain AK-P3 exhibited the highest lipolytic activity of 5.5 U/mL followed by strains AK-P1 and AK-P2 with an activity of 4.5 U/mL and 3.5 U/mL, respectively,  (Figure 1).Figure 1
Lipolytic activities of lipase-producing bacteria isolated from Taptapani hot water spring.


## 3.2. Thermostability of Crude Lipase of AK-P3

The enzyme has shown very good stability up to 65°C for 20 min. Further it decreased considerably at 70°C followed by a sharp decline in enzyme activity after 70°C. The exposure of the enzyme at 75°C for 20 min decreases the enzyme activity to 26% of the total activity (Figure2).Figure 2
Thermal stability of crude lipase produced by AK-P3.


## 3.3. Colony Morphology, Gram Reaction

See Tables1 and 2.Table 1
Colony morphology of isolates.
StrainColony size (mm)Colony morphologyGram reactionCellular morphologyAK-P11Circular, entire edge, Smooth surface−veCoccoidAK-P22Irregular and spreading Raised margin+veRodsAK-P31Circular, entire edge, Smooth surface, Orange pigmented−veOvoid to short rodsTable 2
Results of biochemical tests.
Biochemical testsResultsAK-P1AK-P2AK-P3Carbohydrate fermentationGlucose+ve−ve−veAdonitol−ve−ve+ve, weakLactose−ve−ve−veArabinose−ve−ve+ve, weakSorbitol−ve−ve−veCitrate utilization−ve−ve−veIndole−ve−ve+veMotility−ve−ve+veNitrate reduction+ve+ve−veLysine utilization−ve−ve−veOrnithine utilization−ve−ve−vePhenylalanine deamination−ve−ve−veUrease production−ve−ve−veH2S production−ve−ve+veCatalase+ve+ve+ve, weakOxidase−ve−ve−veMethyl Red−ve−ve−veVoges-Proskauer−ve+ve−veStarch hydrolysis−ve+ve+veGelatin liquefaction−ve−ve−ve+ Positive result; − Negative result.


## 3.4.  16S rRNA Sequence Analysis

More than 1400 bp of the 16S rRNA genes of strains AK-P1, AK-P2, and AK-P3 were sequenced. Analysis of the 16S rRNA sequences confirmed the strain AK-P1 was most similar to Acinetobacter sp. 01B0 (Figure3). The strain AK-P2 was found to be most similar to Brevibacillus sp. B2 (Figure 4) and the strain AK-P3 shares closest homology with Porphyrobacter cryptus (T) ALC-2 (Figure 5).Figure 3
Phylogenetic position based on 16S rRNA gene sequence analysis of strain AK-P1.Figure 4
Phylogenetic position based on 16S rRNA gene sequence analysis of strain AK-P2.Figure 5
Phylogenetic position based on 16S rRNA gene sequence analysis of strain AK-P3.



---

*Source: 452710-2011-04-14.xml*