Characterization of pectin depolymerising exo polygalacturonase by Bacillus sp. HD2 isolated from the gut of Apis mellifera L

Background: Polygalacturonase is an important pectin degrading enzyme. The western honey bee (Apis mellifera L.) collects pollens from different flowers which are rich source of pectin. The microbiota in the gut of honey bee, release polygalacturonase enzyme and help in pectin digestion. This study aims to isolate and characterize novel polygalacturonase producing bacterial strain from honey bee’s gut. Methods: The bacterial strain was isolated by using pectin agar plate assay. The bacterial strain was identified on the basis of morphology and 16S rDNA sequence analysis. The enzyme assay was performed by using pectin as a substrate. Biomass of different fruits/vegetables was also used as a source of carbon during fermentation. The protein gel was run using SDS-PAGE for molecular weight determination of the polygalacturonase. Results: The bacterial strain showed the maximum growth, protein and polygalacturonase production at 72 hours of incubation. This bacterial strain was identified as new Bacillus sp. HD2. The sequence of this strain was successfully uploaded in NCBI Genbank database (Accession no. KP676929). The exo polygalacturonase produced by this strain of Bacillus was optimal at 40°C and exhibited enzyme activity in a wide range of pH from pH 5-12. The polygalacturonase production was enhanced by using yeast extract (3%) in the production medium and the enzyme activity was stimulated by Ca2+ (2 mM) and SDS (200 mM). Biomass of apple’s peel (1%) was found as an excellent source of carbon for the polygalacturonase production in fermentation medium (17.11±0.46 μmol ml-1min-1). In SDS-PAGE gel, the two clear bands of polygalacturonase were found at ~36 kDa and ~72 kDa. Conclusions: A new bacterial strain Bacillus sp. HD2 was isolated from the gut of honey bee. This strain produced the exo polygalacturonase enzyme. This enzyme was characterized under different pH and temperature and found to have maximum activity in pH 11 at 40°C. Apple peel’s biomass was found as a good source of carbon during fermentation for polygalacturonase production. The SDS-PAGE analysis confirmed two bands of protein with polygalacturonase activity at ~36 kDa and ~72 kDa.


Introduction
The natural diet of honey bee is mainly plant nectar and pollen. Pectin is an important polysaccharide which helps to form different layers in pollen wall [1]. The honey bee mid gut bacteria help to facilitate the digestion of pollen by releasing pectin degrading enzymes [2]. Engel et al., (2012) identified the genes which encode pectin degrading enzymes in bacteria of honey bee's gut [3]. Pectin digestion in honey bee gut might be helpful in resulting the release of nutrient from pollen. Also, the pectin has been shown to be toxic to honey bees [4], its digestion by gut bacteria might help the bees by avoiding intoxication. Pectin hydrolysing enzymes are known as pectinolytic enzymes or pectinases. There are three types of pectinases; pectin methyl esterase, pectin lyase and polygalacturonase. Pectin methyl esterase helps in the de-esterification of pectin by breaking ester bond between the methyl group and carboxylic acid of galacturonic residues. Pectin lyase breaks the glycosidic bonds between galacturonic doi: 10.7243/2052-6180-3-2 residues by trans-elimination reaction and polygalacturonase (PG) helps in the hydrolysis of α-1, 4 glycosidic bonds of pectin polymer converting into its galacturonic acid units [5]. Polygalacturonases have industrial importance since they help to decrease the viscosity, increase the fruit juice yield and help to determine the crystalline structure of fibers [6].
In the industrial sector, acidic pectinases are used in the extraction and clarification of fruit juices [7], whereas, alkaline pectinases have great commercial importance in the treatment of effluents discharged from fruit processing units [8], coffee and tea fermentation, oil extraction, processing and degumming of plant fibres such as ramie [9,10]. The alkaline pectinases have also been used in several biotechnological processes, like purification of plant viruses [11] and paper making [12]. The major source of acidic pectinases are fungi. The alkaline pectinases are produced from alkalophilic bacteria, mainly Bacillus spp. Although many fungi produce polygalacturonases [13], they are slow growing. The aim of this study was to isolate the bacterial strain capable of producing polygalacturonase from the gut of western honey bee (Apis mellifera L.) and optimization for maximum polygalacturonase production by the isolated strain.

Isolation of bacteria using pectin agar medium
The bacterial strains were isolated from the gut of western honey bee (Apis mellifera L.). For the isolation of bacterial strains, the gut sample from a honey bee was suspended in 10 ml of sterile potassium phosphate buffer solution (PBS). After vortexing the solution, a 10X serial dilution of the suspension was made in 1X PBS (pH 7). Thereafter, 100 μl of each dilution in the series was spread onto the surface of pectin agar using the standard spread plate technique. The plates were incubated at 28°C for 24 h. The bacterial colonies were selected based on their morphology (size and color) [14]. Pure cultures were repeatedly sub cultured on pectin agar plates and maintained for enzyme studies.

Screening of isolates for polygalacturonase activity
The isolates were screened for polygalacturonase activity by culturing it in the pectin agar medium. The clear zone around colony was detected while testing it with potassium-iodide solution [15]. The strain showing maximum zone of hydrolysis was selected for further assay.

DNA isolation and 16S rDNA amplification
The polygalacturonase producing isolate HD2 was grown in LB broth for 24 h at 28°C. Genomic DNA was isolated using Geneaid DNA extraction kit (Frogga Bio, Canada) by following the manufacturer's protocol. The DNA was amplified by using HAD-1 (5´-GACTCCTACGGGAGGCAGCAGT-3´) and E1115R (5´-AGGGTTGCGCTCGTTGCGGG-3´) primers. The PCR reaction mixture contained Taq buffer (10X), MgCl 2 (25 mM), dNTPs (0.4 mM), primers (10 mM), Taq DNA polymerase (0.25 U/μl), ddH2O (7.5 μl) and DNA template (3 μl). The PCR program used was as follows: primary denaturation 3 minutes at 95°C, followed by 35 amplification cycles consisting of denaturing at 95°C for 1 minute, annealing for 1 minute at 63°C, and extension at 72°C for 1 minute. Upon completion of 35 amplification cycles, a final extension step was done at 72°C for 10 minutes. The amplified DNA was visualized on 1% agarose gel to confirm size, quantity and purity. The PCR product was purified by using Geneaid PCR/Gel purification kit (Frogga Bio, Canada). Then the purified PCR products were sent for sequencing to Eurofins Genomics (U.S).

Isolate identification
BLAST (Basic Local Alignment Search Tool) program of NCBI database (http://blast.ncbi.nlm.nih.gov/) was used for identifying the possible genus of the isolate from the sequencing result.

Enzyme assay and total protein determination
The isolate HD2 was further screened for the quantitative polygalacturonase activity and total protein estimation by transferring 7 μl of an overnight culture to 7 mL of Dubois pectin media (pH 7.0) in a glass culture tube. The cultures were incubated for up to five days. The enzyme in the culture medium was harvested in the 1 st , 2 nd , 3 rd , 4 th and 5 th days of incubation. The enzyme activity was determined by measuring the release of reducing groups using the Dinitrosalicylic acid reagent DNS assay [16] using pectin as substrate for polygalacturonase activity. For this, 10 μl of enzyme supernatant was added to 90 μl of substrate buffer (0.5% pectin in 0.05 M potassium phosphate buffer, pH 6-13) and incubated at 40-50°C for 15 min. The reducing sugar released as galacturonic acid was determined. The cell free supernatant was used to evaluate the total protein by Bradford assay [17] using bovine serum album as standard.

Effect of incubation time on polygalacturonase production
Effect of incubation time was studied by incubating the bac-doi: 10.7243/2052-6180-3-2 teria in production medium for different time intervals (24 h, 48 h, 72 h, 96 h, and 120 h). Also, the growth of bacteria was monitored by measuring the optical density (O. D) at 600 nm and the cell free supernatant was used for the enzyme assay. Effect of pH and temperature on polygalacturonase activity The impact of the pH on enzyme activity was determined by performing the assay at different pH levels from pH 5.0 to 13.0 with cell free supernatant. For this, different pH buffers were used (Citrate buffer pH 5-6, PBS buffer pH 7-8, Glycine+NaOH buffer pH 9-11 and KCl+NaOH buffer pH [12][13]. Similarly, the polygalacturonase activity was assessed at different temperatures ranging from 30°C to 70°C.

Effect of different nitrogen source on polygalacturonase production
In order to study the impact of nitrogen source on enzyme production, bacterial culture was grown in fermentation medium containing various nitrogen sources (0.5% w/v). The different nitrogen sources were yeast extract, peptone, urea and ammonium sulphate. Similarly, the effect of concentration of yeast extract on polygalacturonase production by bacterial strain HD2 was studied by using different concentration of yeast extract ranging from 1 to 5% (w/v) in the production medium.

Effect of metal ions on polygalacturonase activity
The effect of different metal ions Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ and Zn 2+ in their chloride and sulphate salts on the activity of polygalacturonase was determined by performing the enzyme assay in the presence of these metal ions (2 mM) at 40°C for 15 min. For this assay, the reaction mixture contained 10 μl enzyme supernatant, 10 μl metal ion, 30 μl buffer and 50 μl 1% substrate (pectin). Further, the effect of different concentration of Ca 2+ from 1 mM to 5 mM, was determined by performing the polygalacturonase assay at 40°C for 15 min.

Effect of detergents on polygalacturonase activity
The effects of detergents Sodium Dodecyl Sulphate (SDS, 10 mM) and Triton X-100 (10%) were observed on the polygalacturonase activity. The assay conditions were same as that of metal ions except the detergents were used instead of metal ions. Also, different concentrations of SDS were used ranging from 50 mM to 300 mM to determine the effects of these concentrations on polygalacturonase activity.

Effect of biomass on polygalacturonase production
For the assay of polygalacturonase production in fermentation state, different fruit peels (apple, pomengrate and orange) and vegetables peels (potato and squash) were used. The fruit/ vegetables' peels were dried and powdered by a grinding machine. The overnight LB broth grown bacterial strain was inoculated in a powdery biomass (1% w/v) with Dubois salt medium for the fermentation and the polygalacturonase activity was determined at 72 h of incubation.

SDS-Polyacrylamide gel electrophoresis (PAGE)
The enzyme was confirmed by using SDS-PAGE. Ten percent SDS-PAGE was performed on the polygalacturonase by the method described by Laemmli [18]) using Bio-Rad electrophoresis apparatus. The protein marker and enzyme were allowed to run simultaneously to determine the molecular weight of the enzyme. After completion of the electrophoresis, coomassie brilliant blue R-250 was used to stain the gel. The gel containing 0.25% pectin was used for detection of PG activity. The gel was then washed with 2% Triton X-100 for 30 min, it was transferred in pH 11 and then incubated at 40 o C for 30 min. After that the gel was stained with 0.1% Congo Red solution and the stained was removed with 1M NaCl to visualize the clear bands of PG activity.

Statistical analysis
All the experiments were performed in triplicates and the results are expressed in terms of mean±SD (standard deviation). The statistical analysis of data was performed by one way Analysis of Variance (ANOVA) followed by Duncan's multiple comparison test (p<0.05). Tests for normality by Kolmogorov-Smirnov test and Shapiro-Wilk test were performed to check if assumptions of ANOVA were met before the analysis.

Results and discussion
In this study, four bacterial strains were isolated from gut of western honey bee (Apis mellifera L.). For screening purpose, these bacteria were grown in pectin agar plate and pectinolytic activity was detected using plate assay. The isolate HD2 showed maximum pectinolytic activity on pectin agar plate. Sequencing results were successfully obtained for this isolate's 16S rDNA PCR products. The DNA sequences were analysed by the nucleotide BLAST feature of the NCBI database to obtain possible identities based on homology. From the BLAST, the isolate was 99% similar to genus Bacillus. The sequence of this strain was successfully uploaded to NCBI Genbank database (Accession no. KP676929). This strain was further screened for exo polygalacturonase activity.

Bacterial growth, polygalacturonase production and the total protein determination
Bacterial growth is an important factor for the production of the enzymes. The growth factors are also of prime importance in industrial production for high production of enzymes for different applications. Bacteria show high levels of pectinases in pectin supplemented media [19]. The pattern of polygalacturonase production with reference to incubation period was monitored and the results showed that the Bacillus sp. HD2 showed considerable growth and maximum enzyme production (12.44±0.8 μmol ml -1 min -1 ) at 72 h of incubation (Figure 1) which was higher than other strains of Bacillus like B. sphaericus MTCC 7542 [20]. The production of polygalacturonase was decreased gradually (p<0.05) after 72 h of incubation. Also, the total protein in the supernatant

Effect of pH and temperature on enzyme activity
pH plays a significant role in the stability of enzyme activity. The present polygalacturonase from Bacillus strain was stable in the broad range of pH. The maximum polygalacturonase activity was found at pH 11 which was significantly different (p<0.05) than other pH levels (acidic and neutral). The relative enzyme activity showed that the PG was also active even in acidic pH retaining its activity of 56% at pH 5 ( Figure 3A). Similar results of different alkaline polygalacturonase by different species of Bacillus were reported by other researchers [21][22][23][24]. Kapoor (2000) also reported that the polygalacturonase produced by Bacillus sp. MG-cp-2 was stable in alkaline conditions pH 7-12 [25]. Similarly, a polygalacturonase from Klebsiella sp. Y1 was reported to be stable in a wide range of pH (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in digestive tract of sheep [26].
Like pH, temperature stability of enzyme is important for industrial application [27]. The effect of temperature on polygalacturonase production by strain HD2 was studied at different temperature ranging from 30°C to 70°C. The enzyme was stable in a wide range of temperature and an increased polygalacturonase activity was found at 40°C. The polygalacturonase activity was reduced significantly (p<0.05)) at the temperatures below 40°C. The PG was stable even at 70°C retaining 66% of its enzyme activity (Figure 3B). A moderate temperature is important for longer incubation period to reduce the cost of enzyme production [21]. The results were similar with the temperature optimization for polygalacturonase from Bacillus subtilis DT7 [28].

Effect of different nitrogen source (0.5%)
Maximum polygalacturonase (p<0.05) production was achieved when yeast extract (YE) was used in culture indicating 8% increment over the control treatment without any nitrogen source followed by peptone ( Figure 4A). It might be because of the fact that yeast extract has essential vitamins, minerals   and amino acids which are helpful for bacterial growth and enzyme production. Similar reports were found in Bacillus sp. by Rehman (2012) [29]. Yeast extract is helpful for exo pectinase expression [29,30]. Different concentrations of yeast extract were used for enzyme production and it was found that maximum enzyme production was achieved when 3% yeast extract was used in the medium. On increasing the concentration of YE over 3%, the PG relative activity was decreased gradually (Figure 4B).

Effect of metal ions on PG activity
The activity of polygalacturonase was enhanced by Ca 2+ over Co 2+ , Mg 2+ , Mn 2+ and Zn 2+ . The PG activity by Ca 2+ was statistically significant (p<0.05) in comparison to other metal ions. All of these metal ions had no remarkable inhibition effects on PG activity ( Figure 5A). While using different concentrations of Ca 2+ , maximum polygalacturonase was produced at 2 mM of Ca 2+ . Further, the PG activity was decreased with the higher concentrations of Ca 2+ (Figure 5B). Similar results of polygalacturonase stimulation by Ca 2+ were found in Bacillus sp. and Klebsiella sp. Y1 [26,31,32]. The metal ions Mg 2+ , Ca 2+ , Zn 2+ , Co 2+ and Mn 2+ have been found to stimulate the exopectinase activity by Bacillus GK-8 [33].

Effect of detergents
The polygalacturonase was found to be significantly tolerant (p<0.05) to surfactant SDS ( Figure 6A) and was reduced by Triton X-100. The stimulation of PG activity by SDS might be due to the increase affinity of active site of the enzyme to the substrate by lowering the surface tension by this detergent. The polygalacturonase activity was found to be most active when 200 mM of SDS was used ( Figure 6B). The activity of PG from Bacillus sp. was stimulated by SDS as reported by other researchers [25], while Kobayashi (2001) reported 10% reduction in polygalacturonase from Bacillus strain by SDS [31]. In other reports, the polygalacturonase from Sporotrichum thermophile was slightly activated by Triton X-100 but the enzyme activity was completely lost by SDS [34]. The tolerance

Effect of different biomass in PG production during fermentation
When the enzyme production profile of Bacillus sp. was studied by using different fruits and vegetables' peels, the enzyme was assayed at 72 h of incubation. The biomass of apple's peel (1%) was found most significant (p<0.05) for the production of polygalacturonase (17.11±0.46 μmol ml -1 min -1 ) during fermentation (Figure 7). Apple biomass is an excellent source of polygalacturonase and other pectolytic enzymes [35,36]. However, Embaby (2014) reported that orange peel is an effective inducer (carbon source) for alkaline polygalacturonase by Bacillus licheniformis SHG10 [37]. Pectinases could be used to hydrolyse the pectin in pectin rich agro industrial wastes. The bacterial strain in its optimized conditions could be used to increase the polygalacturonase production in industrial scale.

SDS-PAGE analysis
The SDS-PAGE analysis of crude PG showed different protein bands. Out of which, two clear bands were found with positive

Conclusions
In this research, a new strain producing polygalacturonase was isolated from the gut of western honey bee (Apis mellifera L.) and identified as Bacillus sp. HD2 after 16S rDNA sequence analysis. Maximum production of polygalacturonase by this strain was achieved at 40°C after 72 h of incubation (14.31±0.54 μmol ml -1 min -1 ). A novel, alkaline active and temperature stable polygalacturonase has been produced from this Bacillus sp HD2 showing molecular weights of ~36 kDa and ~72 kDa. The enzyme activity was enhanced by metal ions Ca 2+ and detergent SDS. On the basis of these properties, the polygalacturonase from this bacterial strain qualifies for use in the depectinization of pectic wastewaters from industries, as alkalophilic pectinolytic microbes help in the easy removal of pectic material and render it easily decomposed by activated sludge treatment. In addition to these properties, some additional features like enhanced production by yeast extract and apple biomass in the growth medium (17.11±0.46 μmol ml -1 min -1 ) and the activity of enzyme in a wide range of pH, indicate the potential use of this organism at commercial level for animal feedstock, degumming of ramie and fruit juice-processing. Further, these results might be helpful to study the enzymatic pectin degradation mechanism in western honey bee (Apis mellifera L.).