Detection of Disinfectant property of purified Amylopullulanase from Citrobacter freundii SW

Wafaa Hassan Muslem, Sahira Nsayef Muslim,Alaa N. Mahammed Ali, Raghad J. Fayyad

Department of Biology, College of Science, Mustansiriyah University, Baghdad- Iraq

*Corresponding Author E-mail: stln@uomustansiriyah.edu.iq

ABSTRACT:

There are varieties of marketable detergents and disinfectants purchased; in order to choose an effective one; it is very necessary to ensure its decontamination proficiency. So that a screening of pullulanase production from many isolates of Citrobacter freundii have been done using dried water cabbage as inducer of pullulanase production in the culture medium.Pullulanase producing bacteria was isolated and strain SW was designated. Morphological and biochemical tests in addition theanalysis of 16s rRNA observed that the strain SW revealed a 100% sequence identity withsimilarity to that of Citrobacter freundii. The pullulanase was purified by using two-step purification procedure consisting of ammonium sulphate precipitation, ion exchange chromatography followed by gel filtration chromatography with a yield of 29.7%. The pullulanase has an important role in augmentation of disinfectant efficiency. Depending on the current results, the combination between pullulanase and commercial disinfectant resulted to increase the disinfectant activity to greater levels against fungi more than bacteria.

KEYWORDS: Pullulanase, Citrobacter freundii, Disinfectant, 16s rRNA

INTRODUCTION:

Citrobacteris members of Enterobacteriaceae family, it’s a gram-negative bacteria. Citrobacter is commonly found in soil, water, sewage and foods. Citrobacter species are belong to a variety of animals' natural microbial flora. Besides becoming human pathogens, Citrobacter species are significant pathogens in fish ¹. Enterobacteraceae are a sign of sewage contamination and an opportunistic pathogen in fish that causes diarrhea in fish have been recorded². The infection is triggered by the penetration of pathogens to the epithelium resulted to toxins releasing, and then spreads further in the sub mucosal locations.³

Pullulan is a linear maltotriosis polymer used in many industries². Pullulanases are defined as endo-acting debranching enzyme that specifically cleaves a-1,6glycosidic linkages in pullulan, starch, amylopectin, and related oligosaccharides. Pullulanase in plants and bacteriais widely distributed. The special behavior on α-(1,6) glycosidic linkages in pullulan attracts greater interest in Microbial pullulanase. Bacillus acidopullulyticus, Bacillus deramificans and Bacillus cereus are some of the pullulanases producers⁴.

Since Pullulanases possess different applications. The most important industrial application of pullulanase is to the productionof glucose or maltose syrups⁵. This group of enzymes are mainly used for the production of starch that characterized with resistanceduring starch processing industries, detergent, cyclodextrins and baking manufacturing, it can also be used as an agent to control dental plaque⁴. Because of the high cost of production and low yield and for the potential large-scale productionof pullulanase we need to use cheap substrates that are high in starch.

Disinfectants are antimicrobials used to kill microorganisms that live on the surface of non-living objects.They are an essential part of infection control practices and aid in the prevention of laboratory and nosocomial infections. Many gram negative bacteria, gram positive bacteria and yeasts leads to nosocomial infections⁶. As there are widely distributed commercial disinfectants, choosing of effective product is a prime target to ensure its efficiency to decontaminate. The goal of this research was to explore pullulanase production by Citrobacter freundii, purification of pullulanase and using it as disinfectants against pathogenic bacteria and fungi.

MATERIALS AND METHODS:

Sample preparation:

Thirty randomly chosen fish were scarified and the gastrointestinal tract was detached with sterilized dissecting tools from each sample and to removethe contents, anterior and posterior guts were gently shaked for 2-3 minutes in sterilized saline solution, after putting the prepared sections in tubescontaining a mixture of 9.0mLfrom NaCl solution (0.85%) and 15 to 20 glass beads, the tubes shaked for 2 minutes.Serially, the homogenates were diluted to 10^-6 insaline solution (0.85%). A volume of (0.1) ml of each sample was spread onpetri dishes containing blood agar and MacConkeys agar, then incubated at 37°C for 18-24 hour³.

Bacterial Identification:

The purified isolates were subjected to morphological and biochemical identifications guided by Berge's Manual of Systemic Bacteriology. Suspect isolates ofCitrobacterwere identified byGram staining pattern and standard biochemical reaction like catalase test (+), oxidase test(-),i ndole test(+), methyl red test(-),urease test (-),and ability to motile(6). Further, the Citrobacter freundii isolates were verified using the Vitek 2 system with the use of a Vitek GNI card (bio Merieux, France) as directed by the manufacturer.

The extraction of DNA:

The genomic DNA was extracted from the SW bacterial strain using A DNA purification Kit (Promega Kit). The SW bacteria genetic material concentration was 250 ng/µl as calculated by measurement of Nano-Drop analysis, then, the gel electrophoresis for the extracted DNA was performed.

PCR amplification of the 16S rRNA, gene sequencing and analysis of the 16S rRNA.

 As recommended by [7], universal primers Fd1 (5’-AGA GTT TGA TCC TGG CTC AG-3’) and rP1 (5’-ACG GTC ACC TTG TTA CGACTT’) is used to amplify the 16S rRNA of the SW isolate and the PCR produce was visualized by gel electrophoresis. Wizard Genomic DNA Purification Kit (Promega, USA) was used to prepare the chromosomal DNA from the late exponential phase culture. Every amplification reaction volume was 25µL which contained a premix.Then the amplicons have electrophoresed on an agarose gel (0.8%). The sequence found was compared with other sequences in the public databases utilizing the BLAST search program in the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov).

Phylogenetic analysis of 16S rRNA gene.

BLASTn analysis tool was used to align and compare the sequence of 16S rRNA with the sequences deposited in the Gene Bank database from NCBI. Then, BLASTn sequences were selected and added to MEGA6 software directly. The first five sequences gained from the BLAST analysis was aligned using the command of CLUSTAL‒W. Consequently, using CLUSTAL-W and Phylogeny Construction applications MEGA6 the alignment of the multiple sequences have been determined⁸.

Screening of pullulanase producers:

The abilityto produce pullulanase of all bacterial isolateswas evaluated by culturing these isolates in the center of improved pullulan agar medium having) (g/l): (4) dried water cabbage (10), NaCl (2), MgSO₄.7H₂O (0.1), K₂HPO₄ (0.17), KH₂PO₄.7H₂O (0.12) and agar [9], pH 7.0. Then plates were incubated at 30°C till the colonies observed. The colonies with clear zones around, regarded as producers of pullulanase.

Submerged fermentation for pullulanase production:

The chosen isolate has been inoculated to modified pullulan agar medium (water cabbage medium) except that it does not contain agar. The incubation was done at 30°C for 2 days. The culture was centrifuged for 10 min at 8000rpm and as crude enzyme, the supernatant was used.

Pullulanase activity:

This test was assayed through calculating the reduced sugar released from pullulan. A total volume ( 3ml) of reaction mixture consisting of 0.5 ml pullulan (1% w/v) and 0.5ml crude source of enzyme in 2ml of sodium phosphate buffer (0.1M, pH 6.5) and 0.5ml CaCl₂ (0.02% w/v) were added to the reaction mixture. The reaction was stopped after an incubation of 40 ºC for 20 min. when the tubes were cooled in an ice bath and sugar released by pullulan enzyme hydrolysis was determinedby adding 1ml of 3,5-dinitrosalicyclic acid reagent and incubated in boiling water for 5 min.the enzyme activity at 540nm was measured⁹. The enzyme amount that released one micromole of glucose from reducing sugar per minute in standard assay conditions is classified as one unit of pullulanase.

Determination of protein content:

Bradford dye method with BSA was used for the determination of the protein content of the enzyme¹⁰.

Pullulanase extraction and purification:

The pullulanase was purified according to¹¹ with minor modifications. At the final stage of the fermentation process, the supernatant that obtained after the centrifugation was used a source of crude enzyme. Ammonium sulphate precipitation was used to concentrate the crude enzyme at 20-70% saturations. The test was left at 4ºC overnight and the pellet was collected at 1000rpm for 15 min by centrifugation and dissolved in 0.1M sodium phosphate buffer(pH 6.5), then dialyzed against the same bufferovernight. DEAE-cellulose column (2x25cm) equilibrated with the same buffer was used to load the dialyzed protein. The elution of bound proteins was done by gradient mode with increasing concentration of NaCl (0.2to 0.6 M) at a flow rate 3ml/ min. The eluted fractions were checked for pullulanase activity and active fractions were pooled and stored at 4ºC. Pooled samples were loaded on Sephadex G-100 column (1.5x70cm). The protein elution was done with sodium phosphate buffer (0.1M) at a flow rate of (1ml/min). The concentration of protein at 280 nm and pullulanase were assessed and for further studies the active fractions were pooled.

Effect of pullulanse on the efficiency of disinfectants:

Two types of purchased commercial disinfectant were examined to determine their inhibition zone against different types of gram negative bacteria (Acinetobacter sp., P. aeruginosa and salmonella typhimurium), gram positive bacteria (Staph. aureus) and yeast (Candida albicans). A serial dilutions (Twofold) of each tested disinfectant using distilled water to obtain concentrations ranged between 16 to 128μg/ml. A 0.1 ml of 1.5x10⁸ cfu/ml bacterial suspension was spreadered on the surface of Mueller-Hinton agar plates, left to dry for 15 minutes at room temperature. About 50µl from each dilution was located in wells (7mm in diameter) on Mueller-Hinton agar plates. In the other hand, 3.3mg/ml of purified pullulanase was combined with each concentration of each disinfectant separately then 50µl from the mixture was placed in wells on Mueller-Hinton agar plates. After that the plates were incubated at 37°C for 18 to 24hour. The diameter of an inhibition zone for the mixtures and disinfectant alone was measured¹².

RESULTS AND DISCUSSION:

Bacteriological analysis:

The standard biochemical identification findings for all bacterial isolates are compatible with thoseresulted by the Vitek 2 system. Out of 30 fish samples, 8Citrobacterfreundiiisolates were gotten with percentage of 27%.The bacterial species including Enterobactersp, Escherichia coli, Citrobacter sp, Pseudomonas sp and Klebsiella sp, Streptococcus sp ,Staphylococcus sp and Bacillus. The fishes were the source to isolate all the previous isolates, this indicated that these possible human pathogens¹³.The study in [14] found thatCitrobacterfreundiiwas Isolated from the intestines of catfish with a percentage of 10.4%. Moreover, [2] isolated Citrobacter freundii from Oreochromisniloticus fish samples with percentage of 8.1%.

Identification and Sequencing of SW bacteria using analysis of 16S rRNA gene:

With the universal primers Fd1 and rP1, the 16S rRNA gene for the SW isolate was successfully amplified. The PCR product gel electrophoresis showed the amplified gene corresponding to the expected sizing of approximately 1500 bp.The PCR result of the SW isolate was sequenced and the BLASTn method was used to align 1500 bases of the sequenced SW gene with other organisms.The outcomes for the search of BLASTn and the significant alignments of sequences proposethat the SW strain of the genus Citrobacter freundii (Table 1).

Table 1: The top 5 entries species in the NCBI database for SW.

Microorganisms	Accession Number	Maximum Score	Maximum Identity
Citrobacterfreundii isolate MGYG-HGUT-01705	LR698971.1	2177	100%
Citrobacterfreundii strain LDL3-3	CP047247.1	2056	98.14%
Citrobacterfreundii strain FDAARGOS_549	CP033744.1	1998	97.26%
Citrobacterfreundii strain UMH14	CP024680.1	1998	97.26%
Citrobacterfreundii strain UMH19	CP024673.1	1986	97.08 %

Phylogenetic analysis:

For phylogenetic analysis, bacterial species from the same isolate family were chosen.The 16S rRNA sequence for this species have been gotten from the database found in NCBI and aligned using CLUSTAL Wtool in MEGA6software. A phylogeny tree of neighbor-junction with boot strap value has been observed (Figure 1), and it suggests that the bacterial indivisual SW closely designated as Citrobacterfreundii.

Figure 1: Neighbour-Joining phylogeny tree of isolate SW. The numbers before the bacteria names are their respective accession numbers

Screening for pullulanase production:

Throughout this study eight Citrobacter freundii Isolates were screened to determine their ability to produce pullulanase (Figure2) Among these isolates, there was no pullulanase development in three isolates, while the remaining five isolates showed clear areas around their colonies after 24 hour of incubation and This suggests the pullulanase enzyme produced by these isolates and Citrobacter freundii F₈ gave higher pullulanase productivity hence this isolate was selected for another studies.The differences in the diameter of hydrolytic zone among Citrobacter isolates return to differences in genetic expression of pullulanase. Earlier reports showed that pullulanases are mainly extracellular enzymes generated by various bacterial generations, mainly Bacillus sp.^{4, 5}.

The results of submerged fermentation for pullulanase production revealed that water cabbage which verified as a single source of carbon and energy gave maximum pullulanase production achieved since the pullulanase activity approach to 86.8U/ml with Citrobacter freundii F₈ ( Figure2) after incubation at 30ºC for 24 hour.

Figure 2: Pullulanase activity for Citrobacter freundii isolates

The carbon sources are considered the key factor in the bacteria that provide the energy needed for a growth and enzyme production [12]. Pullulanase or also recognized as α-dextrin 6-glucanohydrolase, limit dextrinase, pullulan 6-glucanohydrolase and amylopectin 6-glucanohydrolase is resulting from a number ofbacterial genera like Bacillus deramificans, Bacillus acidopullulyticus, Bacillus cereus FDTA-13, Klebsiella planticola, thermophilic Bacillus sp. AN-7 and Geobacillus stearothermophiles¹⁰.

Purification of pullulanase:

In various stages of purification, the purification profile of Citrobacterfreundii extracted pullulanase is shown in (Table 2). The results show that the precipitate produces the best yield of the enzyme at 60 percent saturation in ammonium sulfate. The sample of DEAE-cellulose column was eluted by using gradient concentrations of NaCl ranging from 0.2 to 0.6M (pH6.5) in the sodium phosphate buffer.In the first protein peak, the eluted fractions had three protein peaks and pullulanase activity (Figure- 3).The fractions which had higher activity of pullulanase were concentrated and passed through the column of Sephadex G-100 with a buffer of sodium phosphate at 0.1M (pH 6.5). After elution, a protein peak occurred, and activity of pullulanase was seen at this peak (Figure 4).The enzyme was purified by a yield of 29.7% and 11.2 fold of purification as reported in (Figure 4).

Lactococcus lactis IBB 500 have been used to purify Extracellular pullulanase by using fractionation by ammonium sulfate and dialysis as alternative to ultrafiltration, I addition, ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step with a yield 14.36 has been achieved¹⁵. The enzyme was purified to homogeneity from Bacillus naganoensis by ion exchange and size exclusion chromatography from cell free culture supernatant¹².

Figure 3: Purification of pullulanase from Citrobacter freundii F₈ by using ion exchange chromatography on DEAE-cellulose column

Figure-4: Purification of pullulanase from Citrobacter freundii F₈ by using gel filtration chromatography on Sephadex G-100column

Effect of pullulanse on the efficiency of disinfectants

The results showed that any one of each type of disinfectant had antimicrobial activity against all the tested microorganisms.The first type of disinfectant reveald higher effectiveness toward fungi since in Candida albicansthe diameter of inhibition zone reached to23 mm also toward Salmonella typhimuriumand Acinetobactersp. with 23and 22 mm while lower effectiveness againstother tested bacteria. But in the combination with the purified pullulanase the effect increased and showed higher level toward Candida albicansin comparison with other bacteria as shown in table3.

Table2: Stepsof pullulanasepurification from Citrobacter freundii F₈

Purification step	Size(ml)	Pullulanase activity (U/ml)	Protein conc. (mg/ml)	Specific activity (U/ mg)	Total activity	Purification fold	Yield (%)
Crude extract	40	86.8	22.1	3.92	3924	1	100
(NH4)2SO4 precipitation	20	98.1	11.2.	8.75	1962	2.23	50
DEAE-cellulose	12	120	6.1	19.6	1440	5	36.6
Sephadex G-100	8	146	3.3	44.24	1168	11.2	29.7

Table 3: Diameter of inhibition zones for differentstrains in plates in presence of disinfectants with and without pullulanase

Diameter of inhibition zone for disinf.2 (mm) at different con. + 3.3 (mg/ml)				Diameter of inhibition zone for disinf.2 (mm) with different con. (mg /ml)				Diameter of inhibition zone for disinf.1 (mm) at different con. + 3.3 (mg/ml)				Diameter of inhibition zone for disinf.1 (mm) with different con. (mg /ml)				Organism
128	64	32	16	128	64	32	16	128	64	32	16	128	64	32	16
18	18	19	17	21	20	17	15	23	23	20	18	21	22	18	16	Acinetobactersp.
19	20	20	18	14	15	18	17	21	22	19	18	14	19	17	16	P. aeruginosa
19	20	22	18	18	19	21	18	18	24	20	18	20	23	20	17	Salmonella typhimurium
20	21	22	20	15	17	19	18	22	23	21	19	21	20	19	17	S. aureus
24	25	26	20	18	18	22	19	24	26	23	20	23	23	19	19	Candida albicans

In the case of using the second disinfectant, this disinfectant had lower effect than the first toward all tested microorganisms.On the other hand, the mixing of this disinfectant andobtained pullulanaseresulted to rise the disinfectantactivity to the maximum levels sincethe diameter of inhibition zone increased to 26 mm against Candida albicansfollowed byS. Aureus and Salmonella typhimurium with 22 mm and lower diameter was 19 mm against Acinetobacter sp. According to these results we can conclude that the pullulanase has significant role in enhancement of disinfectant activity against fungi more than bacteria.The fungal cell wall is uniquely composed of mannoproteins, chitins, and α- and β- linked glucans ¹⁴. Microbe pullulanase becomes more essentialdue to its specific action on α-1,6 linkages in pullulan, a linear α-glucan which is mainly made up ofmaltotriosyl units connected by 1,6-α-bonds ¹¹.

ACKNOWLEDGMENT:

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad- Iraq for its support in the present work.

CONFLICT OF INTEREST:

Authors do not have a conflict of interest.

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Received on 09.04.2021            Modified on 22.05.2021

Accepted on 11.06.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(2):847-852.

DOI: 10.52711/0974-360X.2022.00141