Isolation Of Pectobacterium Carotovorum , Identification With 16s Rrna, Phytase Activity And Characterization Of The Bacteria CURRENT STATUS: POSTED

Background: Phytases can be produced by animals, plants and microorganisms. However, the most promising ones for commercial use and biotechnological applications are those of microbial origin. Phytases are also used in the preparation of myo-inositol phosphates in the food industry, soil remediation and in the paper industry. Biotechnology, along with the increased use of phytase enzymes, is a highly effective technology that is used today and will be used in the future to produce these enzymes and improve their properties. Results: The aim of this study was to conduct the molecular identification of Pectobacterium carotovorum strains isolated from lettuce to produce phytase from a new microbial source and the characterization of the enzyme. The activity and characterization of the phytase obtained from the bacterium was carried out. Isolation of strains was carried out following incubation at 26 ° C for 48 hours using Nutrient agar (Oxoid). The identification was performed using the 16S rRNA method. The phytase produced from Pectobacterium carotovorum showed the best activity at pH 8.0. The optimum temperature of the phytase obtained from Pectobacterium carotovorum was 60 ° C. Conclusions: In this study, enzymatic activity of phytase was investigated in Pectobacterium carotovorum for the first time. The results showed that it can be used in the industry due to the characteristics of the enzyme produced by Pectobacterium carotovorum.

The term "Biotechnology" can single-handedly explain what biotechnology means.
Biology explains its relation to living organisms, while technology means shedding light to existing or emerging problems and developing useful products. Accordingly, biotechnology means using living organisms to shed light to problems and develop more useful products [1]. The application areas of biotechnology are divided into five fields comprising medical, agricultural, food, animal, environmental and industrial biotechnology [2].
In microbial biotechnology, the production of enzymes and organisms that are used in the production of various foods, facilitation of processing and more efficient disposal of industrial wastes has become possible with the manipulation of microorganisms such as bacteria and yeast. The vast number of bacteria and other microorganisms allows various potential biotechnological applications. Microbial biotechnology is a dynamic field that generates a wide range of new applications [3]. The advantages of bacteria including their rapid growth, easy and controlled culture in a small area and simpler structure compared to eukaryotes facilitate their use in the common fields of genetics and biochemistry [4].
Pectobacterium carotovora subsp. carotovora is a phytopathogen bacterium that causes soft rot in many economically valuable plant varieties. [5]. Pectobacterium carotovora subsp. carotovora causes one of the most destructive vegetable diseases worldwide (ECC). Pectobacterium carotovorum subsp. Brasiliense causes serious damage to potatoes and other vegetables due to black spot and soft rot diseases.
When the differences between isolated species and subspecies are considered, Pectobacterium, which are similar to other species from the Enterobacteriaceae family that cause soft rot in plants, are facultative anaerobic, Gram (-), rod-shaped bacteria that have multiple flagella. In the studies, all isolates of the species (P. carotovora. subsp. carotovora, P. carotovora. subsp. atroseptica, P. carotovora. subsp. betavasculorum, P. carotovora. sp. wasabiae, P. carotovora. subsp. odorifera) were determined to be catalase positive and oxidase negative and have the ability to carry out glucose fermentation, reduce nitrate to nitrite, produce ß-galactosidase and H 2 S and use L-arabinose, D-galactose, D-glucose, glycerin, D-mannose, D-ribose and sucrose, do not have the ability to produce urease and have the ability to produce acid from adonitol. Majority of the isolates can use L-rhamnose and Dmannitol but lack the ability to use dextrin. The Pectobacterium species in this group cannot be tested using every test used for Enterobacteriaceae and, thus, an ambiguity emerges in their taxonomy. The general methods used in the isolation of phytopathogen bacteria from soil and plant samples can also be used for these bacteria. Enriched or selective media should be used in the isolations from rotten plant tissues and soils [6].
A new standard for the identification of the bacteria was developed in the 1980s.
We have come to understand that the comparison of certain regions of the genetic codes of bacteria can help determine the phylogenetic similarity between bacteria [7,8]. The genes in the bacteria genome used for this purpose include 5S, 16S and 23S rRNA and the regions between these genes. Today, the most commonly used DNA region in the classification of bacteria is the 16S rRNA gene region [9,10,11].
16S rRNA gen sequence region is around 1550 base pairs (bp) long and consists of hypervariable and conserved regions. Using the 500 bp-long region is more costfriendly and applicable and, thus, the 500 bp-long region is widely used in sequence analyses. 16S rRNA gene sequence analysis steps comprise DNA isolation, PCR, purification, sequence cycle, sequence analysis, evaluation of the electropherogram and data analysis, respectively [12].
Enzymes have an important role in bacteria metabolism and various enzyme systems are needed for metabolic activity. Enzymes are catalyzer activators, i.e.
has an accelerating effect on a reaction. Reactions can start without enzymes.
Enzymes allow a significant amount of freedom for substrates. They show better activity under optimum pH and temperature conditions. The purity of industrial enzymes is usually low and, thus, their cost is lower, whereas scientific, analytic and medical enzymes have high purity and, thus, cost more.

3.1.3.26)
. This classification is based on the first phosphate group the enzyme attaches to. 3-phytase is reactive for microorganisms and 6-phytase is reactive for plants [13]. Phytases catalyze the gradual removal of phosphate groups from the myo-inositol ring of phytate. These enzymes can be widely found in nature and more than 2000 soil isolates have been reported. According to their catalytic mechanisms, phytases can be called histidine acid phytases, cysteine phytases and purple acid phytases [14]. Although phytase was detected in plants, microorganisms and some animal tissues, recent studies have shown that microbial phytase yielded successful results in biotechnological applications [15. 16].
The study investigates the molecular identification of the P. carotovorum strain that was isolated from lettuce and procured from the Agricultural Biotechnology Culture Collection, phytase production from a new microbial source and characterization of phytase.

Results
In the study, the stock culture of the bacteria strain No. M7 available in the Enzyme and Microbial Biotechnology Science Department was used.

Morphological Properties of the Microorganism
Certain morphological properties of the isolate were examined. The sizes, endospore formation and Gram properties of the isolates were determined. Table 1 shows the results obtained for the morphological properties of the isolates.

Determination of the Properties of the Culture
In the investigation of the culture properties of the bacteria, the results were obtained when mature colonies of each isolate were formed in the agar medium (Nutrient agar). Table 2 shows the culture properties of the isolates in detail.
Furthermore, Fig. 1 shows the colony and micron morphologies. The bacteria were isolated using streak plate method. Pure colony structures in the culture were examined and the pure colonies were used in further studies.

Determination of Some Biochemical Properties
To support the identification with the molecular method, some biochemical tests were performed to obtain a precise identification. Table 3 shows the biochemical test results. The genomic DNA isolation from the P. carotovorum isolate was carried out by following the protocol described in Sect. 3.2.5.a with no modification. Figure 2 shows the DNA isolation results.

Gel image of the PCR results
The PCR reactions that were carried out using the genomic DNA obtained from the P. carotovorum isolate yielded PCR products of desired sizes of 1400-1500 bp. Figure 3 shows the results. The marked region indicates 1400 bp on the marker.

Sequence analysis results
Gene sequencing was carried out to confirm whether the PCR result was the 16S rRNA gene region. Figure 4 shows the sequences obtained by sequence analysis.
The purpose of the tree is to show the relationship between close species and similarities between the samples that were determined to be species and their subspecies.
The tree shown in Fig. 5 was obtained from NCBI and the accession number was given. The examination of Section A revealed three systematic main groups.
Pectobacterium atrosepticum was the main taxon and identified as the outgroup.
There were major similarities between the outgroup and other groups. The second important group was Pectobacterium carotovorum, the subject of the article. These serve as two examples of sister taxa. The group showed a high similarity. The other group is currently called Erwinia in mass. They had a high similarity among each other. E. persicinus and E. rhapontici were identified as different species and showed a similarity of 94% and, thus, had a higher similarity even when compared to their subspecies.

Phytase Activity Results
The activity of isolated and identified P. carotovorum was measured at different temperature and pH values and the results are given below.

Optimum pH results for phytase
Measurements were carried out to determine the optimum pH for phytase that was produced and purified from P. carotovorum. For optimum pH, 10 different pH values ranging from pH 2.0 to pH 11.0 were investigated. For pH 2.0-6.0, acetate, for pH 7.0-9.0, Tris HCl and for pH 10.0-11.0, carbonate were used as buffers. The activity of the enzyme was measured using the results and "Activity-pH" graphs were drawn. Figure 6 shows the results.

Optimum temperature results for the phytase obtained from P . c a r o t o v o r u m
Measurements were carried out to determine the optimum temperatures for phytase that was produced and purified from P. carotovorum. Its activity was calculated using the results and the "Activity-°C" graphs were drawn. The results showed that the optimum temperature for the phytase obtained from P. carotovorum was 60 °C. Figure 7 shows the optimum temperature results for the phytase obtained from P. carotovorum.

Discussion
The individual morphologies of bacteria can only be examined under a microscope due to their small sizes. For this purpose, the pure preparations of the bacteria grown in liquid or agar media are stained with specific dyes. In microscopic examinations, the properties of bacteria such as size, endospore formation and staining (Gram (-) or (+), vb.) are examined [17,18] P. carotovorum is a rodshaped, non-endospore-forming, Gram (-) bacterium with a diameter of 0.5-0.6 µm and size of 2 µm. The isolates are inoculated into agar media either with spot or streak plate isolation methods and, then, colony formation is waited; then, colony morphology is examined under a microscope to determine their appearance from above, height, edge shape and color [19]. The colony structure of P. carotovorum is oval, cream-colored and dry. Certain biochemical tests were carried out for P.
carotovorum subps. carotovorum. Our results agree with the results obtained in previous studies [6]. The results showed that P. carotovorum subps. carotovorum tested positive for citrate and catalase and negative for indole and phosphatase.
The bacteria were Gram (-) in terms of cell wall structure.
In the study, phytase activity of P. carotovorum, which is used in various areas, temperature and pH values that can affect phytase activity and optimum pH and temperature values were investigated. To determine the optimum pH of the phytase purified from P. carotovorum, activity measurements were carried out in the pH range of 2.0-11.0. For pH 2.0-6.0, 0.1 M acetate buffer, for pH 7.0-9.0,10 mM Tris/HCl buffer and for pH 10.0-11.0, 10 mM carbonate buffer were used to measure the activity of the enzyme with spectrophotometry. The phytase that was extracellularly produced and purified from P. carotovorum had the highest activity at pH 8.0 [28]. Since there were no previous studies on phytase from P.
carotovorum, the results of the study were compared by using the activities of phytases from other bacteria species as reference. Tran et al. [29] found that the optimum temperature for the Bacillus Sp. MD.2 strain was 70 °C. In their study, In their study, Erkan [30] determined that the optimum pH for Bacillus subtilis was 6.5.
The study is an important contribution to the literature as it is the first study on phytase production from P. carotovorum. There were no studies on phytase production from P. carotovorum in the literature. With phytase production from P.
Carotovorum, we are of the opinion that the study will both add to biotechnology in terms of its R&D potential and add diversity to global enzyme industry in terms of production. The phytase activity of P. carotovorum differs depending on temperature. In their study, Tran et al. [29] found that that the optimum temperature for the Bacillus Sp. MD.2 strain was 70 °C. Erkan [30] determined the optimum temperature for Bacillus subtilis as 60 °C. Since there were no previous studies on the phytase activity of P. carotovorum, we were not able to compare the optimum temperature of 60 °C as found in our study to the literature. The lowest activity was measured at 20 °C. Phylogenetic tree was drawn to determine the position of Pectobacterium carotovorum on the tree. Finally, three systematic main groups were generated and our bacteria were highly similar to these groups. In conclusion, in the study, the optimum pH of P. carotovorum was determined to be 8.0, while it had the lowest activity at pH 4.0. Optimum temperature was found to be 60 °C, while the lowest activity was measured at 20 °C. The phytase activity of

Funding
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.    Sequence analysis results of the 16S rRNA gene region from the P. carotovorum isolate Figure 5 Determination of the location of P. carotovorum on the phylogenetic tree Determination of the location of P. carotovorum on the phylogenetic tree Effects of pH on the enzymatic activity of the extracellular phytase obtained from P. carotov Effects of pH on the enzymatic activity of the extracellular phytase obtained from P. carotov Effects of temperature on the enzymatic activity of the phytase that was obtained extracellu

4.4.b. Citrate test
The test is used to determine the ability of microorganisms to use citrate as the carbon source and ammonium salts as the nitrogen source and identify the genera and species of bacteria. The degradation of citrate (citrate metabolism) by bacteria is carried out by their enzyme systems.
After dilution with physiological water or buffer, the pure cultures to be treated were inoculated into the media and the tubes were incubated at 37 °C for 2-7 days. Inoculation with needle on the

4.4.c. Indole test
The test is used to examine the indole production of microorganisms by the degradation of tryptophan, an amino acid. After inoculation into a liquid medium or peptone-containing water, the microorganisms were incubated at 37 °C for 1-5 days. A total of 0.5 ml kovacs (or Ehrlich) was added to the cultures and thoroughly stirred. Formation of a red ring at the top of the tubes indicates a positive reaction (indole formation). Presence of a yellowish ring indicates that indole formation did not occur (negative indole test). The color is due to the pyrroles in indole [6].

4.4.d. Phosphatase test
The test is used to determine the presence of phosphatase especially the phosphatase synthesized by staphylococcus (coagulase positive). Microorganism cultures were inoculated into phenolphthalein phosphate agar and incubated at 37 °C for 1-2 days. At the end of this period, the colonies were exposed to ammonia vapor (1 ml NH 4 OH is put on the lid of the petri dish and culture is closed upside down). If a liquid medium is used, one drop of sodium hydroxide is dropped on the tubes. Red coloration in the colonies or tubes indicates a positive reaction. No change in color is observed if the reaction is negative [6].

4.4.e. Determination of Gram properties
The test was used to determine the differences between the cell walls of the isolates. Two different methods can be used for this purpose.
In the first method, 1-2 drops of 3% KOH solution is dropped on the microscope slide. Then, the bacteria collected with a loop is mixed with the KOH solution for 5-10 seconds and the loop is lifted up. If the microorganism is Gram (-), the peptidoglycan layer in the cell wall has fewer layers and does not contain teichoic acid and thus, is easily disintegrated by KOH and cytoplasm and cell content are released. This leads to viscous stretching. While in Gram (+) bacteria, KOH-bacteria mixture is an aqueous liquid and no stretching is observed when the loop is lifted up [19].
A drop of pure water is dropped on the microscope slide; then, a loopful of the bacteria that were grown on the medium for 24 hours is thoroughly streaked and fixed by fire. The preparation is first

Measurement of enzymatic activity
In the phytase activity measurements, activity measurement was performed for all supernatants obtained in each stage. The ranges in which phytase was active were determined. In the activity measurements, 250 µL substrate was added to100 µL enzyme solution and the mixture was vortexed. Then, the mixture was placed in the water bath at 50 °C for 10 minutes to allow reaction.
To stop the reaction, 500 µL 10% TCA was added. After a 15-minute waiting period at room temperature, 500 µL coloring solution was added to the tubes. After incubation for 15 minutes, the tubes were centrifuged at 3200 rpm for 10 minutes. Following centrifugation, the absorbance values of the samples at 700 nm were measured using spectrophotometry and a blind sample.

Funding
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable. Gel image after DNA isolation Figure 3 Gel image of the PCR results *Marked region indicates 1400 -1500 bp.

Figure 3
Gel image of the PCR results *Marked region indicates 1400 -1500 bp.

Figure 4
Sequence analysis results of the 16S rRNA gene region from the P. carotovorum isolate Figure 4 Sequence analysis results of the 16S rRNA gene region from the P. carotovorum isolate Figure 5 Determination of the location of P. carotovorum on the phylogenetic tree Determination of the location of P. carotovorum on the phylogenetic tree Effects of pH on the enzymatic activity of the extracellular phytase obtained from P. carotovorum Effects of pH on the enzymatic activity of the extracellular phytase obtained from P. carotovorum Effects of temperature on the enzymatic activity of the phytase that was obtained extracellularly Effects of temperature on the enzymatic activity of the phytase that was obtained extracellularly