Production and Purification of Laccase Enzyme by Klebsiella pneumoniae K7

Sixty-four isolate were klebsiella pneumoniae. Fourteen bacteria isolates “ Kelbsiella species” were taken from soil and water hospital in the period between October to December 2018, those isolated were cultured on a blood agar to test their ability to hydrolytic due to formation the inhibition zone . Twenty one isolates of K. pneumoniae were selected to be cultured in mineral salt agar for testing their efficiency to produce laccase enzyme .The efficient isolate was diagnosed depending on phenotypic, microscopic and biochemical tests to be Klebsiella pneumoniae K7. Laccases (benzenediol: oxygen oxidoreductases; EC: 1.10.3.2) are subfamily of multicopper oxidases (MCOs) from Klebsiella pneumoniae K7 has been partially characterized by many researchers. In this paper, we purified laccase to homogeneity from Klebsiella pneumoniae K7 with about 10.82 ; 5.12 purification fold and a 34.14; 21.46% recovery by ion-exchange and gel-filtration chromatographic respectively. The molecular weight of the Laccase as determined by gel filtration chromatography using Sephacryl S-200 gel was 120 KDalton.


INTRODUCTION
Laccases (benzenediol: oxygen oxidoreductases ; EC: 1.10.3.2) are subfamily of multicopper oxidases (MCOs), which catalyze the one electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccase was first described from the sap of the Japanese lacquer tree Rhus vernicifera. Also laccases are wide spread in fungi, bacteria and insects (Rezaei et al., 2017) [1]. Laccases have a good potential for industrial and environmental applications (Viswanath et al., 2014) [2].
The utilization of bacterial laccases are growing rapidly and became outstanding biocatalysts that applied in different fields. This is because they have many features included: the bacterial systems are easier to handle than fungal ones , work in a broad range of temperature and pH with enormous stability against various inhibitory agents (Guan et al. , 2018) [3]. Liu et al. , (2017) [4] purified thermostable and pH-stable laccase from K.pneumoniae which isolated from soil, the laccase cloned and expressed in E. coli., rLac purified with the 6-His tag, the cells were harvested by centrifugation , the supernatant was collected by centrifugation and applied to a nickelnitrilotriacetic acid (Ni-NTA) agarose gel column. The purity and apparent molecular mass of the rLac were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Laccases are exist in a different forms: monomeric, dimeric and tetrameric glycoprotein with molecular mass ranges from 50 to 140 kDa, depending on the organism (Jaiswal et al. , 2015) [5] , The molecular weight of bacterial laccases are within the range 60 to 112 kDa (monomer was 32 to 44kDa). The bacterial laccases were found to be thermostable and alkaline stable (Sharma et al., 2007) [6]. Baldrian (2006) [7] suggested that the location of laccase depends on the substrate and its physiological function. Therefore, the aim of the study were to isolate and identify a local laccase producing strain of Klebsiella pneumoniae, and to purify and characterize the laccase produced by the bacterium.

MATERIALS AND METHODS Isolation and identification of bacteria
The Samples of soil and water were collected from different sites in Baghdad city: Tigris , Al Rustamiya Sewage Station, Dora Refinery, Gas station, Electricity generators, Gardens and Water pools at Baghdad University and Car repair places. The isolation of bacteria was based on serial dilution method technique. In this, one gram soil or 1ml of water was added to a tube containing 9ml of PBS solution and mixed vigorously for 30s. The suspension was serially diluted from 10 -1 to 10 -8 dilution factors and 0.1ml of each dilution was spread on the surface of MacConkey medium by spread plate method, then the plates were incubated at 37°C for (24-48) hrs and then examined for bacterial growth. After the growth the bacteria isolates, they subculture by streaking on MacConkey agar, incubated at 37°C for 24hrs and take colony for identifying it.

Screening of laccase producing bacteria
The screening of bacteria that have ability to produce laccase were done by using: Trace Element Solution -1 : 0.10g ZnSO4. 7H2O  These components were dissolved in 900ml of distilled water (D.W) , pH was adjusted to (7±0.2) , and the volume completed to 1000ml . The media then sterilized by autoclave at 121 o C and 15 psi for 15 min.

Methyl orange plate screen assay
This prepared in by adding 0.1g/l of Methyl Orange (after filtering it) in sterilized Mineral Salt Agar. Drop 5μL of overnight culture onto the plate (when this dries it will form a little circle), incubated at 37 o C for 24hrs. A clear zone of methyl orange hydrolysis around the drop was an indication of laccase secretion.

ABTS plate screen assay
The isolates showing methyl orange degradation were further screened for enzyme laccase, on this media which prepared by adding 0.2 mM ABTS and 0.1 mM CuSO4 (after filtering them) in Mineral Salt Agar. Blue green oxidation zone around the bacteria colony indicated the presence of laccase.

Laccase assay
Laccase activity was monitored by measuring the maximum absorption of oxidation of ABTS at 25ºC as a substrate. The reaction mixture containing 1ml of crude enzyme and 1ml of ABTS (0.2mM). The oxidation of ABTS was determined by measuring the absorbance at 420 nm using a spectrophotometer . The blank mixture containing 1ml of sodium acetate buffer (0.1mM, pH5) and 1ml of ABTS . Laccase activity was calculated as follows: Laccase Where: A: Absorbance at 420nm ; V: Total volume of reaction mixture in (ml); € : molar extinction coefficient of ABTS =36000M -1 cm -1 ; t:incubation time (1min) and v: volume of enzyme used in (ml) One unit of enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 1 μmol of substrate per minute. Protein concentration was assayed by the method of Bradford (1976) [8] and Stoscheck (1990) [9], with bovine serum albumin as standard.

Crude enzyme
The isolate , that have the highest enzyme activity, was incubated into fifty hundred milliliter of Luria bertani broth in sterile Erlenmeyer flasks at 37°C for 24hrs on a shaker incubator with 120 rpm. After 24hrs, the broth culture was filtrate by using cooling centrifuge at 15000rpm and 4°C for 5min to obtain supernatant, this supernatant used as crude laccase, which stored at 4˚C for further purification steps. The laccase activity in the supernatant was determined spectrophotometrically with the ABTS assay.

Ammonium Sulfate Precipitation
The ammonium sulfate was added in different saturation ratios (0-100%) into crude enzyme. To reach the optimum ratio of ammonium sulfate, the amount of salt was add gradually to crude enzyme solution in ice bath with gently magnetic stirrer , to avoid foaming, for 1hr. The resulting solution was kept stationary overnight and centrifuged at 10000 rpm for 20 min, dropped the supernatant and take the precipitate , dissolved it in 5ml Tric-HCl buffer (0.05M, pH=8.0), and calculated the activity and protein concentration. The supernatant was then subjected to ammonium sulfate precipitation. The precipitate obtained was dialyzed and then loaded onto a DEAE-Cellulose anion-exchange column.

Ion exchange chromatography
The exchanger DEAE-cellulose was prepared and packed into column following the method described by Whitaker, (1972) [10]. A column with a diameter of ( 7.5 x 2.5 cm) was equilibration with 0.05 mM Tris-HCl buffer pH 8.0. Partially purified concentrated laccase enzyme (12 ml) were separately passed after loaded onto the column carefully. Then (100 ml) of (0.05mM) Tris-HCl buffer pH (8.0) was added. Proteins were eluted by using (200 ml) of a stepwise salt concentration from (0.125-1.0 M) in 0.05mM Tris-HCl buffer (pH 8.0). Fractions of (5 ml) were collected and absorbency was monitored at (280 nm). The presence of the Laccase enzyme were estimated from each fraction of the major peaks then protein concentration and specific activities were determined for the collected active fractions. company. The column was equilibrated with (0.05 mM) Tris-HCl buffer (pH-8.0) at a flow rate of (50 ml/hour). A (5 ml) sample of each concentrated partially purified laccase enzyme was added to the column, carefully using pasture pipette. A (5 ml) fraction were collected for each laccase then protein contents were estimated by measuring the absorbance at (280 nm). Laccase activity was determined for each fraction of the major peaks. Protein concentrations and specific activities were also determined for the collected fractions of the major peaks of the Laccase enzyme.

Gel filtration chromatography
Determination of the molecular weight Molecular weight was determined by gel filtration chromatography by using Sepharose 6B, (Laue and Rhodes, 1990) [11] and the standard proteins used for the standard curve and their relevant molecular weights were (Catalase, 230000; Arginine deaminase , 125000; Alkaline phosphatase, 80000; Bovine serum albumin, 67000; Pepsin 34000).

RESULTS AND DISCUSSION Isolation and Identification of the Bacteria :
Fifty eight samples that collected of soil and water samples were used for isolated klebsiella. , several morphological, physiological and biochemical tests were made to identify the isolates. The results of bacterial subculture on MacConkey and biochemical test showed that 64 isolate were k. pneumoniae. (Table 1).

Screening of Klebsiella pneumoniae Isolates for Laccase Production :
Out of sixty four Klebsiella pneumoniae isolates , there were thirty isolates showed a clear zone around the colony on methyl orange plate, these isolate further screened for enzyme laccase on ABTs plate , it found that twenty one give a positive results . The activity of the enzyme of the twenty one isolates of K. pneumoniae , that have ability to produce laccase , was measured to select the highest isolation activity for choose the isolate that have the highest activity ( Table 2). The laccase enzyme extraction then the laccase activity was monitored by measuring the maximum absorption of oxidation of ABTS as a substrate. Crude Extraction: The laccase enzyme was extracted from the local isolation of K. pneumonia after incubated in Luria broth in the shaker incubator at 37° C for 24 hrs . The production medium contains the laccase enzyme that was excreted outside the cell bacterial to the medium of culture production. After centrifuge , the volume of crude enzyme was 300ml with enzyme activity 146 U/ml and specific enzyme 0.695 U/mg protein, as shown in Table (3).

Ammonium Sulfate Precipitation:
The crude enzyme extract was fractionated with different percentage of ammonium sulphate (0-100%) . The results of specific activity of supernatant were showed in Table (3) , the higher specific activity was (1.21U/mg) at (60-80%) than other saturation of ammonium sulphate. Table (

Wash Eluation
Chemistry and Materials Research www.iiste.org ISSN 2224-3224 (Print) ISSN 2225-0956 (Online) Vol. 10 No.5, 2020 Gel filtration Chromatography: Purification carried out by a gel filtration using Sephacryl S-200. Enzymes fraction from DEAE cellulose were pooled and passed through gel filtration column. The fractionation yielded two protein peaks as absorbance reading at 280nm (wave length), only one peak where appeared when reading absorbance at wavelength of 280 nm and when determined for enzyme activity in resulting parts enzyme activity recorded in 16-20, the specific activity reached 7.52U/mg ,fold of 10.82 and a yield 21.46% as mentioned in table (3) and figure (2). Laccase from T. versicolour is purified with Ion Exchange chromatography followed by gel filtration with specific activity of 101UmL−1 and 34.8 fold purification. (Cordi et al,, 2007) [12].  The molecular weight was estimated by gel filtration depending on the size of the separated molecules with their charge .It was possible that the different methods of estimation may be used.(Segal,1992) [13]. Sephacryl S-200 for gel filtration was used for estimation the molecular weight of Laccase enzyme that purified from Klebsiella pneumoniae K4 illustrated in figure (3). The logarithm molecular weight standard protein versus elution volume / void volume ratio (Ve/ V0) was the basis to estimate the molecular weight. By using this relationship, the molecular weight of the enzyme was estimated with 120000 Dalton.