Proteomic analysis and characterization of a novel glucansucrase from a newly isolated strain of Leuconostoc mesenteroides AA1
A Aman, S Ul Qader, S Bano, A Khan, A Azhar
characterization, dextransucrase, glucan, glucansucrase, leuconostoc mesenteroides, purification
A Aman, S Ul Qader, S Bano, A Khan, A Azhar. Proteomic analysis and characterization of a novel glucansucrase from a newly isolated strain of Leuconostoc mesenteroides AA1. The Internet Journal of Genomics and Proteomics. 2008 Volume 5 Number 1.
E.C. 22.214.171.124 (glucosyltransferase)
Glucansucrase is a glucosyltransferase (E.C. 126.96.36.199) that catalyzes the transfer of glucosyl residues from sucrose to glucan polymer and liberates fructose. Glucan is a high-molecular-mass polymer (10 7 to 10 8 Da) and composed of a linear chain of glucosyl residues all linked through α (1→6) glucosidic bond and several α (1→2), α (1→3), or α (1→4) branched linkages . The frequency and nature of the branch points mainly depend on the origin of the glucansucrase (i.e., the producing microorganism) . Glucansucrase can catalyze the synthesis of several types of linkages that leads to the formation of a branched polymer . The enzyme responsible for the production of glucan (dextran) from sucrose is a glucansucrase, which belong to the family 70 of the glucosidases and transglycosidases in the CAZy classification .
Glucansucrase have broad applications in the biotechnology industries. They have made a remarkable impact in the world of biotechnology because of their applications in the food, cosmetic, agricultural, photography and fermentation industries. The most promising application of glucansucrase and glucan is their use as protective colloid in blood plasma volume expander, flocculation, stabilization, lyopholization and cosmetic ingredient formulation . Other applications include its use as gel permeation matrices in research and various industries for the separation purposes of various products.
Several species of genera
To satisfy the industrial need of glucansucrase, it is imperative to explore new microbial strains. In this study a new strain of
Materials And Methods
DEAE Sephadex A50, Sepharose CL-6B, Sephacryl 300 HR and all other reagents of analytical grade were purchased from Sigma (Sigma Chemicals Co., St. Louis, MO, USA). Molecular weight markers were purchased from Promega (Promega Corporation, USA). Media used in this study were purchased from Oxoid Ltd., Hampshire, UK.
Isolation and screening of strain
Bacterial culture was isolated from
Culture media and growth conditions
For fermentation purpose, the organism was grown at 25C in a medium containing (g l –1 ): Sucrose, 25.0; Bacto-peptone, 5.0; yeast extract, 5.0; K2HPO4, 15.0; MnSO4.H2O, 0.01; NaCl, 0.01; MgSO4.7H2O, 0.01; CaCl2.2H2O, 0.1. The pH of the medium was adjusted to 7.5 before sterilization at 121ºC for 15 minutes.
Enzyme assay & protein quantification
Glucansucrase activity was determined by measuring the reducing sugar by Nelson Somogyi method as described earlier . Units of glucansucrase activity are represented in DSU . “One unit of enzyme activity was defined as the enzyme quantity that converts 1.0 milligram of sucrose into fructose and glucan in 1.0 hour using 0.1M citrate phosphate buffer of pH 5.00 at 35C”. Total protein content of the samples was estimated using the standard method described with bovine serum albumin as a standard .
Cultivation and crude enzyme preparation
Sterile sucrose broth medium was inoculated by a growing culture of
Isolation and purification of glucansucrase
PEG 4000 precipitations
The cell free supernatant was first subjected to partial purification by treating it with PEG 4000. A solution of 30 % (w/v) PEG 4000 was added to the crude enzyme to give 25 % saturation at 4C and stirred for 10 minutes. The suspension was left for 24 hour and centrifuged at 35,000 x
DEAE Sephadex A50 ion exchange chromatography
LKB gel filtration system was used for this purpose. The sample was applied through automatic sample applicator on XK16/70 glass column packed with DEAE Sephadex A50 column. The fractions were collected through automatic fraction collector Ultro Rac II (Model LKB 2070).
The dialyzed sample was applied to a DEAE Sephadex A50 column (1.6 x 40), pre-equilibrated with 0.1M citrate phosphate buffer (pH 5.0). After washing the column with 500 ml of equilibration buffer, bound proteins were eluted with the same buffer containing 0-4M guanidine-HCl. The flow rate was adjusted to 20ml/hr and each fraction of 0.5 ml was collected. The colleted fractions were assayed for enzyme activity. The fractions showing glucansucrase activity were pooled and concentrated using freeze-drying method.
Sepharose CL6B gel filtration chromatography
The concentrated fraction was then further applied on a Sepharose CL6B gel filtration column (1.6 x 49) earlier equilibrated with 0.1M citrate phosphate buffer (pH 5.0). The proteins were eluted at a flow rate of 20 ml/hr with the same buffer and fractions (1.0 ml each) were collected. Active fractions showing enzyme activity was pooled, dialyzed against the same buffer and then freeze dried. This freeze-dried gel filtration fraction was stored at 20C and was used as the purified enzyme preparation for the characterization studies.
Assessment of homogeneity
The homogeneity of the purified enzyme was analyzed by HPLC using C18 column (Teknokroma HPLC Column, Kromasil, 5 m 25 x 0.46). Protein was eluted isocratically using 0.1 M citrate phosphate buffer (pH 5.0) at a flow rate of 0.5ml/minute. The protein profile was monitored at 280nm using a UV detector (Perkin Elmer, USA). Purified dextransucrase from
Molecular mass determination by SDS PAGE and In-situ electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) of the enzyme was resolved on 7.5 % polyacrylamide gel according to the standard protocol . The proteins were stained with Coomassie brilliant blue R-250. The molecular mass of the purified glucansucrase was estimated from its position relative to those with the broad range protein molecular weight markers (Promega, Promega Corporation, USA).
Catalytic properties of glucansucrase
Initial rate of sucrose utilization was determined at various substrate concentrations. The kinetic constants Km and Vmax were estimated by the method of Lineweaver and Burk plot using sucrose as substrate and plotting the values of 1/v as a function of 1/S .
Effect of pH & temperature
The activity of purified glucansucrase was measured in a pH range of 3.0 to 7.0 at 35C for 15 minutes.
The glucansucrase assay mixture was incubated with substrate at different temperatures ranging from 15C to 45C for a period of 15 minutes followed by the standard assay procedure.
Amino acid analysis
Acid hydrolysis of the purified enzyme was carried out using 6N HCl. The sample was sealed in a Pyrex test tube under vacuum. The test tube was kept at 110C for 24 hours. After 24 hours the sample was washed thrice with deionized water, concentrated and filtered through 0.45 μm membrane filter. Derivatization of the sample carried out using o-phthalaldehyde (OPA). The amino acid analysis of the purified enzyme was carried out using an amino acid analysis system (Shimadzu LC – 10A/C – R7A, USA). For fluorescence detection and for calibrating amino acid analyzer amino acid standard solution was purchased from Sigma (Sigma Chemicals Co., St. Louis, MO, USA). Amino acids are in 0.1 N HCl at the indicated concentration of ± 4 %. Amino acid standard solution was stored at 4C.
Analysis of N-terminal protein sequence
Purified enzyme was resolved by SDS-PAGE and was electronically blotted onto the polyvinylidene difloride membrane (PVDF), using a semi-dry blotting device . Blots were stained with Coomassie brilliant blue R-250, protein band of interest was excised and amino terminal amino acid sequence was determined using an automated protein sequencer (ABI Procise 491 Protein Sequencer, USA).
The EMBL accession of the sequence reported in this paper is UniProtKB P85080.
Results And Discussion
In the present study isolation, purification and characterization of extracellular glucansucrase and glucan from a newly isolated strain of
Purification of glucansucrase
The purification of 162.5 folds was achieved using the following chromatographic procedures (Figure 1 & 2). The active fractions were pooled and freeze dried for further characterization analysis. Previously gel permeation chromatography on Bio-gel A5m and Sepharose 6B  and ion-exchange chromatography on a DEAE type gel have frequently been used for glucansucrase purification .
Assessment of homogeneity
The apparent purity of the enzyme was further demonstrated by HPLC for purity check analysis. HPLC profile obtained from the standard shows a single peak with a retention time of 4.24 minutes (Figure 3A). When glucansucrase purified sample was injected, the profile also showed a single peak with retention time of 4.24 minutes (Figure 3B) confirming that glucansucrase has been purified to homogeneity.
Catalytic properties of glucansucrase
Effect of substrate concentration on glucansucrase activity
Sucrose was used as a substrate for the determination of substrate saturation kinetics. A wide range of sucrose concentration was used to measure the initial rate of reaction. Km and Vmax values at 35ºC were calculated as 69.88 mM and 61.75 DSU/ml/hr, respectively. Substrate saturation kinetic showed that after reaching to the maxima, the substrate inhibitory effect started due to higher substrate concentration and no further increase in glucansucrase activity was detected. Dextransucrase from
Effect of temperature & pH on glucansucrase activity
Glucansucrase activity was determined at various temperatures ranging from 15C to 45C. The purified enzyme displays an optimal activity at 35C (Figure 4A). Glucansucrase from
The pH optimum, determined at optimal temperature (35C) was found at pH 5.0 with a rapid decline in the activity as pH moved to either extreme (Figure 4B). The results showed that the activity of the glucansucrase depends,
Molecular mass determination
SDS PAGE profile showed three protein bands that were detected in the partially purified sample (Figure 5, Lane B). While, after purification a single protein band of extracellular glucansucrase (Figure 5, Lane C) was detected confirming that the glucansucrase has been purified to homogeneity with a approximate molecular mass of 177,000 Da which is similar to the molecular mass of glucansucrase isolated from
Amino acid analysis
Amino acid analysis of purified glucansucrase was performed after acid hydrolysis. Standard amino acid solution (Sigma) was used for the detection purpose with reference to retention time. Amino acid analysis profile of standard solution is shown in Figure 6A, where as amino acid analysis profile of glucansucrase is presented in Figure 6B. Compositional analysis data provides a unique distribution profile of all amino acids in the protein (data not shown). Amino acid analysis of the purified extracellular glucansucrase shows that the enzyme is rich in both the basic (His, Lys, & Arg) and polar/hydrophilic amino acid and is less rich in acidic amino acids (Asp & Glu).
N- terminal sequence
The first six amino acid residues obtained from the sequencing of the N-terminus of the purified glucansucrase are: Asp-Ser-Thr-Asn-Thr-Val (D-S-T-N-T-V) (
A comparison of the N-terminus sequence of the glucansucrase AA1 showed no strong homology with the previously characterized glucansucrase from
A new strain taxonomically identified as
Acknowledgement is due to Dr. Nuzhat Ahmed, Centre for Molecular Genetics, University of Karachi Pakistan, for assistance during the N-terminal protein sequencing. Authors are also thankful to Mrs. Askari Begum, FMRRC, PCSIR Laboratories Complex Karachi Pakistan, for her assistance during amino acid analysis.