Improving the Chitinolytic Activity of Bacillus pumilus SG2 by Random Mutagenesis

Abstract

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2-9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

Introduction

Chitinase (E.C.3.2.1.14) is a glycoside hydrolase that catalyzes the degradation of chitin, the linear polymer of β-1,4-linked N-acetyl glucosamine (GlcNAc). Chitin is the second most abundant biopolymer in nature next to cellulose [14,18]. Chitinases have been discovered in various organisms, including bacteria, fungi, higher plants, insects, crustaceans, and some vertebrates. Bacteria, in particular, produce various chitinases to digest chitin and utilize it as an energy, carbon, and nitrogen source [11]. Chitinases confer a variety of potential advantages to producer organisms, including morphogenesis, defense, pathogenesis, and substrate utilization capabilities. They have also found otential applications in agriculture and different industries. A great deal of interest has been generated on chitinase because of its applications in the biocontrol of plant pathogenic fungi [8,20]. Mutation induction and/or selection techniques, along with cloning and protein engineering strategies, have been used extensively to develop enzymes with higher activity [16,20,25]. Physical mutagenesis using different irradiation methodologies has been adopted as one of many strategies to mutate bacteria. UV irradiation as a physical mutagenic is one of the well-known and most commonly used mutagens, which is generally used to induce genetically improved strains. This method has been used in Bacillus to overproduce the enzyme protease [19]. Mutagenesis of the Bacillus strains has been carried out using a modificaton of the method described by Courcelle et al. [13].

Chitinases are of interest in the development of efficient biocontrol agents that inhibit the growth of various phytopathogenic fungi [10,12,24,27]. The utilization of these useful enzymes for the protectin of crops and livestock, instead of chemical agents, reduces the harmful side-effects on the environment. The production of chitinase enzymes by various bacteria, including Bacillus sp., and archaea has been well documented [6,15,21]. However, less attention has so far been paid to the isolation and characterization of halotolerant Bacillus strains with chitinase activity, at high salt concentrations [16]. To date, no effort has been made to track the detailed genetic characteristics of chitinase produced by the mutant strains. This study sought to apply this technology to obtain mutant strains with improved chitinase activity. An earlier study demonstrated the production of two chitinase enzymes by a Bacillus pumilus strain isolated from saline environments [8]. The aim of this study was to provide an economical methodology to enhance the chitinolytic activity of this native strain by using a combination of ultraviolet rays and nitrous acidinduced mutation. This investigation attempted to establish a cost-effective and simple approach, in order to concomitantly increase the rate of chitinase activity and improve product characteristics in a proficient manner. This work also presents evidence of a correlation between the modulation of chitinase ChiL activity and change at the genetic level in the corresponding gene of the mutant strain. This may shed some light onto the structure-function relationship of the enzyme. The change in the structure of the mutant enzyme due to this mutation and comparison with that of the wildtype enzyme are discussed.

 

Materials and Methods

Bacteria and Culture Media

Bacillus pumilus strain SG2 (1) was used throughout this research project. This strain, previously isolated from salty soils of Gavkhooni marsh located in central Iran, is a halotolerant bacterium capable of producing chitinase [1]. Bacterial cultures were grown in LB broth and in Bacillus subtilis minimal medium (BSS), but with some modifications for chitinase production, whereby chitin was used as the sole carbon source. BSS was prepared by mixing 5 g of colloidal chitin containing the following salts (g/l): KH2PO4, 14, K2HPO4, 6, (NH4)2SO4, 2, Na3C6H5O7, 1, and MgSO4, 0.12. The pH of the medium was adjusted to 7.0 before sterilization at 121℃ for 20 min. Trace elements, MgCl2, ZnSO4, FeCl3, and CaCl2 were added to give a final concentration of 10-6 mol/l. Casamino acids solution was filter-sterilized and added to a final concentration of 0.1% )undefined(w/v). The pH of the medium was again adjusted to 7.0. For BSS agar plates, BSS medium was made as mentioned above, but with the addition of agar at a final concentration of 1.5 % (w/v).

Colloidal Chitin Preparation

One gram of purified chitin (Sigma-Aldrich, USA) was dissolved in 12 ml of concentrated HCl while stirring overnight at 4℃. A 400ml sample of pure cold ethanol was then added to the mixture, which was left to stir overnight at room temperature. The chitin solution was centrifuged at 6,000 ×g for 20 min, and washed three times with distilled water. The pH of the solution was then adjusted to 7.0, resulting in a 5% (w/v) colloidal chitin preparation.

Mutagenesis by Treatment of Cells with Ultraviolet Rays and Nitrous Acid

A single colony of the wild-type strain B. pumilus SG2 was inoculated into 3 ml of LB medium [27], and incubated overnight at 37℃ with shaking at 180 rpm. A 0.5 ml sample of this culture was diluted into 50 ml of fresh LB medium and incubated at 37℃ until an optical density (OD600) of 0.5 was reached after approximately 6 h of incubation. When the culture reached the mid-log phase, serial dilutions of the broth were carried out to adjust the cell density to 4.4 × 108/ml. Subsequently, mutagenesis with short wavelength ultraviolet (UV) irradiation (240 nm) was carried out using a Philips tube (15 W). The cell suspension was distributed in 3 cm petri plates (2-3 ml in each plate) on a magnetic stirrer and exposed to UV radiation for variable time periods (1.5, 10, 20, or 40 sec) and (1, 2, 5, or 10 min). The appropriate UV dose was determined as that dose in which the viable cell population was 0.01% (4.4 × 106/ml), by keeping the distance of the UV source fixed at 40 cm. After exposure, the sample was incubated for 45 min in the dark.

The UV-irradiated cells were used for the next mutation, stage which involved treatment with nitrous acid. Different concentrations of fresh nitrous acid solution consisting of 0.2 to 0.2 M of NaNO2 in acetate buffer (0.2M,pH4.5) were added to the washed and centrifuged cells so that a final cell density of approximately 1.1 × 105 cells/ml was obtained. The solution was thoroughly shaken and then incubated at 37℃ with shaking at 180 rpm for varying periods of time (8, 11, 15, 25, and 40 min). A 0.2 ml sample of NaOH (0.2 M) was used to stop the reaction. The cultures were then spread onto LB agar plates and incubated overnight to determine the survival and evaluate the chitinase activity. Each colony was then carefully evaluated for its ability to produce chitinase using the enzyme assay method, with the wild-type strain being used as the control. Finally, a mutant with the highest chitinase activity was selected and compared with the wild type.

Screening of Mutants with Increased Chitinolytic Activity

A plate assay for primary screening of mutants was employed by culturing the survivors onto BSS agar plates, so as to screen a large number of mutants for higher chitinase activity. Plates were incubated at 37℃ and examined after 48 h for the formation of clear halos around the colonies, which were the result of chitin degradation.

Native Enzyme Production and Purification

A single colony of the wild-type and mutant B. pumilus strain was inoculated into 5 ml of LB medium and grown at 37℃ on a rotary shaker overnight. The resulting overnight culture was then transferred into a 200 ml flask containing 500 ml of BSS minimal medium supplemented with 5 g of colloidal chitin as the sole carbon source, and incubated for 18 h at 37℃. The culture broth was centrifuged for 15 min at 8,000 ×g (4℃) and the supernatant was used for enzyme purification.

Ammonium sulfate was added to the supernatant at 80% (w/v) saturation. The solution was kept at 4℃ for 2 h. After centrifugation for 20 min at 9,000 ×g, the protein pellet was dissolved in 25 mM phosphate buffer so that the final enzyme concentration was 10 times higher than normal. The protein solution was then dialyzed against 25 mM phosphate buffer (pH 7) (molecular cutoff was 12 kDa) to obtain crude chitinase. In general, cell disruption leads to the release of proteolytic enzymes, which could lower the overall yield. To control this undesirable proteolysis, it may be necessary to add a cocktail of protease inhibitors to the cell suspension. To this end, a stock solution of 100 mM PMSF in isopropanol was prepared and diluted into the buffer immediately before use.

Identification of Mutation in the Chitinase Operon of Wild-Type and Mutant Strains

To compare chitinase genes from both wild-type and mutant strains, different primers were used in the PCR and DNA sequencing to find the possible mutation in the chitinase operon of the AV2-9 strain. The entire chitinase operon of the wild-type B. pumilus SG2 strain had previously been identified and its complete nucleotide and amino acid sequences were deposited in GenBank (Accession No. DQ859055.1). The complete DNA sequence of the ChiSL operon of 5,792 bp in length, spanning two open reading frames (Supplementary Fig. S1A), was determined. The complete nucleotide sequences and the deduced amino acid sequences of the mutant chitinase, chiL, were also deposited in GenBank (Accession No. DQ490987.1). Alignment of the sequences of the chitinase operons from the wild-type and mutant strains was carried out using programs available at the NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) database.

Table 1.Primers, plasmids, and strains used in this study.

DNA Manipulation

The wild-type and mutant ChiL were cloned into the pQE-30 expression vector (Qiagen, Germany). Forward and reverse primers (Table 1) were designed according to the coding region of the genes without the signal sequence, so as to amplify the nucleotide sequence, using PCR with the purified wild-type and mutant genomic DNAs as templates. In this way, the signal sequences of the genes were removed and the chitinase genes could be expressed in the cytoplasm of the Escherichia coli M15 strain. The PCR products of the chil gene were digested with BamHI and SalI and ligated into the pQE-30 vector previously digested with the same enzymes. The nucleotide sequences of the plasmid constructs were determined (MWG Biotech, Germany) to confirm the cloning. All recombinant proteins were expressed as an N-terminal fusion protein carrying the 6×His tag, in the E. coli M-15 host strain.

Expression of Recombinant Wild-Type and Mutant Chitinases

E. coli M-15 transformants containing different plasmid constructs were grown overnight in 5 ml of LB supplemented with 100 μg/ml of ampicillin (Sigma-Aldrich). The bacteria were grown in a shaker incubator (37℃, 180 rpm) for 4 h until an OD600 of 0.6 was reached. Isopropyl-β-D-galactopyranoside (IPTG) (1mM) (Sigma, USA) was then added to the medium. Thereafter, the cultures were centrifuged and the pellets resuspended in 10 ml of lysis buffer that contained 50 mM NaH2PO4 (pH 8), 250 mM NaCl, 10 mM imidazole, and 0.05% (w/v) Tween 20. The cells were disrupted by sonication and the induced proteins were analyzed by 10% (w/v) SDS-PAGE. In all of the experiments, the E. coli M15 cells harboring plasmid pQE-30 without the insert were grown in the same conditions (as described above), and used as a control.

Purification of Recombinant Chitinases

The cell-free supernatants from the E. coli M15 cultures harboring plasmid pQE-30 expressing chiL, and its derivatives, were applied to a Ni-nitrilotriacetic (NTA) acid (10 ml column volume, Ni-NTA; Qiagen, Germany) column equilibrated with 10 mM imidazole lysis buffer. The column was then washed twice with approximately 12 ml of 20 mM imidazole lysis buffer. Chitinases were eluted with 500 ml of 250 mM imidazole lysis buffer. The final elution containing the isolated protein was dialyzed against 25 mM sodium phosphate buffer (pH 7) at 4℃ and stored at -70℃ until further analysis.

Chitinase Assay

Chitinase activity was measured with colloidal chitin as a substrate. The reaction mixture containing 0.9 ml of 1% (w/v) colloidal chitin and 0.1 ml of the crude enzyme was incubated at 55℃ for 1 h. The reaction was stopped by the addition of 3 ml of DNS followed by heating at 100℃ for 5min. Following centrifugation, the concentration of the reducing sugar in the supernatant was determined using the modified DNS method [6]. The absorption of the appropriately diluted test sample was measured at 530 nm using a UV spectrophotometer (Beckman DU530, USA) along with substrate and enzyme blanks.

Antifungal Activity Assay

A culture assay was performed to assess and compare the potential biocontrol and antifungal activities of the wild-type and mutant of B. pumilus SG2 against those of Fusarium graminearum, Bipolaris sp., Alternaria raphani, Alternaria brassicola, and Sclerotinia sclerotiorum. The growth of the five fungi was examined daily for the formation of an inhibition zone. Antifungal activity of wildtype and mutant forms of chitinase (mixed ChiS and ChiL) was assessed by the hyphal extension inhibition assay [22]. Briefly, a disc of each fungal strain was placed at the center of a fresh PDA plate. Sterile paper discs (5 mm diameter) containing 4 U of both purified wild-type and mutant chitinases and one control disc (phosphate buffer) were placed 25 mm from the perimeter of the fungal culture. The plates were then kept at a temperature of 28℃. When growth of fungi mycelium reached 10 mm, 5 mm sterile paper discs containing 4 U of purified chitinases and a control disc were placed 25 mm from the perimeter of the fungal culture. Owing to the reduced enzymatic activity that was maintained over time, 30 μl of the enzyme was added to the disc (total of 100 μl of enzyme was added to each paper disc) once every 24 h. Plates showing a zone of growth inhibition were observed for a few days for evidence of breakthrough growth. Inhibition of hyphal extension was detected as a crescent-shaped clear zone around the peripheral discs, as the fungus grew out from the central discs. The zones of inhibition were determined by measuring the distance between the margin of the hyphal growth and the border of the paper disc. Antifungal activity was classified as absence of inhibition demonstrated by the negative control (phosphate buffer), medium inhibition, and strong inhibition. The experiment was repeated three times. The experiments were continued until the inhibitory effects of the enzymes were abolished.

Effects of Temperature, pH, and NaCl Concentration on Chitinase Activity of Wild-Type and Mutant Cells

The activity of the enzyme at different temperatures was determined by incubating the reaction mixture at temperatures ranging from 30℃ to 70℃. The optimum pH of the enzyme was determined by incubating the assay reaction mixture containing recombinant chitinases using the following buffers (all at a concentration of 0.1 M): sodium acetate (pH values 4 and 5), sodium phosphate (pH values 6 and 7), Tris-HCl (pH values 8 and 9), and glycine- NaOH (pH 10). The optimal pH for enzyme activity was determined by incubating the purified enzyme at different pH values (3-9) and measuring the activity under standard assay conditions using colloidal chitin as the substrate. To determine the salt-tolerance property of the enzyme, different concentrations of NaCl (0.1-3 M) were used for this purpose.

Effect of Temperature on Enzyme Stability

Thermal stability was investigated by incubating the wildtype (ChiL) and mutant (ChiLm) enzymes at temperatures of 55℃ and 70℃ for 1 and 6 h at optimum pH, after which those from the higher temperatures were cooled rapidly by immersing in ice before the residual activities of each were measured at 55℃.

Appropriate buffers were used to maintain the optimum pH of the enzyme-substrate reaction mixtures, which were incubated at different temperatures within the stability range of the enzymes, for 1 and 6 h. Afterwards, the enzyme activity of each reaction mixture was measured, employing appropriate substrate controls.

SDS-PAGE Analysis of Chitinases

The crude enzymes derived from the SG2 and AV2-9 strains were precipitated with 10% (v/v) TCA at 4℃. The mixture was incubated for 1.5 h at 4℃ and centrifuged at 13,000 ×g for 15 min at 4℃. Then, the supernatant was removed and 2 ml of absolute acetone was added. The resulting samples were centrifuged twice at 13,000 ×g for 10 min at 4℃. The samples were resolved by a 12% gel and stained with Coomassie blue [26].

Bioinformatics Analysis

The ChiL sequence from the wild-type B. pumilus strain was used in the BLASTP program to find homologous proteins. Multiple alignments were then carried out using the ClustalW program. Three-dimensional structures of wild-type and mutated proteins were built by homology modeling using the SWISS-MODEL software [3]. Prediction of the effect of this mutation on protein stability using the SDM [30] and PoPMuSiC [9] servers was also investigated.

 

Results and Discussion

Effects of UV Irradiation and Nitrous Acid on Chitinolytic Activity of B. pumilus

The chitinolytic Bacillus pumilus SG2 was subjected to random mutagenesis by a combination UV irradiation and nitrous acid. A mutant of B. pumilus, tentatively named AV2-9, with higher chitinolytic activity was isolated after extensive screening.

A mutant colony with the highest chitinase activity relative to that of the wild type was selected for further studies. In other words, the above-mentioned strain consumed chitin at a higher rate, and produced a clear zone with larger diameters (Fig. 1). Equal numbers of wild-type and mutant strains were used in the primary screening of the mutants by a halo formation test using chitin-agar (Fig. 1). Subsequent enzyme assays of the extracts showed maximum chitinase activity in a strain designated AV2-9. Among 105 colonies that were screened, only a few mutants with increased chitinolytic activity were found and a mutant with highest activity was selected. Surprisingly, all other mutants showed either a lower or decreased chitinase activity, when compared with the wild type; the strain SG3, as demonstrated in Fig. 1, is an example of these mutants. There have been several reports of different mutagenesis methods [23,29] and their effectiveness for improving the efficacy of microbial enzymes. We first used Gamma and He-Ne irradiation to develop mutants with higher chitinase activity. Surprisingly, after screening a large number of colonies, no mutants with higher chitinolytic activity were detected; in fact, radiation resulted in mutants with lower or no chitinolytic activity (data not shown). The reason for this is not clear, but it could be due to the unusual length of the chitinase operon, which is approximately 5 kb long. Therefore, other more efficient methods of mutagenesis, which included UV irradiation and nitrous acid random mutagenesis, were subsequently considered. Consequently, this investigation has provided a different and simple approach regarding the improvement of enzyme yield and characteristics. Aside from overall efficacy, microbial modifications using various types of physical mutagenesis methods need the broader attention of investigators in the field. The results of this study demonstrate the clear advantage in using a combinatorial method of random mutagenesis that improves enzyme activity. During the two stages of mutagenesis, lethal doses and the frequency of selected mutants were estimated, as presented in Fig. 2A. As shown, the best UV dose (time of irradiation), calculated as the ratio of surviving cells to the total number of cells at the beginning of treatment, was observed after 600 sec of exposure to UV light. This was the point at which 99.75% of the initial cells were killed. Fresh nitrous acid solution at a concentration of 0.2 M (pH4.5) was used for a cell density of approximately 1.1 × 105 cells/ml. After a 15 min incubation, there were less than 5% of survivors (Fig. 2B).

Fig. 1.AV2-9 produced a clear zone with larger diameters, as compared with wild type and other mutant cells. 1: SG3; 2: SG2 (wild type); 3: AV2-9.

Fig. 2.Survival and mutation frequency curves of bacterial cells following UV irradiation at 235 nm.

Identification of Mutation in the Chitinase Operon of Wild-Type and Mutant Strains

To find the possible changes that occurred by random mutagenesis, the genome of the mutant B. pumilus AV2-9 was extracted and the sequencing of the entire chitinase operon (including the promoter, the coding region of the two chitinases ChiS) and ChiL, and the intergenic region) was performed to find possible variations in the sequence. The whole chiSL operon was then amplified using relevant primers (listed in Table 1) to cover the complete operon. Sequences were then compared with that of the wild-type chiSL operon previously sequenced in our laboratory. Alignment of the sequences from the wild-type and mutant strains showed a point mutation at position 432 (GGA changed to GAA). This changed a Gly residue to Glu. This residue is located in the catalytic region of second chitinases (chiL) in the operon. No other mutation was found in the whole operon.

The wild-type and mutant chitinases (ChiL and ChiLm) were cloned, expressed, and purified in E. coli. Both chitinases were able to produce active chitinolytic enzymes, but the mutant cells showed a higher enzyme activity of approximately 30% (Fig. 3). To investigate whether the increase in chitinolytic activity was due to changes in the level of secreted chitinases, the supernatant chitinases from both strains were analyzed by SDS-PAGE, NanoDrop spectrophotometry, and protein assay using the Bradford method [4] (data shown in Supplementary Fig. S1B). The results of this study did not show any changes at the level of protein expression and secretion. Meanwhile, this result was confirmed by sequencing the promoter region, which did not show any mutation in this region.

Fig. 3.Chitinase assay for wild-type ChiL and mutant ChiLm. ChiLm showed an increase in activity (up to 30%) when compared with the wild-type strain (SG2).

Antifungal Effect

The present study focused on enhancing the chitinase activity of B. pumilus SG2 by random mutagenesis in order to develop a potential biocontrol agent against certain important phytopathogens. The nitrous acid- and UV-derived mutants of B. pumilus SG2 were assessed for their ability as a biopesticide. The antifungal activity of the recombinant wild-type and mutant forms of chitinase (ChiL and ChiLm) against different fungi, including F. graminearum, Biopolaris sp., A. brassica, S. sclerotiorum, and Aspergillus parasiticus ATCC 15517, revealed the ability of the mutant form of the enzyme to inhibit the growth of fungal hyphae more efficiently than the wild-type enzyme (Fig. 4). Substitution of glutamic acid in the mutant resulted in inhibition of the hyphal growth of F. graminearum, Biopolaris sp, and S. sclerotiorum, more more effectively than the wild-type enzyme. These results were supported by the change in the minimum inhibition concentration (MIC) of the mutant protein enzyme (0.005 mg/ml), when compared with that for the wild-type enzyme (0.01 mg/ml). The MIC of the wild-type enzyme against A. parasiticus ATCC 15517 was two times lower than those of its respective mutant.

Fig. 4.Antifungal activity of purified recombinant chitinases. Disc number 1 is SG3 (0.01 mg/ml), disc number 2 is SG2 (0.005 mg/ml), and disc number 3 is AV2-9 (0.005 mg/ml). (A) F. graminearum; (B) Biopolaris. sp; (C) A. raphnmi; and (D) A. parasiticus.

Effects of Temperature, pH, and NaCl Concentration on Activity of Recombinant Chitinases from the Wild-Type and Mutant Cells

Enzyme activity was measured at temperatures ranging from 30℃ to 70℃ (Fig. 5). Although the chitinase was active at temperatures from 30℃ to 60℃, the optimum temperature for enzyme activity was 55℃, for both wildtype and mutant cells. In comparison to the activity of the wild-type strain, the chitinase enzyme of the mutant cells, AV2-9, was about 25% more active at 55℃ (Fig. 5).

Although the enzyme was active at a pH range of 4-10, it displayed the highest activity at pH 7, under both conditions. In fact, enzyme activity was found to increase by up to 27% at pH 7 in the case of the mutant cells (Fig. 6).

Salts and ions may have many different effects on the activity of an enzyme and this could alter the rate of enzyme activity. Fig. 7 shows the profile of chitinase activity in the presence of different salt concentrations (1-3 M NaCl). Results showed that the chitinase enzyme from both the wild-type and the mutant cells exhibited highest activity in the presence of 0.5 M NaCl. However, the mutant ChiLm enzyme activity was enhanced by 26% in 0.5 M NaCl, when compared with that of the wild-type SG2 strain.

Fig. 5.Effect of different temperatures on chitinase activity of both wild-type and mutant strains. The optimal temperature for enzyme activity was determined by incubating the purified recombinant chitinases at pH 7 and the enzyme sample was incubated with 1% (w/v) chitin in 0.1 M sodium phosphate buffer (pH 7) at 30℃, 40℃, 50℃, 55℃, 60℃, and 70℃ for 60 min, and then measuring the activity under standard assay conditions using colloidal chitin as the substrate.

Fig. 6.Effect of different pH values on chitinase activity of both wild-type and mutant strains. The optimal pH for enzyme activity was determined by incubating the purified recombinant chitinases at different pH levels (3-9) and then measuring the activity under standard assay conditions using colloidal chitin as the substrate.

Fig. 7.Influence of different NaCl concentrations on chitinase enzyme activity. The effect of salt concentration on enzyme activity was determined by incubating the purified recombinant wild-type and mutant chitinases at pH 7, optimum temperature, and different concentrations of NaCl (0.1-3 M), and then measuring the chitinase activity under standard assay conditions using colloidal chitin as the substrate. The enzymes of both wild-type and mutant cells exhibited greatest activities in the presence of 0.5 M NaCl.

The ability to maintain the mutational changes in the genome of the B. pumilus AV2-9 strain with regard to chitinolytic enzyme production was determined by successive subculturing on chitin-containing agar plates over a 6- month period. After each subculture, the mutant was examined for its ability to produce chitinolytic enzyme under aerobic conditions. Bacillus pumilus AV2-9 was found to consistently produce the enzyme at approximately 30% higher chitinolytic activity. The information obtained in this research can be used in bacterial strain development for industrial purposes. Additionally, precise examination of the products synthesized by irradiated cells, using highly developed analytical techniques, can lead to a better understanding of the molecular basis of the structurefunction relationship of mutant enzymes.

Effect of Temperature on Enzyme Stability

Observations noted in Fig. 8 indicate that the temperature stability of the mutant enzyme was more than that of the wild type at the temperature of 70℃. However, both the mutant and wild-type strains exhibited optimal working temperatures at 55℃, but the mutant strain had almost equal enzymatic activity at temperatures between 55℃ and 70℃ over a 6-h period. As mentioned, the stability of the ChiLm mutant for higher chitinolytic activity was confirmed by sequential subculturing of the strain, and extracting the relevant wild-type and mutant chitinases, over a 6-month period. Our data on the thermal stability of the enzymes by this mutation was further confirmed using bioinformatics analysis, as discussed below.

Bioinformatics Analysis

The B. pumilus wild-type ChiL sequence was run on BLASTP to find homologous proteins, which were then aligned using the ClustalW multiple sequence alignment programs. As shown in Fig. 9, surprisingly, at position 432 of the wild-type sequence, all members of the ChiL family were shown to have Glu, and only in the B. pumilus ChiL sequence was Gly observed to be located at this position. It seems that Glu is conserved in this region of most chitinases and in the wild-type ChiL, it has been substituted by Gly, and in the mutant ChiLm, this residue was mutated to Glu. Accordingly, the stability of the mutant enzyme was enhanced owing to the presence of the conserved Glu residue.

Three-dimensional structures of the wild-type and mutated proteins were built using SWISS-MODEL, the homology modeling server. Fig. 10 shows the superimposed structures of the mutant (part A) and the wild-type (part B) enzymes. As shown in the figure, substitution of Gly by Glu has affected the local structure of the mutated enzyme. Prediction of the effect of this mutation on protein stability using the SDM and PoPMuSiC servers also showed that the G432E mutation stabilizes protein structure. Stabilization of charged transition states can be created by residues in the catalytic domain forming ionic bonds. These bonds can come from acidic side chains found on glutamic acid. It is known that glutamic acid is involved in stabilizing the internal isopeptide bonds of proteins and has a key role in maintaining the correct protein fold, as found using site-specific mutagenesis and molecular dynamics simulations studies. Mutation of this residue can strictly affect the protein folding [5,7]. Meanwhile, our bioinformatics analysis showed that, by this mutation, an alpha-helix secondary structure is changed to an antiparallel beta-sheet arrangement. It is known that this arrangement produces the strongest stability as it allows the formation of planar interstrand hydrogen bonds between carbonyl and amine groups. The hydrogen bonds of alpha-helices are considered slightly weaker than their counterpart in beta-sheets, because they are attacked by the water molecules around them [2,18,28]. The presence of a Glu residue in this region of the evaluated chitinases stabilizes the structure of this enzyme by forming a betasheet, and the presence of other amino acids like glycine promotes formation of the alpha-helix, which makes it less stable. It looks like that the presence of Glu in this position in most strains is a natural selection for the stability of this enzyme in other strains, and our mutant enzyme returned to the naturally selected version by this point mutation. In addition to the main object of this research regarding developing a mutant strain with improved chitinase activity, we obtained another interesting result about the structurefunction relationship of the chitinase. Our experiments also showed an advantage of random mutagenesis, as our knowledge is limited and most of our views and ideas can come from nature.

Fig. 8.Effect of different temperatures on chitinase stability in the wild-type and mutant enzymes. Thermal stability was carried out by incubating the wild-type and mutant chitinases at temperatures of 55℃ and 70℃ for 1 and 6 h at optimum pH. Afterwards, the enzyme activity of each reaction mixture was measured, employing appropriate substrate controls. The mutant enzyme showed more stability than the wild type.

Fig. 9.BLASTP and ClustalW multiple sequence alignment of the homologous region of the wild-type ChiL and other Bacillus-originated chitinases.

Fig. 10.Superimposed structures of the mutant (part A) and the wild-type (part B) chitinases. Substitution of Gly by Glu has changed an alpha-helix to a beta-sheet structure in the mutant enzyme.