|Year : 2019 | Volume
| Issue : 4 | Page : 341-355
Genetic identification and optimization of novel β-glucosidase-producing Lysinibacillus sphaericus QS6 strain isolated from the Egyptian environment
Ahmed F El-Sayed1, Nivien A Abo-Sereih1, Abeer E Mahmoud2, Tahany M El-Kawokgy1, Abbas A El-Ghamery3
1 Department of Microbial Genetic, Genetic Engineering and Biotechnology Division, National Research Center, Giza, Egypt
2 Department of Biochemistry, Genetic Engineering and Biotechnology Division, National Research Center, Giza, Egypt
3 Department of Botany and Microbiology, Faculty of Sciences, Al-Azhar University, Nasr city, Egypt
|Date of Submission||30-Sep-2019|
|Date of Acceptance||21-Oct-2019|
|Date of Web Publication||28-Jan-2020|
Ahmed F El-Sayed
Master’s Degree in Microbial Genetics, Assistant Researcher at Department of Microbial Genetics, Genetic Engineering and Biotechnology Division, National Research Center, 12311 Dokki, Giza
Source of Support: None, Conflict of Interest: None
Background and objective β-Glucosidase-producing bacteria are potential sources for biotransformation of lignocellulose biomass and agricultural wastes into biofuels. The aim was the isolation, screening, molecular identification, and optimization of highly efficient β-glucosidase-producing bacteria under different growth conditions.
Materials and methods Cellulose-degrading bacteria were isolated and screened for β-glucosidase enzymes. Then, they were identified by phenotypic and genotypic identification. Optimization for β-glucosidase production was studied under different culture conditions.
Results and conclusion Highly efficient β-glucosidase-producing strain QS6 was selected and identified morphologically and biochemically as Lysinibacillus sp. using 16 s rDNA gene sequencing approach and bioinformatics analysis. Strain QS6 was most similar to Lysinibacillus sphaericus, with similarity of 98%. Phylogenetic analysis was done to determine the relationship of strain QS6 with different strains of genus Lysinibacillus sp. It indicated that the suitable culture conditions of producing β-glucosidase were the culture temperature of 35°C, the initial pH of 7.0, the incubation time of 24 h, and 1% inoculum size. While studying the effect of carbon sources on β-glucosidase production, it was found that cellobiose (1%w/v) was the best carbon source for inducing β-glucosidase production. Moreover, the nitrogen source peptone at 0.5% w/v was optimum for β-glucosidase production by this bacterium. L. sphaericus QS6 was found to be sensitive to antibiotics (amoxicillin, streptomycin, tetracycline, cefadroxil, kanamycin, chloramphenicol, ampicillin, erythromycin, and tobramycin). Moreover, in-vitro antibacterial bioassay of the most potent β-glucosidase-producing strain (QS6) showed high antimicrobial activity against Escherichia coli (1.9 cm) and Pseudomonas aeruginosa 1.0 cm). A promising Lysinibacillus sp. completely identified as L. sphaericus QS6 (GenBank MN493725.1) is an efficient source of β-glucosidase production.
Keywords: 16S rDNA gene, celluloses, Lysinibacillus sphaericus, optimization, β-glucosidase
|How to cite this article:|
El-Sayed AF, Abo-Sereih NA, Mahmoud AE, El-Kawokgy TM, El-Ghamery AA. Genetic identification and optimization of novel β-glucosidase-producing Lysinibacillus sphaericus QS6 strain isolated from the Egyptian environment. Egypt Pharmaceut J 2019;18:341-55
|How to cite this URL:|
El-Sayed AF, Abo-Sereih NA, Mahmoud AE, El-Kawokgy TM, El-Ghamery AA. Genetic identification and optimization of novel β-glucosidase-producing Lysinibacillus sphaericus QS6 strain isolated from the Egyptian environment. Egypt Pharmaceut J [serial online] 2019 [cited 2020 Feb 22];18:341-55. Available from: http://www.epj.eg.net/text.asp?2019/18/4/341/272375
| Introduction|| |
Lignocellulose biomass (all plants and plant-derived materials) is the Earth’s most abundant and renewable organic material with great potential for production of bioenergy and commodity chemicals . Certain microbes are categorized as cellulolytic owing to their capability of growing on cellulose as sole carbon and energy source and secreting cellulases . Three kinds of cellulases are required to carry out cellulose hydrolysis: first, endoglucanases locate surface sites randomly along the cellodextrin creating a new reducing and nonreducing chain end; second, exoglucanases cleave cellulose chains at the ends to release cellobiose; and third, β-glucosidases convert cellobiose to glucose, and thus relieve the system from end-product inhibition . Cellulases are widely produced by various microorganisms such as bacteria, fungi, molds, and microbes present in the animal gut. Microbial cellulases are used in various industries such as food and animal feed, laundry and detergents, pulp and paper, textiles, and biofuel. Moreover, they have some applications in the pharmaceutical industry, genetic engineering, waste treatment, and protoplast production . Therefore, worldwide research has been focused on isolation and exploitation of new microbial resources for the extraction of cellulolytic enzymes with desirable catalytic potential . Cellulase production is inducible in bacteria and is significantly influenced by nutritional composition and physical process parameters such as incubation period, temperature, pH and agitation speed . Optimization of both nutritional and production parameters can considerably improve cellulase production in bacteria and plays a significant role in development of industrial bioprocess for enzyme production . An ideal practice to optimize such parameters is response surface methods (RSM). This method employs a statistical experimental design and provides statistically validated predictions to ease the optimization process . The 16S ribosomal RNA (rRNA) genes have been the most predominantly used molecular markers for bacterial classification. The bacterial 16S rRNA gene is ∼1500bp long and contains both conserved and variable regions that evolve at different rates. The slow evolution rates of the former regions enable the design of universal primers that amplify genes across different taxa, whereas fast-evolving regions reflect differences between species and are useful for taxonomic classification . The relatedness between bacterial species can be studied by the construction of phylogenetic tree or dendrogram using freely available tree-making software such as MEGA7.0. The phylogenetic tree confirms the genus to which the query sequence strain belongs and its closest neighbors by comparing with other sequences from database followed by which further genotypic, chemotaxonomic, and phenotypic analysis platforms are designed . The objective of this work is to isolate and provide molecular identification of some high β-glucosidase-producing microorganisms from soil in the Egypt, and to experimentally find out the favorable conditions required in one of them to produce maximum amount of β-glucosidase, so that it could be used on an industrial scale.
| Materials and methods|| |
Screening of cellulolytic producing strains
For isolation of cellulolytic microorganisms, 1 g of different soil samples was collected from the Qalubia governorate, Egypt, and was transferred to the fresh 100 ml salt medium [(g/l): CMC, 10; NaNO3, 0.5; K2HPO4, 1.0; MgSO4. 7H2O, 0.5; KCl, 0.5; FeSO4; 7H2O, 0.001], containing CMC as the sole carbon source in 500 ml sealed bottles for incubation at 37°C for 48 h. At the end of the incubation, the agar medium was flooded with an aqueous solution of Congo red (0.1% w/v) for 15 min. The excess Congo red solution was poured off, and the plates were further treated by flooding with 1 mol/l NaCl for 15 min. The ratio of the clear zone diameter was measured to select the highest cellulase-producing bacterium. Isolates were maintained on CMC plates for additional experiments and also stored in 15% glycerol at −80°C for future use .
Identification of selected cellulolytic producing strains
The isolated microorganism was further identified based on morphological, biochemical, and physiological characteristics as described in Bergey’s Manual of Systematic Bacteriology .
Preparation of crude enzyme
The highest cellulase-producing strain was inoculated in production medium at pH 7 and incubated at 37°C for 48 h. After incubation, the supernatant obtained after centrifugation served as a crude enzyme source for cellulases assay .
Determination of filter paper assay
Filter paper activity of the culture filtrates was determined according to the method of Miller . Whatman filter paper containing 50 mg weight was suspended in 1 ml of 0.05 mol/l sodium citrate buffer (pH 4.8) at 50°C in a water bath. Suitable aliquots of enzyme source were added to the aforementioned mixture and incubated for 60 min at 37°C. After incubation, the liberated reducing sugars were estimated by the addition of 3, 5-dinitrosalicylic acid. One unit of filter paper unit was defined as the amount of enzyme releasing 1 μmol of reducing sugar from filter paper/ml/h.
Determination of endoglucanase activity
Endoglucanase activity was quantified by carboxymethylcellulose method . The reaction mixture with 1.0 ml of 1% carboxymethylcellulose in 0.2 mol/l acetate buffer (pH 5.0) was preincubated at 37°C in a water bath for 20 min. An aliquot of 0.5 ml of culture filtrate was added to the reaction mixture and incubated at 37°C for an hour. The reducing sugar produced in the reaction mixture was determined by 3, 5-dinitrosalicylic acid method. One unit of enzyme was defined as the amount releasing 1 μmol of reducing sugar/ml/h.
Determination of β-glucosidase activity
The β-glucosidase activity was determined with 100 µl of enzymatic extract, 250 µl of sodium acetate buffer (0.1 mol/l, pH 6.5), and 250 µl of p-nitro phenyl-β-D-glucopyranoside (5 mmol/l, pNPβG; Sigma Chemical Co., USA) during 30 min reaction at 37°C. The reaction was stopped with 2 ml of sodium carbonate (2 mol/l), and the liberated product was spectrophotometrically quantified at 410 nm. One unit of enzyme was defined as the amount of enzyme required to release 1 µmol of nitro phenol per minute of reaction .
Determination of total protein
Soluble proteins in the culture supernatant were estimated by dye-binding method of Bradford using bovine serum albumin (Sigma Chemical Co.) as a standard .
Molecular identification of QS6 strain using 16s rDNA gene sequencing
Isolation of bacterial genomic DNA
For molecular identification of most potent β-glucosidase-producing strain (QS6), DNA extraction was performed using Promega wizard Genomic DNA purification Kit (Promega Company, WI, USA) according to the manufacturer’s protocol.
PCR amplification of 16s rRNA gene
Amplification was done using forward primer 8F (5′-CAG GCC TAA CAC ATG CAA GTC-3′) and reverse primer 1492R (5′-GGG CGG GGT GTA CAA GGC-3′). The PCR mixture was carried out in a volume of 50 µl, contained 22 µl of MQ, 25 µl of DreamTaq Green DNA Polymerase (Thermo Fisher Scientific, USA), 1 µl of each forward and reverse primer (10 µmol/l each, IDT synthesized), and 1 µl of template. The PCR amplification conditions were 4 min of preheating at 95°C, 30 s denaturation at 95°C, 45 s of primer annealing at 50°C, 1 min extension step at 72°C, and postcycling extension of 10 min at 72°C for 35 cycles. The reactions were carried out in a thermal cycler (Applied Biosystem Thermal Cycler, USA) .
Sequence alignment, phylogenetic analysis, and bioinformatics analysis
Amplified PCR product was purified and sequenced at Macro gene, Korea. Raw data of sequencing were edited (contig and peak chromatogram verification) using the Finch T.V 1.4.0 program. Analysis of 16 s rRNA sequences of QS6 strain was performed using the BLAST (N) program of the National Center of Biotechnology Information (NCBI) (Rockville Pike, Bethesda MD, USA). Multiple sequence alignment was done using the ClustalX 2.1 program. The phylogenetic trees were constructed using neighbor-joining method by MEGA. X .
Nucleotide sequence accession number
The 16s rRNA gene sequence of the isolated Lysinibacillus sphaericus QS6 was determined, edited. and submitted to GenBank database under accession number MN493725.1.
Optimization of cultural condition for β-glucosidase production
Effect of incubation time
Incubation period was an important parameter for enzyme production. To determine the optimum incubation period of the isolate L. sphaericus QS6 for maximum β-glucosidases production, the supernatant was collected after 6, 12, 18, 24, 48, 72, 96, and 120 h of incubation.
Effect of temperature
To determine the effective temperature for β-glucosidase production by L. sphaericus QS6, the experiment was carried out at 25, 35, 40, 45, 50, 55, and 60°C for 24 h.
To determine the effective inoculum size for β-glucosidase production by L. sphaericus QS6, aliquots (50 ml) of the production medium were inoculated with different inoculum size of the selected strain (0.5, 1, 2, 4, 6, 10% v/v) at 35°C for 24 h.
Effect of pH
The effect of pH on β-glucosidases production was studied by varying pH levels (3, 4, 5, 6, 7, 8, 9, and 10). Selected medium of different pH was inoculated with the L. sphaericus QS6 isolate at 35°C for 24 h. with 1% inoculum size .
Different carbon sources
Different carbon sources, such as glucose, fructose, mannitol, sucrose, lactose, cellobiose, salicin, CMC, cellulose, starch, and avice (1 g/l), were separately added as a sole carbon source. At the end of incubation, the β-glucosidase activity was assayed. Carbon sources were autoclaved separately and added to the medium under aseptic conditions .
Different of nitrogen source
The liquid medium was separately supplemented with different nitrogen sources, such as yeast extract, beef extract, peptone, soymeal, urea, sodium nitrate, ammonium chloride, potassium nitrite, ammonium sulfate, and ammonium nitrate (1 g/l), at the end of incubation, the β-glucosidase activity was assayed as studied by Jahangeer .
The examination of antimicrobial activity was based on the in-vitro agar well diffusion method according to Hindler et al. . The prepared crude was tested against standard microorganisms of gram-positive bacteria (Enterococcus faecalis, Bacillus cereus, Bacillus. lichniforms, Bacillus thuringiensis, Enterobacter ludwigii, and Bacillus subtilis) and gram-negative bacteria (Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumonia, and Escherichia coli) at a concentration of 100 μl per well. The diameter of the inhibition zones was measured (mm) at 37°C after 24 h.
Strain L. sphaericus. QS6 was tested for its susceptibility to some antibiotic. The test was carried out by diffusion in Müller-Hinton agar. Antibiotic discs included amoxicillin (AX, 25 µg), streptomycin (S, 10 µg), tetracycline (TE, 30 µg), cefadroxil (CFR, 30 µg), kanamycin (K, 10 µg), chloramphenicol (C, 30 µg), ampicillin (AM, 10 µg), erythromycin (E, 10 µg), nalidixic (NA, 30 µg), and tobramycin (TOB, 10 µg) .
| Results and discussion|| |
Identification of producer organism
For isolation of cellulose-degrading bacteria, samples were collected from agricultural soil in Egypt, as it contains cellulose-degrading bacteria in it, because of the soil being rich with lignocellulose biomass. The colony morphology was studied in detail, and the results are presented in [Figure 2] and [Table 3], followed by biochemical tests performed for the isolated microorganisms ([Table 3]). The collected samples were then subjected to serial dilution technique and were inoculated onto a plate containing CMC agar medium and incubated at 37°c for 24–48 h ([Figure 1]).
|Figure 1 (a) Secondary screening (quantitative) for cellulolytic bacteria by evaluating glucosidase activity; (b) primary screening (qualitative) for cellulolytic bacteria by covering the petri dishes with Congo red solution.|
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|Figure 2 Morphological characteristics of QS6 bacterial isolate: (a) morphological characteristics of purified colony; and (b) microscopic characterization of microorganism.|
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After incubation, Cong red test was performed for screening of the cellulolytic organisms. The strains showing maximum clear zones around the colonies were selected for further studies. A total of eight bacterial isolates were found to be positive on screening media (cellulose Cong red agar) by producing clear zone around their colonies ([Figure 2] and [Figure 3] and [Table 1]). Furthermore, QS6 was selected for enzyme production and their respective cellulolytic activity was estimated.
|Figure 3 Secondary screening (quantitative) for cellulolytic bacteria by evaluating the endoglucanase, FPase activity, and glucosidase activity.|
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|Table 1 Primary screening (qualitative) for cellulolytic bacteria by zone of clearance surrounding the colonies is indicative of hydrolysis by secreted CMCase|
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The β-glucosidase activity was measured by PNPG method. Among the potent β-glucosidase producers, strain QS6 showed the highest β-glucosidase activity (33.79±0.33 U/ml) at 24 h after incubation. Therefore, strain QS6 was used for future study and identification through 16 s rRNA sequence analysis and phenotypic characterization. Based on the results of primary screening and secondary screening, QS6 isolate was selected for further study because it had the highest cellulase endoglucanase activity and β-glucosidase activity as presented in [Table 2].
|Table 2 Secondary screening (quantitative) for cellulolytic bacteria by evaluating the endoglucanase, FPase activity, and β-glucosidase activity|
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Morphological, physiological, and biochemical characterization
The isolated QS6 strain was initially identified and tested for primary characterization. According to morphological characteristics, the cells were rod shaped, single, gram positive, motile, and strictly aerobic, and the colonies were pale white colored, spore forming, as shown in [Figure 2], catalase negative, and produced ammonia from arginine hydrolysis, which is the main characteristic feature, as observed with all bacilli. The culture could grow at different temperatures 30–37°C, pH (5.0–8.0), and NaCl concentrations (1–10%) ([Table 3]).
|Table 3 Morphological, biochemical, and physiological characteristics of QS6 bacterial isolate|
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To identify the isolated strains, we used the microscopic examination techniques, and biochemical test. Considering a previous report , the stain QS6 showed positive results in catalase test, oxidase test, gelatin test, nitrate reduction, and esculin test.
These results correlate that the organisms resemble L. sphaericus strain QS6. The isolates were identified as belonging to member of the genus Bacillus sp. QS6 strain was identified as Lysinibacillus spp. ([Table 3]). All the biochemical and physiological tests were found to be Lysinibacillus spp. The result is in consistent with previous reports .
Phylogenetic analysis of the producer microorganism
To identify the QS6, a fragment of 16S rDNA was generated and sequenced. The sequencing result was aligned online in the nucleotide BLAST tool through the (NCBI) database to identify the possible genera of the isolates base on homology. From BLAST search results, QS6 isolate has 97% homology to 16S rDNA fragment of Lysinibacillus spp. CMJ2-5 (accession number NR 042073.1). Then, 16S rDNA sequences of QS6 strain was deposited in NCBI database with an accession number MN493725.1. Evolutionary analyses were conducted in MEGA X . Based on the homology and phylogenetic analysis ([Figure 4]), it was concluded that the isolated QS6 culture was L. sphaericus.
|Figure 4 (a) Neighbor-joining phylogenetic tree showing relationship of strain QS6 with different types of strain of the genus Lysinibacillus spp. The tree was generated using MEGA X software; (b) 1.5% agarose gel electrophoresis of PCR product of the isolated QS6. Lane 1: DNA marker 1 kb, and lane 2: isolate QS6.|
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The alignment of the 16S rRNA nucleotide sequence of QS6 bacterial isolate comprised 1440 bp. The 16SrRNA stated gene sequence was coordinated in the gene bank database through the NCBI BLAST (http://www.ncbi.nlm.nih.gov). A correlation between the 16S rRNA sequence of this strain and those individuals in the genomic database bank was accomplished. This correlation demonstrated an extraordinary level of sequence similarity (98%) with Lysinibacillus sp.
Effect of incubation period on β-glucosidase production
The effect of incubation time on enzyme production was studied from 6 to 120 h. using cellobiose as substrate. The production increased with increase in incubation period, and for L. sphaericus QS6, β-glucosidase activity reached maximum (35.17±0.026 IU) at 24 h after incubation. The β-glucosidase activity and protein content determined ranged from 14.48±0.087 IU/ml and 81.67±0.881 μg/ml at 6 h. to maximum 35.17±0.026 IU/ml and 86.33±0.881 μg/ml at 24 h. The optimum incubation time for the β-glucosidase production was found at 24 h with the higher 88 µg/ml of protein content and enzyme activity of 35.12 U/ml. It was also observed that the enzyme activity and protein showed decreasing pattern after 24 h. However, the minimum yield of the protein content of 74.01±2.081 µg/ml and enzyme activity of 12.91±0.043 IU/ml was observed at 120 h ([Figure 5] and [Table 4]). At the same incubation period (24 h) , much lesser FPase (0.011 IU/ml) and CMCase (0.079 IU/ml) activities by Bacillus pumilus EB3 were recorded. Decrease in enzymatic activity with time might be owing to the depletion of nutrients and production of other by-products in the fermentation medium . Our results revealed that, the enzyme production was increased until 24 h, and with further increase in the incubation period, decreased enzyme activity was seen. Thus, 24 h was considered as the optimum incubation period for the production of β-glucosidase by L. sphaericus strain QS6.
|Figure 5 Effect of incubation time on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6.|
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Effect of temperature on β-glucosidase production
Temperature is one of the most important parameters essential for the success of a fermentation process. For β-glucosidase production by L. sphaericus strain QS6, 35°C was the most effective. The production started declining after further increase in temperature. As shown in [Figure 6] and [Table 4] and [Table 5], β-glucosidase activity was recorded as 27.49±0.690, 37.25±0.161, 32.79±0.588, and 32.79±0.588 IU/ml at 30, 35, 40, and 45°C, respectively. The activity of β-glucosidase changes slightly with temperature, but there is marked increase in the activities of β-glucosidase at incubation temperature of 35°C. Our results revealed that 35°C was considered as the optimum temperature for the production of β-glucosidase by L. sphaericus strain QS6. Similar observations was observed by Acharya and Chaudhary , who reported similar kinds of results for Bacillus licheniformis and Bacillus spp. Moreover, Gondalez et al.  reported that higher β-glucosidase activities was obtained from Trichoderma reesei GM9414 on wheat straw as carbon source. Kato  reported optimum temperature for growth and cellulose degradation of C. straminisolvens at 50 to 55°C, and Immanuel  recorded maximum endoglucanase activity in Cellulomonas, Bacillus, and Micrococcus spp. at 40°C at neutral pH.
|Figure 6 Effect of temperature on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6 for 24 h.|
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Effect of pH on β-glucosidase production
Like temperature, hydrogen ion concentration of the production medium strongly affects many enzymatic processes and transport of compounds across the cell membrane. It has been shown that growth medium pH strongly influences many enzymatic reactions by affecting the transport of a number of chemical products and enzymes across the cell membrane . The effect of pH on β-glucosidase production by L. sphaericus strain QS6 was investigated by growing culture at different initial pH between 3 and 10. A high level of β-glucosidase activity was obtained in the culture medium with pH 7 with β-glucosidase production at 36.38±0.281 IU/ml and specific activity at 366.33±0.881 U/mg. As presented in [Figure 7], there was a decrease in β-glucosidase activity from 36.38±0.28 to 12.54±0.313 IU/ml on increasing the pH from 7 to 10. The obtained results are inconsistence with several researchers, such as Immanuel , who reported that the cellulolytic enzyme, endoglucanase, from Cellulomonas, Bacillus, and Micrococcus spp., isolated from the estuarine coir netting effluents hydrolyzes substrate in the pH range of 4.0–9.0, with maximum activity at pH 7.0. Contrary to that, Lee et al  observed optimal cellulase production at pH 9.0 by Clostridium acetobutylium. Our results revealed that pH 7.0 was considered as the optimum pH for the production of β-glucosidase by L. sphaericus strain QS6.
|Figure 7 Effect of pH on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6 for 24 h. at 35°C.|
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Effect of inoculum size on β-glucosidase production
For optimum enzyme production in fermentation medium, inoculum size was optimized, and 1% inoculum size was found to be better for maximum enzyme production ([Figure 8]). Highest levels of β-glucosidase activity and protein (38.13 IU/ml and 90 μg/ml, respectively) were produced when strain QS6 was grown on Lb. along with cellobiose at 1% for 48 h at 40°C. For production of raw starch-hydrolyzing amylase by Bacillus spp., 2% inoculum was recommended  Inoculum size (2–3%) was also found to be optimum for cellulases produced by Bacillus subtilis CY5 and Bacillus circulans TP3 .
|Figure 8 Effect of inoculums size on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6 for 24 h at 35°C, pH 7.|
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Effect of different carbon sources on β-glucosidase production
The results of the effect of various carbon sources on β-glucosidase production by L. sphaericus QS6 are illustrated in [Figure 9]. Among all the carbon sources tested, cellobiose gave maximum β-glucosidase activity when used as a sole carbon source.
|Figure 9 Effect of carbon sources on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6 for 24 h at 35°C, pH 7.|
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In our results, different carbon sources were examined to study their effects on L. sphaericus strain QS6 β-glucosidase production under identical conditions (temperature, 35°C; 1% inoculum size; incubation time, 24 h). The production of β-glucosidase in L. sphaericus strain QS6 was found to be maximum protein content of 89.23±0.577 µg/ml and enzyme activity at 35.18±0.527 U/ml with cellobiose at 24 h of incubation. Starch and cellulose yielded the least enzyme activity of 23.05±0.504 and 13.17±0.172 U/ml, respectively, with protein content of 50 and 40 µg/ml, respectively, after 48 h of incubation period.The present results suggested that optimum β-glucosidase production was observed with cellobiose as a carbon source ([Figure 10]).
|Figure 10 Effect of cellobiose concentration on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6 for 24 h at 35°C, pH 7.|
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β-glucosidase production by L. sphaericus strain QS6 was studied by testing β-glucosidase secretion in the culture medium using different substrate cellobiose concentrations at 35°C for 24 h at pH.7. Cellobiose is used as a substrate for β-glucosidase production owing to its less complexity and easy digestion by the microbes . The different concentrations of cellobiose were tested for β-glucosidase production, among which 1% cellobiose was optimum for this strain ([Figure 10]). Above this concentration, β-glucosidase production was inhibited. Similarly, cellulase production was inhibited by 1% cellulose in Thermomonospora curvata isolated from municipal solid waste compost .
Effect of different nitrogen sources on β-glucosidase production
The nitrogen source in the control was better over other treatments for both CMCase and FPase production for both wheat and rice straw. The present results showed lower β-glucosidase activity with inorganic nitrogen sources, which suggested the reduced utilization of inorganic nitrogen by aerobic bacteria. These data were in accordance with the results of Ray et al.  who reported that organic nitrogen sources were more suitable for optimizing the cellulase production by B. subtilis and B. circulans than inorganic sources. On the contrary, Spiridonov and Wilson found that NH4 compounds were the most favorable nitrogen sources for cellulase synthesis
The results showed that strain QS6 can utilize organic nitrogen sources efficiently, and the maximum β-glucosidase activity (27.71±0.202) was observed when peptone was used as the sole nitrogen source. However, the β-glucosidase activity was almost zero when organic nitrogen sources (Soymeal or urea) were used as the sole nitrogen sources ([Figure 11] and [Figure 12]). This could be because the metabolism of inorganic nitrogen contributes to medium acidification, which in turn affects β-glucosidase production. Similar observations were observed by Rajoka et al. . Moreover, KNO3 and NH4NO3 were used as the best N sources for cellulose production in Cellulomonas flavigena. NH4 compounds were considered as the most favorable N sources for cellulase synthesis as noted in Thermomonospora fusca .
|Figure 11 Effect of nitrogen sources on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6 for 24 h at 35°C, pH 7.|
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|Figure 12 Effect of Peptone concentration on protein content and glucosidase production in Lysinibacillus sphaericus strain QS6 for 24 h at 35°C, pH 7.|
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Antibiotic susceptibility and antibacterial activity
The antibiotic susceptibility of the L. sphaericus strain QS6 strain was assessed by disk diffusion using 10 antimicrobial agents ([Table 6]). Our data evidenced that L. sphaericus strain QS6 was sensitive to amoxicillin, streptomycin, tetracycline, cefadroxil, kanamycin, chloramphenicol, erythromycin, and tobramycin, but was resistant to nalidixic antibiotics, with the exception of ampicillin, where the strain showed intermediate resistance. Moreover, in-vitro antibacterial bioassay of the most potent β-glucosidase-producing strain (QS6) showed high antimicrobial activity against Escherichia coli (1.9 cm) and Pseudomonas aeruginosa (1.0 cm).
|Table 6 Antibiotic susceptibility and antibacterial activity on β-glucosidase-producing Lysinibacillus sphaericus QS6 strain|
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| Conclusion|| |
β-glucosidase is commercially produced by several industries globally and is widely being used in food, animal feed, fermentation, agriculture, pulp and paper, and textile applications. For this reason, we have screened β-glucosidase-producing bacteria from different ecological niches and obtained this strain of L. sphaericus strain QS6. The culture conditions like pH, temperature, carbon sources, and nitrogen sources were optimized. The optimum conditions found for β-glucosidase production are 35°C at pH 7 on 24-h incubation period with cellobiose (1%) as the carbon source and peptone (0.5) as the nitrogen source.
The financial assistance received from the Department of Microbial Genetics, Genetic Engineering and Biotechnology Division, National Research Centre, Giza, Egypt.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Somerville C. The billion-ton biofuels vision. Am Assoc Adv Sci 2006; 312:1277.
Schwarz W. The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 2001; 56:634–649.
Himmel ME. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 2007; 315:804–807.
Yin L-J, Huang P-S, Lin H-H. Isolation of cellulase-producing bacteria and characterization of the cellulase from the isolated bacterium Cellulomonas sp
.YJ5. J Agric Food Chem 2010; 58:9833–9837.
Harshvardhan K, Mishra A, Jha B. Purification and characterization of cellulase from a marine Bacillus
sp.H1666: a potential agent for single step saccharification of seaweed biomass. J Mol Catalysis B 2013; 93:51–56.
Yang W. Isolation and identification of a cellulolytic bacterium from the Tibetan pig’s intestine and investigation of its cellulase production. Electron J Biotechnol 2014; 17:262–267.
Singh S, Moholkar VS, Goyal A. Optimization of carboxymethylcellulase production from Bacillus amyloliquefaciens
SS35. 3 Biotech 2014; 4:411–424.
Srinivasan R. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS One 2015; 10:e0117617.
Ciccarelli FD. Toward automatic reconstruction of a highly resolved tree of life. Science 2006; 311:1283–1287.
Lo YC. Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production. Enzyme Microbial Technol 2009; 44:417–425.
Brown JH. Bergey’s manual of determinative bacteriology. Washington: American Public Health Association; 1939.
Shaikh N. Isolation and screening of cellulolytic bacteria inhabiting different environment and optimization of cellulase production. Univ J Environ Res Technol 2013; 3:39–49.
Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959; 31:426–428.
Acharya S, Chaudhary A. Optimization of fermentation conditions for cellulases production by Bacillus licheniformis
MVS1 and Bacillus
sp.MVS3 isolated from Indian hot spring. Brazil Arch Biol Technol 2012; 55:497–503.
de Palma-Fernandez E, Gomes E, Da Silva R. Purification and characterization of two β-glucosidases from the thermophilic fungusThermoascus aurantiacus
. Folia microbiologica 2002; 47:685–690.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–254.
Rastogi G. Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA. J Ind Microbiol Biotechnol 2009; 36:585.
Kumar S. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 2018; 35:1547–1549.
Jahangeer S. Screening and characterization of fungal cellulases isolated from the native environmental source. Pak J Botany 2005; 37:739.
Hindler J, Howard B, Keiser J. Antimicrobial agents and antimicrobial susceptibility testing. In: Howard BJ (Ed). Clinical and Pathogenic Microbiology. 2nd ed. St. Louis: Mosby 1994.
Tuomela M. Biodegradation of lignin in a compost environment: a review. Bioresource Technol 2000; 72:169–183.
Ariffin H. Production and characterization of cellulase by Bacillus pumilus
EB3. Int J Eng Technol 2006; 3:47–53.
Ikram-ul-Haq UH. Cotton Saccharifying activity of cellulases by Trichoderma harzianum
UM-11 in shake flask. J Bot 2005; 1:19–22.
Gonzalez G, Caminal G, Lopez-Santin J. Cellulase production on lignocellulosic materials and performance of a packed-bed reactor for enzymatic hydrolysis. J Bot 1986; 3:120–128.
Kato S. Clostridium straminisolvens sp. nov., a moderately thermophilic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community. Int J System Evol Microbiol 2004; 54:2043–2047.
Immanuel G. Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. Int J Environ Sci Technol 2006; 3:25–34.
Liang Y. Optimization of growth medium and enzyme assay conditions for crude cellulases produced by a novel thermophilic and cellulolytic bacterium, Anoxybacillus sp. 527. Appl Biochem Biotechnol 2010; 160:1841–1852.
Lee SF, Forsberg CW, Gibbins L. Cellulolytic activity of Clostridium acetobutylicum
. Appl Environ Microbiol 1985; 50:220–228.
Avendano M, Cornejo I. Formation of a raw starch-hydrolyzing alpha-amylase by Clostridium
2021: effect of carbon sources. Biotechnol Lett 1987; 9:123–124.
Ray A. Optimization of fermentation conditions for cellulase production by Bacillus subtilis
CY5 and Bacillus circulans
TP3 isolated from fish gut. Acta Ichthyologica Piscatoria 2007; 1:47–53.
Shanmugapriya K. Isolation, screening and partial purification of cellulase from cellulase producing bacteria. Int J Adv Biotechnol Res 2012; 3:509–514.
Rajoka MI, Malik KA. Cellulase production by Cellulomonas biazotea
cultured in media containing different cellulosic substrates. Bioresource Technol 1997; 59:21–27.
Spiridonov NA, Wilson DB. Regulation of biosynthesis of individual cellulases in Thermomonospora fusca. J Bacteriol 1998; 180:3529–3532.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]