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3PCR AMPLIFICATION OF CELLULOSE-DEGRADING ENDOGLUCANASE AND B-GLUCOSIDASE GENES OF BACILLUS SUBTILIS STRAINS
1Safarov Kh., 2Kutlieva G., 3Turaeva B.
1,2,3Institute of Microbiology of Uzbekistan Academy of Sciences, Tashkent, Uzbekistan
https://doi.org/10.5281/zenodo.13685135
Abstract. The given article presents that the main part of animal feed is plant products. The decomposition of cellulose in the cell shell of plants and the assimilation of the resulting products are considered one of the factors that determine the productivity of the organism. In this restriction, genes involved in cellulose degradation were screened in 8 strains of B. subtilis isolated from the digestive system of animals. The fi-glucosidase gene was detected by PCR in all B. subtilis strains. Endoglucanase gene was found to be present in B. subtilis 1, B. subtilis 2, B. subtilis 3, B. subtilis 7 and B. subtilis 8 strains.
Keywords: B. subtilis, endoglucanase, fi-glucosidase, cellulose, fi-1,4-glucoside, PCR,, marker.
It is important to inform that cellulose constitutes the majority of plant biomass on Earth. It constitutes about 35-50% of the dry weight of plants, while hemicellulose and lignin constitute 20-35% and 5-30% of the dry weight of plants, respectively. Cellulose is a linear polysaccharide consisting of monomers of glucose units linked by P-1,4-glycosidic bonds [1]. Cellulase is important as food for ruminants, poultry, pigs, fish, and domestic animals. Cellulose is also used to increase milk yield and live weight of ruminants. [2]. Undigested starch in the large intestine can serve as a substrate for bacterial fermentation. Cellulases have a positive effect on intestinal fermentation processes by increasing the production of propionic acid, act as a bacteriostatic material, and thereby reduce the colonization of pathogenic bacteria [3]. Three main enzymes are involved in the complete breakdown of cellulase: 1) Endoglucanase: hydrolyzes the internal P-1,4-glucosidic bonds in the cellulose chain. 2) Exoglucanases (cellobiohydrolases): hydrolyze the cellulose chain to form cellobiose units. 3) P-glucosidase (cellobiase): breaks the glycosidic bonds between disaccharides and converts them into glucose [4], [5].
Definitely, 15 Bacillus subtilis strains showed high cellulase activity among isolates collected from the sedimentary soil layer of the Mahanadi River, India [7]. Jose A. et al. isolated bacteria living in symbiosis with plants. This bacterium showed endoglucanase activity in 62% of isolates, and among the isolates, Bacillus sp. showed the highest activity [8].
The aim of the research was to identify the genes of cellulose-degrading enzymes of B. subtilis strains isolated from the digestive system of animals. Eight B. subtilis strains isolated from animals and endoglucanase (EN1) and P-glucosidase (bglP) primers were used in the study. B. subtilis strains (LP: tryptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l; sterilization at 121 °C, 20 min) were grown in liquid nutrient medium for 24 h at 37 °C and shaker at 180 rpm. 1.5 ml of B. subtilis strains were centrifuged at 1400 rpm for 10 min. Genomic DNA was isolated using a modified Marble method [9]. The extracted DNA samples were dissolved in 100 pl of TE buffer and their concentration was measured using an Implen NanoPhotometer® (Table 1).
Glukoza
SeLLobioza voki SeUotetroza
Endogluconase, Cellulose, P-glucosidase (cellobiase), Glucose Figure 1. Decomposition of cellulose by enzymes [6].
Table 1
Concentration of nucleic acids isolated from B.subtilis strains.
№ Samples Concentration Ed
1 TE buffer 0,0000 ng/ul
2 B.subtilis 1 43,400 ng/ul
3 B.subtilis 2 44,200 ng/ul
4 B.subtilis 3 152,60 ng/ul
5 B.subtilis 4 101,30 ng/ul
6 B.subtilis 5 87,450 ng/ul
7 B.subtilis 6 58,900 ng/ul
8 B.subtilis 7 32,200 ng/ul
9 B.subtilis 8 17,000 ng/ul
Genomic DNA was visualized by electrophoresis in 0.8% agarose gel stained with ethidium bromide (Bio-rad, USA) (Fig. 2).
Figure 2. Genomic DNA of B.subtilis strains DNA marker M-1kb.
It should be mentioned that in the genome of the given strains, primers for the P-glucosidase and endoglucanase genes, which break down cellulose, were developed and synthesized by Integrated DNA Technologies (IDT) [10].
Primer Sequenced nucleotides Gene name PCR product size
bglP-F 5' TGC TGA GTG CGG TCT TTG AT 3' ß- glucosidase 991 bp
bglP-R 5' AGG CCG ATCA TCG CGT AAA T 3'
EN1-F 5' CCA GTA GCC AAG AAT GGC CAG C 3' endoglucanase 1311 bp
EN1-R 5' GGA ATA ATC GCC GCT TTG TGC 3'
The nucleotide sequence of 16S rRNA of B. subtilis shows high similarity compared to other species belonging to the genus Bacillus. For isntance, B. atropaeus was found to be 99.3% similar to B. licheniformis, and B. amyloliquefaciens was 98.3% similar [11]. It has now been shown that EN1, a specific primer for the detection of the endoglucanase gene, can be used to detect B. subtilis [12].
Figure 3. Design of primers for endoglucanase and P-glucosidase genes (ncbi.nlm.nih.gov
gene ID: HQ000093.1; identifier: Z34526.1).
Besides that, PCR amplification of the extracted DNA samples was performed in GenPak® PCR MasterMix. PCR products were prepared from 20 |l (10 |l masterMix, dis-H2O 8.2 |l, primer-F 0.4 |l, primer-R 0.4 |l, DNA 1 |l). Denaturation consists of 94 Co 3 min, 58 Co 50 sec, 72 Co 2 min, 30 cycles. After amplification, PCR products were visualized in 0.8% agarose gel
A B
Figure 4. PCR results for the A-P-glucosidase and B-endoglucanase genes in 8 B. subtilis
strains.
The conducted study showed that all of the above B. subtilis strains have the gene for the cellulose-degrading P-glucosidase enzyme. The primer developed for the masker gene is bglP-991 bp. It was found to be the same size as the PCR amplification product (Fig. 4). PCR analysis of the endoglucanase enzyme gene in B. subtilis strains showed that this gene is present in B. subtilis 1, B. subtilis 2, B. subtilis 3, B. subtilis 7, and B. subtilis 8 out of 8 strains. A primer was designed for a 1311 bp region of the EN1 gene and the PCR amplification product was found to match it. Some samples also had a DNA sample of the wrong size for the EN1 gene replicated, possibly due to suboptimal temperatures during this process.
By summarizing it should be suggested that based on the above, B. subtilis strains live symbiotically in the digestive system of animals and participate in the process of breaking down cellulose contained in feed. Mixing these strains with animal feed in livestock, cattle and poultry farming can lead to an increase in their productivity. The method of PCR amplification of endoglucanase and P-glucosidase genes of Bacillus subtilis strains obtained in the experiment creates opportunities for determining the synthesis of cellulose-degrading enzymes (endoglucanase, P-glucosidase) in bacteria isolated from different sources and for effective screening.
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