{"id":496,"date":"2025-12-25T17:43:25","date_gmt":"2025-12-25T10:43:25","guid":{"rendered":"https:\/\/dnape.online\/?p=496"},"modified":"2026-03-19T17:00:57","modified_gmt":"2026-03-19T10:00:57","slug":"emi-i","status":"publish","type":"post","link":"https:\/\/dnape.online\/en\/2025\/12\/emi-i\/","title":{"rendered":"The restriction endonuclease EmiI, an isoschizomer of Ksp632I, recognizes the non-palindromic DNA sequence 5\u2032-CTCTTC(1\/4)-3\u2032"},"content":{"rendered":"\n<\/span>

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Authors<\/strong><\/p>\n

M.A. Abdurashitov, D.A. Gonchar, V.A. Chernukhin, V.S. Dedkov, N.A. Mikhnenkova, A.A. Nikonova, S.Kh. Degtyarev<\/p>\n

Affiliation<\/strong><\/p>\n

SibEnzyme Ltd., Novosibirsk, Russia<\/p>\n

*<\/strong> Corresponding author:\u00a0 <\/em>M.A. Abdurashitov, SibEnzyme Ltd., 2\/12 Ak. Timakova Street, Novosibirsk 630117, Russia Tel.: +7 (383) 333-4991Fax: +7 (383) 333-6853 E-mail: abd@sibenzyme.ru<\/a><\/p>\n

Abstract<\/h3>\n

A bacterial strain, Exiguobacterium mexicanum<\/em> 6, was identified as a producer of a novel restriction endonuclease designated EmiI. The enzyme recognizes the non-palindromic hexanucleotide DNA sequence 5\u2032-CTCTTC-3\u2032 and cleaves DNA outside the recognition site at positions 1\/4, generating three-nucleotide 5\u2032-protruding ends: 5\u2032-CTCTTC(N)\u2081\u2193-3\u2032 \/ 3\u2032-GAGAAG(N)\u2084\u2193-5\u2032.
Thus, EmiI is an isoschizomer of the restriction endonucleases Ksp632I [1] and Bst6I [2]. The producer strain was identified based on morphological and biochemical characteristics as well as analysis of the primary structure of a fragment of the 16S rRNA gene. A preparation of restriction endonuclease EmiI with an activity of 5000 U\/ml was obtained through purification using three chromatographic steps. Optimal reaction conditions for EmiI include SE buffer Y (33 mM Tris\u2013acetate, pH 7.9; 10 mM Mg\u2013acetate; 66 mM potassium acetate; 1 mM DTT) at a temperature of 37 \u00b0C.<\/p>\n

Abbreviations<\/strong><\/p>\n

BSA, bovine serum albumin; DTT, dithiothreitol; \u03bb DNA, bacteriophage \u03bb DNA; T7 DNA, bacteriophage T7 DNA; U, units of activity; Tris, tris(hydroxymethyl)aminomethane; PAGE, polyacrylamide gel electrophoresis; bp, base pairs; RE, restriction endonuclease; EDTA, ethylenediaminetetraacetic acid.<\/p>\n

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Keywords:<\/strong>
producer strain; enzyme purification; type IIS restriction endonuclease; isoschizomer.<\/p>\n

DOI:10.26213\/3034-4301.2025.7.4.002<\/strong><\/p>\n

Citation:<\/strong>
Abdurashitov M.A., Gonchar D.A., Chernukhin V.A., Dedkov V.S., Mikhnenkova N.A., Nikonova A.A., Degtyarev S.Kh. (2025) The restriction endonuclease EmiI, an isoschizomer of Ksp632I, recognizes the non-palindromic DNA sequence 5\u2032-CTCTTC(1\/4)-3\u2032. <\/em>DNA-Processing Enzymes<\/strong>, 2025, Vol. 4. DOI:10.26213\/3034-4301.2025.7.4.002<\/p>\n

 <\/p>\n

\"\u041b\u0438\u0446\u0435\u043d\u0437\u0438\u044f<\/a><\/p>\n

This article is distributed under the terms of the Creative Commons Attribution\u2013NonCommercial\u2013NoDerivatives 4.0 International License (CC BY-NC-ND 4.0)<\/a>.<\/strong>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

Introduction<\/h2>\n

Type IIS restriction endonucleases [3] cleave DNA at defined positions outside their recognition sites, generating unique cohesive ends, which enables their application in a range of genetic engineering and molecular biology technologies. The present study describes a novel restriction endonuclease, EmiI, which recognizes the non-palindromic hexanucleotide DNA sequence 5\u2032-CTCTTC and cleaves DNA outside the recognition site at positions 1\/4, as indicated by arrows:
5\u2032-CTCTTC(N)\u2081\u2193-3\u2032 \/ 3\u2032-GAGAAG(N)\u2084\u2193-5\u2032.<\/p>\n

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Experimental Conditions<\/h2>\n

Reagents used in this study were purchased from Sigma-Aldrich (USA), Fisher (Germany), Panreac (Spain), Dia-M and Helicon (Russia). The following chromatographic media were used for enzyme purification: phosphocellulose P11 and heparin\u2013Sepharose (Sigma-Aldrich, USA), and hydroxyapatite (Bio-Rad, USA). Components of the culture media were obtained from Organotechnie (France). Restriction endonucleases, T4 polynucleotide kinase, bacteriophage \u03bb and T7 DNA preparations, DNA molecular weight markers, and SE reaction buffers for restriction endonucleases (B100, B, G, O, W, Y, ROSE) were purchased from SibEnzyme Ltd. (Russia).<\/p>\n

Morphological and physicochemical properties of the strain were examined using standard methods [4]. Taxonomic identification of the microorganism was performed according to Bergey\u2019s Manual of Systematic Bacteriology [5] and based on sequencing of a PCR fragment of the 16S rRNA gene.<\/p>\n

Assay of EmiI Restriction Activity<\/h3>\n

Bacteriophage \u03bb DNA was used as a substrate for analysis of EmiI activity. During screening of chromatographic fractions, 1 \u03bcl aliquots from each fraction were added to 20 \u03bcl of reaction mixture containing 1 \u03bcg of \u03bb DNA in SE buffer Y and incubated for 15 min at 37 \u00b0C.<\/p>\n

For activity testing of the purified EmiI preparation, either 1 \u03bcl of undiluted enzyme or 1\u20132 \u03bcl of enzyme diluted tenfold in SE buffer B100 (10 mM Tris-HCl, pH 7.6; 50 mM KCl; 0.1 mM EDTA; 200 \u03bcg\/ml BSA; 1 mM DTT; 50% glycerol) were used. The enzyme was added to 50 \u03bcl reaction mixtures containing 1 \u03bcg of \u03bb DNA and one of six reaction buffers for restriction endonucleases. The mixtures were incubated for 1 h at 37 \u00b0C. Reaction products were analyzed by electrophoresis in a 1% agarose gel using Tris\u2013acetate buffer (50 mM Tris\u2013acetate, pH 8.0; 20 mM sodium acetate; 2 mM EDTA) at 180 V. After staining with ethidium bromide, gels were visualized under UV light.<\/p>\n

One unit of restriction activity was defined as the minimum amount of enzyme required to completely digest 1 \u03bcg of \u03bb DNA in SE buffer Y in a 50 \u03bcl reaction volume at 37 \u00b0C within 1 h.<\/p>\n

To determine the cleavage position of EmiI within DNA, a [\u00b3\u00b2P]-labeled oligonucleotide duplex containing the EmiI recognition site and sites for control enzymes was used. Products of enzymatic cleavage were separated by electrophoresis in 20% polyacrylamide gel containing 8 M urea.<\/p>\n

Cultivation of E. mexicanum<\/em> 6 Cells<\/h3>\n

The E. mexicanum<\/em> 6 strain was cultivated in a 20-liter fermenter (New Brunswick, USA) in LM broth containing 1% tryptone (Organotechnie, France), 0.5% yeast extract (same supplier), 0.5% NaCl, 0.05% MgCl\u2082, and 0.001% thiamine at 30 \u00b0C with agitation and aeration for 5 h until an optical density at 550 nm (OD\u2085\u2085\u2080) of 3.0 was reached. Cells were harvested by centrifugation using a Beckman J2-MI centrifuge (USA) with a JA-10 rotor at 8000 rpm for 20 min and stored at \u221220 \u00b0C.<\/p>\n

Purification of Restriction Endonuclease EmiI<\/h3>\n

Purification conditions and buffers<\/h4>\n

All purification steps were carried out at +4 \u00b0C using the following buffers:<\/p>\n

Buffer A:<\/strong> 10 mM Tris-HCl, pH 7.6; 0.1 mM EDTA; 7 mM \u03b2-mercaptoethanol.
Buffer B:<\/strong> 10 mM potassium phosphate, pH 7.3; 0.1 mM EDTA; 7 mM \u03b2-mercaptoethanol.<\/p>\n

Extraction<\/h4>\n

Seventeen grams of biomass were suspended in 60 ml of buffer A containing 0.1 M NaCl, 1 mg\/ml lysozyme, and 0.1 mM PMSF (phenylmethylsulfonyl fluoride). After stirring for 2 h on ice, cells were disrupted by ultrasonication at maximum power with an amplitude of 22\u201324 \u03bcm using a Soniprep 150 device (MSE, UK) equipped with a 2-cm probe, applying seven 60-s pulses with 2-min intervals. The lysate was clarified by centrifugation in a Beckman J2-MI centrifuge with a JA-20 rotor at 15,000 rpm for 30 min.<\/p>\n

Phosphocellulose P11 chromatography<\/h4>\n

The clarified extract was loaded onto a 60-ml phosphocellulose P11 column equilibrated with buffer A containing 0.1 M NaCl. The column was washed with 120 ml of the same buffer. Bound proteins were eluted with a linear gradient of NaCl (0.1\u20130.8 M) in buffer A over a total volume of 600 ml. Sixty fractions (10 ml each) were collected. Fractions containing maximal activity (fractions 42\u201354) were pooled.<\/p>\n

Hydroxyapatite chromatography<\/h4>\n

The pooled fraction was applied to an 8-ml hydroxyapatite column equilibrated with buffer B containing 50 mM NaCl and washed with 15 ml of the same buffer. The enzyme was eluted with a linear gradient of potassium phosphate (20\u2013300 mM) in buffer B containing 50 mM NaCl over a volume of 300 ml. Fifty fractions (6 ml each) were collected. Fractions with maximal activity (fractions 24\u201329) were pooled and dialyzed against 2 liters of buffer A for 1.5 h.<\/p>\n

Heparin\u2013Sepharose chromatography<\/h4>\n

After dialysis, the pooled fraction was loaded onto a 5-ml heparin\u2013Sepharose column equilibrated with buffer A containing 50 mM NaCl and washed with 10 ml of the same buffer. Bound proteins were eluted with a linear NaCl gradient (50\u2013800 mM) in buffer A over a volume of 80 ml. Forty fractions (2 ml each) were collected. Fractions exhibiting maximal enzyme activity (fractions 18\u201323) were pooled.<\/p>\n

Concentration and Storage of the Enzyme Preparation<\/h3>\n

BSA was added to the pooled fraction to a final concentration of 0.2 mg\/ml. The preparation was then dialyzed against 200 ml of concentration buffer (10 mM Tris-HCl, pH 7.6; 0.1 mM EDTA; 7 mM \u03b2-mercaptoethanol; 0.2 M NaCl; 50% glycerol) with gentle stirring for 16 h. The enzyme preparation was stored at \u221220 \u00b0C.<\/p>\n

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Results and Discussion<\/h2>\n

Characterization and Taxonomic Identification of the Producer Strain<\/h3>\n

The producer strain was isolated from pond water. When cultivated on LM agar at 24 \u00b0C for 3 days, it formed circular colonies up to 4 mm in diameter. The colonies were convex to conical, shiny, opaque, and pale orange in color. Cells were rod-shaped, measuring approximately 0.6 \u00d7 3\u20134 \u03bcm, motile, Gram-positive, catalase-positive, and oxidase-negative. The strain grew aerobically at temperatures ranging from 8 to 40 \u00b0C but did not grow at 55 \u00b0C. The genomic DNA G+C content of the strain, determined according to [6], was 53 \u00b1 5%.<\/p>\n

For strain identification based on 16S rRNA gene analysis, genomic DNA was isolated from the cells, and PCR amplification of the 16S rRNA gene was performed using the primers 5\u2032-AGAGTTTGATCMTGGCTCA-3\u2032 and 5\u2032-TACGGYTACCTTGTTACGACT-3\u2032, which are modified versions of primers fD1 and rP1 [7]. A 721-bp PCR fragment corresponding to a portion of the 16S rRNA gene was sequenced (Fig. 1).<\/p>\n

[\/vc_column_text][\/vc_column][\/vc_row][vc_row content_placement=”middle” css=”.vc_custom_1763458482852{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]Figure 1.<\/strong>
Nucleotide sequence of a fragment of the 16S rRNA gene of the producer strain.[\/vc_column_text][vc_row_inner][vc_column_inner][vc_raw_html]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[\/vc_raw_html][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

Based on the determined nucleotide sequence of the 16S rRNA gene fragment, the producer strain was assigned to Exiguobacterium mexicanum<\/em> of the order Bacillales<\/em>, Family XII Incertae Sedis<\/em>. Strain identification was performed using Nucleotide BLAST [8], the SILVAngs identification service [9], and a taxonomic classifier [10]. The microbiological characteristics of the strain were consistent with those of the assigned genus [11]. The strain was designated Exiguobacterium mexicanum<\/em> 6, and the restriction endonuclease isolated from this strain was named EmiI in accordance with established nomenclature.<\/p>\n

Preparation of EmiI and Characterization of Its Properties<\/h3>\n

Cultivation of E. mexicanum<\/em> 6 cells in 20 liters of medium yielded 64 g of biomass with an EmiI activity of 50,000 U\/g. Following three chromatographic purification steps, 3 ml of a restriction endonuclease EmiI preparation with a concentration of 5000 U\/ml was obtained from 17 g of biomass.<\/p>\n

Further experiments were carried out to determine the optimal reaction conditions for EmiI activity. Reaction mixtures (50 \u03bcl) contained 1 \u03bcg of bacteriophage \u03bb DNA, one of six reaction buffers for restriction endonucleases, and 1 or 2 \u03bcl of the EmiI preparation diluted tenfold in SE buffer B100. The mixtures were incubated at 37 \u00b0C for 1 h. The results are shown in Figure 2.<\/p>\n

[\/vc_column_text][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1580187576648{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]Figure 2.<\/strong>
Activity of the EmiI preparation on bacteriophage \u03bb DNA at 37 \u00b0C in different SE buffers (B, G, O, W, Y, and ROSE). Electrophoresis was performed in a 1% agarose gel.[\/vc_column_text][vc_row_inner][vc_column_inner width=”2\/3″][vc_single_image image=”503″ img_size=”large”][\/vc_column_inner][vc_column_inner width=”1\/3″][vc_column_text]Lanes:<\/strong>
1, undigested \u03bb DNA;
2, 4, 6, 8, 10, 12, \u03bb DNA + 1 \u03bcl of EmiI diluted tenfold;
3, 5, 7, 9, 11, 13, \u03bb DNA + 2 \u03bcl of EmiI diluted tenfold;
lanes 2\u20133, SE buffer B;
lanes 4\u20135, SE buffer G;
lanes 6\u20137, SE buffer O;
lanes 8\u20139, SE buffer W;
lanes 10\u201311, SE buffer Y;
lanes 12\u201313, SE buffer ROSE;
14, SE 1 kb DNA molecular weight marker.[\/vc_column_text][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]As shown in Figure 2, the enzyme exhibits maximal activity in SE buffer Y (33 mM Tris\u2013acetate, pH 7.9; 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT), as complete digestion of the substrate DNA was observed upon addition of both 1 and 2 \u03bcl of the tenfold-diluted enzyme preparation (lanes 10\u201311). In all other cases, 1 \u03bcl of the diluted EmiI preparation was insufficient for complete DNA digestion. The results of EmiI activity assays in six SE buffers at 37 \u00b0C are summarized in Table 1.[\/vc_column_text][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1580187576648{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]<\/p>\n

Table 1.<\/strong>
Activity of the restriction endonuclease preparation EmiI in six standard SE buffers for restriction endonucleases (expressed as a percentage of maximal activity).<\/p>\n

[\/vc_column_text][vc_column_text]<\/p>\n\n\n\n\n
SE buffer<\/strong><\/td>\nB<\/strong><\/td>\nG<\/strong><\/td>\nO<\/strong><\/td>\nW<\/strong><\/td>\nY<\/strong><\/td>\nROSE<\/strong><\/td>\n<\/tr>\n
EmiI activity, %<\/strong><\/td>\n75\u2013100<\/td>\n50\u201375<\/td>\n25\u201350<\/td>\n25\u201350<\/td>\n100<\/strong><\/td>\n25<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

Comparison of the DNA cleavage pattern of bacteriophage \u03bb DNA obtained using EmiI with theoretically predicted cleavage patterns based on known restriction endonuclease recognition sites led to the conclusion that EmiI is an isoschizomer of the restriction endonucleases Ksp632I [1] and Bst6I [2], which cleave this substrate at the site 5\u2032-CTCTTC-3\u2032 \/ 3\u2032-GAGAAG-5\u2032.<\/p>\n

To confirm the specificity of the novel enzyme, comparative digestion of bacteriophage \u03bb and T7 DNA with the restriction endonucleases EmiI and Bst6I was performed in 50 \u03bcl reaction mixtures containing 1 \u03bcg of DNA in SE buffer Y and 1 \u03bcl of either Bst6I or EmiI preparation. After incubation for 1 h at 65 \u00b0C (for Bst6I) or 37 \u00b0C (for EmiI), 15 \u03bcl aliquots were loaded onto a 1% agarose gel and subjected to electrophoresis. The results are shown in Figure 3.<\/p>\n

[\/vc_column_text][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1580187576648{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]Figure 3.<\/strong>
Determination of the specificity of the restriction endonuclease EmiI using bacteriophage \u03bb and T7 DNA. Reaction products were separated in a 1% agarose gel.[\/vc_column_text][vc_row_inner][vc_column_inner width=”2\/3″][vc_single_image image=”498″ img_size=”large”][\/vc_column_inner][vc_column_inner width=”1\/3″][vc_column_text]Lanes:<\/strong>
1, undigested bacteriophage \u03bb DNA;
2, \u03bb DNA + 1 \u03bcl of Bst6I preparation;
3, \u03bb DNA + 1 \u03bcl of EmiI preparation;
5, undigested bacteriophage T7 DNA;
6, T7 DNA + 1 \u03bcl of Bst6I preparation;
7, T7 DNA + 1 \u03bcl of EmiI preparation;
4, SE 1 kb DNA molecular weight marker.[\/vc_column_text][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

As shown in the figure, the DNA cleavage patterns produced by the restriction endonucleases Bst6I and EmiI for both DNA substrates are identical, confirming that the two enzymes share the same cleavage specificity.<\/p>\n

To determine the optimal temperature for EmiI activity, 50 \u03bcl reaction mixtures containing 1 \u03bcg of bacteriophage \u03bb DNA in SE buffer Y and either 1 \u03bcl of the undiluted enzyme preparation or 1 \u03bcl of the preparation diluted tenfold were incubated for 1 h at 25, 30, 37, or 55 \u00b0C. The results are presented in Figure 4.<\/p>\n

[\/vc_column_text][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1580187576648{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]Figure 4.<\/strong>
Activity of the EmiI preparation on bacteriophage \u03bb DNA in SE buffer Y at different temperatures. Electrophoresis was performed in a 1% agarose gel.[\/vc_column_text][vc_row_inner][vc_column_inner width=”2\/3″][vc_single_image image=”499″ img_size=”large”][\/vc_column_inner][vc_column_inner width=”1\/3″][vc_column_text]Lanes:<\/strong>
1, undigested bacteriophage \u03bb DNA;
2 and 3, \u03bb DNA + 1 \u03bcl of undiluted or 1 \u03bcl of tenfold-diluted EmiI, 25 \u00b0C;
4 and 5, \u03bb DNA + 1 \u03bcl of undiluted or 1 \u03bcl of tenfold-diluted EmiI, 30 \u00b0C;
6 and 7, \u03bb DNA + 1 \u03bcl of undiluted or 1 \u03bcl of tenfold-diluted EmiI, 37 \u00b0C;
8 and 9, \u03bb DNA + 1 \u03bcl of undiluted or 1 \u03bcl of tenfold-diluted EmiI, 55 \u00b0C;
10, SE 1 kb DNA molecular weight marker.[\/vc_column_text][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

As shown in Figure 4, complete digestion of the substrate DNA by 1 \u03bcl of the undiluted EmiI preparation was observed at all tested temperatures (lanes 2, 4, 6, and 8). In contrast, when the enzyme was diluted tenfold, exhaustive digestion of the substrate was observed only at 37 \u00b0C (lane 7). Therefore, this temperature was determined to be optimal for EmiI restriction activity. Complete inactivation of EmiI was observed after heating the reaction mixture to 80 \u00b0C for 20 min (data not shown).<\/p>\n

Based on these results, the activity of the EmiI preparation was determined. Figure 5 shows an electropherogram illustrating analysis of the target enzyme activity at the optimal temperature (37 \u00b0C) in the optimal SE buffer Y.<\/p>\n

[\/vc_column_text][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1580187576648{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]Figure 5.<\/strong>
Determination of EmiI activity under optimal conditions. Reaction mixtures (50 \u03bcl) contained 1 \u03bcg of bacteriophage \u03bb DNA in SE buffer Y and either 1 \u03bcl of undiluted EmiI (control) or 0.5, 1, or 2 \u03bcl of EmiI diluted tenfold in buffer B100. The mixtures were incubated for 1 h at 37 \u00b0C and analyzed by electrophoresis in a 1% agarose gel.[\/vc_column_text][vc_row_inner][vc_column_inner width=”2\/3″][vc_single_image image=”500″ img_size=”large”][\/vc_column_inner][vc_column_inner width=”1\/3″][vc_column_text]Lanes:<\/strong>
1, undigested bacteriophage \u03bb DNA;
2, \u03bb DNA + 0.5 \u03bcl of EmiI diluted tenfold;
3, \u03bb DNA + 1 \u03bcl of EmiI diluted tenfold;
4, \u03bb DNA + 2 \u03bcl of EmiI diluted tenfold;
5, \u03bb DNA + 1 \u03bcl of undiluted EmiI;
6, SE 1 kb DNA molecular weight marker.[\/vc_column_text][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

As shown in Figure 5, exhaustive digestion of bacteriophage \u03bb DNA by the restriction endonuclease EmiI was observed upon addition of either 2 \u03bcl of the enzyme preparation diluted tenfold (lane 4) or 1 \u03bcl of the undiluted enzyme preparation (lane 5). Thus, the activity of the EmiI preparation was determined to be 5 U\/\u03bcl (5000 U\/ml). One unit of EmiI restriction activity was defined as the minimum amount of enzyme required for complete digestion of 1 \u03bcg of bacteriophage \u03bb DNA in a 50 \u03bcl reaction mixture within 1 h in SE buffer Y at 37 \u00b0C.<\/p>\n

To confirm the recognition site of EmiI and to determine the position of DNA cleavage by the new enzyme, two 5\u2032-end radiolabeled oligonucleotide duplexes, Emi1*\/Emi2 and Emi2*\/Emi1 (the labeled strand is indicated by an asterisk), were used. These duplexes were formed from two oligonucleotides containing the EmiI recognition site (underlined):<\/p>\n

Emi1: 5\u2032-CCTTTGACGCTCTTCGGCGCCATGATGCAACC-3\u2032
Emi2: 5\u2032-GGTTGCATCATGGCGCCGAAGAGCGTCAAAGG-3\u2032<\/pre>\n
The duplexes were digested with the novel enzyme EmiI and, separately, with control restriction endonucleases Bst6I (5\u2032-CTCTTC(1\/4)-3\u2032), HgaI (5\u2032-GACGC(5\/10)-3\u2032), HspAI (5\u2032-G\u2193CGC-3\u2032), FaeI (5\u2032-CATG\u2193-3\u2032), and SfaNI (5\u2032-GCATC(5\/9)-3\u2032). Reactions were carried out using 1 pmol of the Emi1*\/Emi2 and Emi2*\/Emi1 duplexes in a 10 \u03bcl reaction volume for 1 h at 37 \u00b0C (or at 55 \u00b0C for Bst6I) in SE buffer Y. The cleavage products were separated by electrophoresis in a 20% polyacrylamide gel containing 8 M urea using Tris\u2013borate buffer. An autoradiograph of the gel after electrophoresis is shown in Figure 6A.<\/p>\n
[\/vc_column_text][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1580187576648{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]Figure 6A.<\/strong>
Determination of the EmiI cleavage position. Electrophoresis was performed in a 20% polyacrylamide gel containing 8 M urea.[\/vc_column_text][vc_row_inner][vc_column_inner width=”2\/3″][vc_single_image image=”509″ img_size=”large”][\/vc_column_inner][vc_column_inner width=”1\/3″][vc_column_text]Lanes:<\/strong>
1, untreated Emi1*\/Emi2 duplex;
2, Emi1*\/Emi2 duplex + 1 \u03bcl of HgaI;
3, Emi1*\/Emi2 duplex + 1 \u03bcl of EmiI;
4, Emi1*\/Emi2 duplex + 1 \u03bcl of Bst6I;
5, Emi1*\/Emi2 duplex + 1 \u03bcl of HspAI;
6, untreated Emi2*\/Emi1 duplex;
7, Emi2*\/Emi1 duplex + 1 \u03bcl of FaeI;
8, Emi2*\/Emi1 duplex + 1 \u03bcl of EmiI;
9, Emi2*\/Emi1 duplex + 1 \u03bcl of Bst6I;
10, Emi2*\/Emi1 duplex + 1 \u03bcl of SfaNI.[\/vc_column_text][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Figure 6B shows the structure of the Emi1\/Emi2 duplex with the recognition sites and cleavage positions of the restriction endonucleases used for analysis indicated. For clarity, the recognition sites and arrows denoting cleavage positions for each restriction endonuclease are highlighted in different colors. For each strand of the duplex, the enzymes were selected such that their cleavage positions either coincided with the predicted EmiI cleavage position (as in the case of Bst6I) or differed by one nucleotide from the 5\u2032 end (HgaI for the upper strand and FaeI for the lower strand) or by one nucleotide from the 3\u2032 end (HspAI for the upper strand and SfaNI for the lower strand).[\/vc_column_text][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1580187576648{background-color: #f4f4f4 !important;}”][vc_column][vc_column_text]Figure 6B.<\/strong>
Schematic representation of the Emi1\/Emi2 oligonucleotide duplex indicating the recognition sites and cleavage positions of the control restriction endonucleases.[\/vc_column_text][vc_row_inner][vc_column_inner][vc_single_image image=”502″ img_size=”large”][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n
The data presented in Figure 6A indicate that the EmiI cleavage position coincides with that of Bst6I (5\u2032-CTCTTCN\u2193-3\u2032 \/ 3\u2032-GAGAAGNNNN\u2193-5\u2032) and differs by one nucleotide in either direction from the cleavage positions of the control enzymes.<\/p>\n
Thus, the restriction endonuclease EmiI from Exiguobacterium mexicanum<\/em> 6 recognizes the non-palindromic hexanucleotide DNA sequence 5\u2032-CTCTTC-3\u2032 and cleaves DNA outside the recognition site at positions 5\u2032-CTCTTC(N)\u2081\u2193-3\u2032 \/ 3\u2032-GAGAAG(N)\u2084\u2193-5\u2032, generating three-nucleotide 5\u2032-protruding ends, similarly to the previously described restriction endonucleases Ksp632I and Bst6I. The novel enzyme may be applied in research in the fields of genetic engineering, molecular biology, biotechnology, and DNA diagnostics.<\/p>\n
[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n
References<\/h2>\n
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      Ksp632I, a novel class IIS restriction endonuclease from Kluyvera<\/em> sp. strain 632 with the asymmetric hexanucleotide recognition sequence 5\u2032-CTCTTC(N)\u2081-3\u2032 \/ 3\u2032-GAGAAG(N)\u2084-5\u2032.
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      [\/vc_column_text][\/vc_column][\/vc_row]<\/p>","protected":false},"excerpt":{"rendered":"
      A bacterial strain, Exiguobacterium mexicanum 6, was identified as a producer of a novel restriction endonuclease designated EmiI. The enzyme recognizes the non-palindromic hexanucleotide DNA sequence 5\u2032-CTCTTC-3\u2032 and cleaves DNA outside the recognition site at positions 1\/4, generating three-nucleotide 5\u2032-protruding ends: 5\u2032-CTCTTC(N)\u2081\u2193-3\u2032 \/ 3\u2032-GAGAAG(N)\u2084\u2193-5\u2032.
      \nThus, EmiI is an isoschizomer of the restriction endonucleases Ksp632I [1] and Bst6I [2]. The producer strain was identified based on morphological and biochemical characteristics as well as analysis of the primary structure of a fragment of the 16S rRNA gene. A preparation of restriction endonuclease EmiI with an activity of 5000 U\/ml was obtained through purification using three chromatographic steps. Optimal reaction conditions for EmiI include SE buffer Y (33 mM Tris\u2013acetate, pH 7.9; 10 mM Mg\u2013acetate; 66 mM potassium acetate; 1 mM DTT) at a temperature of 37 \u00b0C.<\/p>\n","protected":false},"author":4,"featured_media":521,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[5,45],"tags":[51],"coauthors":[10,9,8,12,13,52,16],"class_list":["post-496","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-new-enzymes","category-45","tag-emii"],"_links":{"self":[{"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/posts\/496","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/comments?post=496"}],"version-history":[{"count":17,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/posts\/496\/revisions"}],"predecessor-version":[{"id":524,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/posts\/496\/revisions\/524"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/media\/521"}],"wp:attachment":[{"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/media?parent=496"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/categories?post=496"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/tags?post=496"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/dnape.online\/en\/wp-json\/wp\/v2\/coauthors?post=496"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}