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Ann Microbiol (2012) 62:129–137 DOI 10.1007/s13213-011-0237-2 ORIGINAL ARTICLE Cloning and heterologous expression of the manganese superoxide dismutase gene from Lactobacillus casei Lc18 Qiulin Liu & Xiaomin Hang & Xianglong Liu & Jing Tan & Daotang Li & Hong Yang Received: 26 October 2010 /Accepted: 15 February 2011 /Published online: 3 March 2011 Springer-Verlag and the University of Milan 2011 Abstract Many of the species of Lactobacillus can be chromatography and found to consist of a single band, as considered to be “probiotics” with a variety of benefits, determined by SDS-PAGE analysis, with an activity of including imparting antioxidative effects to the host. 39.97 U/mg. N-terminal amino acid sequence analysis Lactobacillus species evolve different mechanisms to indicated that it may be a manganese-containing SOD. defend themselves against oxygen toxicity, such as super- This is the first report of a sod gene from Lactobacillus spp. oxide dismutases (SODs), hydroperoxidases and high being expressed in other prokaryotes. intracellular levels of metal ions. The SODs provide a cellular defense mechanism against oxidative stress by Keywords Lactobacillus casei Lc18 Antioxidative . . scavenging O . Most Lactobacillus species appear to lack activities Manganese superoxide dismutase Heterologous a sod gene. To date, only two species of Lactobacillus expression (Lactobacillus casei and L. sakei) may have sod genes, as evidenced by sequence analysis of the genome, but no experimental verification, including cloning and heterolo- Introduction gous expression of the Lactobacillus sod gene, has been reported. It is therefore unknown whether these sod genes Probiotics are living microorganisms that impart health can express SOD or are functional. We have PCR-amplified benefits to the host beyond inherent basic nutrition upon the gene from L. casei Lc18 that encodes the SOD using ingestion in certain numbers (Guarner and Schaafsma primers designed according to the genome of L. casei 1998). Lactobacilli are the most widely used probiotics ATCC 334 and ligated it into the vector pET-28a(+) for and are considered to be key commensals involved in the heterologous expression in Escherichia coli BL21(DE3). promotion of the host’s health. Various studies have After being induced with IPTG, the fusion protein was indicated that lactobacilli may alleviate lactose intolerance, efficiently expressed in a soluble form. The superoxide have a positive influence on the intestinal flora of the host, radical scavenging activity of the recombinant strain was stimulate or modulate mucosal immunity, reduce blood found to be increased relative to that of the control strain. cholesterol and impart antioxidative effects to the host The SOD was also purified by nickel ion affinity (Vizoso Pinto et al. 2006; Wang et al. 2009). Among these potential health-promoting benefits, the antioxidative effect : : : : Q. Liu X. Liu J. Tan D. Li H. Yang (*) is one of the most interesting properties. Key Laboratory of MOE for Microbial Metabolism and School Oxidative stress is the adverse effect of oxidants on of Life Science and Biotechnology, Shanghai Jiao Tong University, physiological function. Oxidative stress, which has been No. 800 Dongchuan Rd., implicated in the progression of aging and disease, occurs Shanghai 200240, People’s Republic of China e-mail: hongyang@sjtu.edu.cn when abnormally high levels of reactive oxygen species (ROS) are generated, resulting in DNA, protein and lipid X. Hang damage (Kullisaar et al. 2002). During the past decade, Institute of Bio-medicine, oxidative stress and antioxidative potency have been Shanghai Jiao Da Onlly Company Limited, identified as key points in the molecular regulation of Shanghai 200233, People’s Republic of China 130 Ann Microbiol (2012) 62:129–137 cellular stress responses. Free radicals, such as the three-dimensional structures, molecular weights of approx- superoxide radical anion (O ) and hydroxyl radical imately 40 kDa (2×20 kDa) and similar metal-binding (OH ), and other ROS are considered to be highly potent ligands in the active site of the enzyme (Meile et al. 1995). oxidants that can react with all biomacromolecules in living MnSODs are cambialistic (functionally active with either cells and are associated with carcinogenesis and mutagenesis metal) SODs of anaerobic microorganisms (Brioukhanov and (Kodali and Sen 2008). The radicals may cause oxidative Netrusov 2004); they were first isolated from Escherichia damage by oxidizing biomolecules, thereby resulting in cell coli and found to be unlike the corresponding enzymes of death and tissue injury. eukaryotes with respect to all parameters except their Various synthetic and natural antioxidants have been catalytic activity (Keele et al. 1970). reported (Brioukhanov and Netrusov 2004; Sánchez-Venegas Until recently, most lactobacilli were believed to lack et al. 2009); however, the safety and long-term effects of SODs (Bruno-Bárcena et al. 2004); however, Lactobacillus synthetic antioxidants on health are uncertain. Accordingly, plantarum and several other aerotolerant lactobacilli have antioxidants from natural sources are both more desirable been identified as possessing a nonenzymatic dismutation (Lin and Yen 1999) and more valuable. Among natural system based on the accumulation of high intracellular 2+ − antioxidants, live microorganisms have the highest applica- levels of Mn , which can stoichiometrically remove O bility. Lactobacillus spp., which are generally recognized as (Archibald and Fridovich 1981). Analyses of genome safe microorganisms with respect to human health (Adams sequences indicate that L. casei and L. sakei likely contain and Marteau 1995), are commonly used in the fermentation the sod gene, but the presence of these genes and the of foods, including dairy, meat, vegetable and bakery expression and activity of SOD have not yet been products. Some of them are reported to have excellent demonstrated by experimental methods, such as cloning antioxidative activities in the host, where they are able to or heterologous expression. decrease the risk of an accumulation of ROS during the In this study, the sod gene from Lactobacillus casei Lc18 ingestion of food, while others apparently lack these was heterologously expressed in E. coli BL21(DE3). The activities (Korpela et al. 1997). fusion protein (SOD enzyme) was then purified and its Some protection against ROS in aerobes and facultative antioxidative activity detected. anaerobes (such as Lactobacillus) is provided by antioxidative defense enzymes, one of the most important of which is superoxide dismutase (SOD) (Brioukhanov and Netrusov Materials and methods 2004). The SODs (EC 1.15.1.1), which provide a cellular defense mechanism against the toxicity of oxygen by Bacterial strains and plasmids catalytically scavenging O (Archibald and Fridovich 1981), are metalloenzymes that catalyze the conversion of The bacterial strains, plasmids and primers used in this the superoxide anion into hydrogen peroxide and dioxygen. study are listed in Table 1. Escherichia coli DH5α was This significant antioxidative activity is the basis for the used for gene cloning and E. coli BL21(DE3) was used as a increased resistance of some Lactobacillus strains to toxic host for the expression of the recombinant proteins. L casei oxidative compounds, allowing them to survive in the host, Lc18 and Lc2 and L. acidophilus LA11 and LA12 were and to serve as defensive components in the intestinal provided by Shanghai Jiao Da Onlly Co. (Shanghai, PR microbial ecosystem (Kullisaar et al. 2002). China. The L. casei and L. acidophilus strains were The SOD enzymes can be distinguished into three types cultivated in De Man, Rogosa and Sharpe (MRS) medium based on the composition of their metal center: manganese, without shaking at 37°C for 18 h to reach the stationary phase. copper–zinc or iron. SODs are found across a broad range The E. coli strains were cultivated in Luria–Bertani (LB) of organisms that utilize one, two, or all three types to meet broth with shaking (200 rpm) at 37°C for 12 h to reach the their antioxidant needs (Hassan 1997). The majority of stationary phase. E. coli transformants were selected with SODs comprise two identical subunits, each containing a 100 μg/ml of ampicillin or 50 μg/ml of kanamycin. As soon metal ion, a disulfide bridge, a sulfhydryl group and an as the OD of recombinant E. coli BLSod reached 0.6, acetylated terminal amino group. The molecular weights of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to Cu,ZnSODs are approximately 32 kDa, and they are 1 mM to induce protein expression. homodimers. Prokaryotes were initially believed to possess FeSODs and/or MnSODs which, unlike the Cu,ZnSODs of Chemicals and enzymes eukaryotes, are insensitive to the cyanide radical (CN ). However, Cu,ZnSODs have since also been identified in Restriction enzymes (NdeI and BamHI), Thermus aquaticus prokaryotes, and these are also inhibited by CN . FeSODs DNA Polymerase, LA Taq polymerase and T4 DNA ligase and MnSODs are highly homologous and have similar were purchased from TaKaRa (Shiga, Japan). All chemicals Ann Microbiol (2012) 62:129–137 131 Table 1 Bacterial strains, plasmids and primers used in this study Strain, plasmid or primer Characteristic(s) Reference or source Strains Escherichia coli DH5α rec cloning strain TaKaRa (Shiga, Japan) E. coli BL21(DE3) Protein expression strain Novagen (Madison, WI) E. coli BLSod Recombinant BL21(DE3) harboring pETsod This study Lactobacillus casei Lc18 This study L. casei Lc2 This study Plasmids pMD19-T TA-cloning TaKaRa pET-28a(+) Expression vector, T7 promoter, His-tag Novagen pETsod 0.69-kb sod PCR amplicon from L. casei Lc18 cloned into pET-28a(+) This study Primers SS (NdeI restriction site underlined) 5’-CAAACAAGAAAGGTTGATTCATATG-3’ This study SR (BamHI restriction site underlined) 5’-AAATTTACGGATCCTTTTTCGG-3’ This study M13-47 5’-CGCCAGGGTTTTCCCAGTCACGAC-3’ TaKaRa RV-M 5’-GAGCGGATAACAATTTCACACAGG-3’ TaKaRa and antibiotics used were purchased from Sigma (St. Louis, PCR product, which was 700 bp, as expected, was purified MO) unless otherwise specified. by using the Agarose Gel DNA Purification kit ver.2.0 (TaKaRa), ligated into the vector pMD19-T and used to Construction of the expression vector transform competent cells of E. coli DH5α with heat shock according to the manufacturer’s instructions. Transformants Lactobacillus genomic DNA was isolated according to were identified using primers M13-47 and RV-M. The Sambrook et al. (2001) with some modifications. Briefly, nucleotide sequences were determined by AmlipTaq FS 50 μl of proteinase K was added during the lysing step, DNA polymerase fluorescent dye terminator reactions by after which the mixture was incubated for 30 min at 37°C, means of an Applied Biosystems 3730 automated sequencer followed by the addition of 600 μl of phenol/chloroform/ (Foster City, CA). isoamyl alcohol (25:24:1). The extracted DNA was electro- The purified PCR products were digested with NdeI and phoresed on a 1% agarose gel, quantified spectrophotomet- BamHI and ligated to the pET-28a(+) expression vector, rically at 260 nm and diluted to 50 ng/μl for PCR. yielding the pETsod plasmid (Fig. 1). Restriction enzymes, The coding sequence of SOD was subjected to PCR T4 ligase and other DNA-modifying enzymes were used amplification from the chromosomal DNA template of L. according to the protocols of the manufacturers. The casei Lc18 using a forward primer containing a NdeI recombinant plasmid pETsod was introduced into competent restriction site SS and a reverse primer containing a BamHI E. coli BL21(DE3) according to Tu et al. (2005). Three restriction site SR. These two primers were derived from positive transfomants were grown in liquid LB medium the whole-genome sequences of L. casei ATCC 334 using containing 50 μg/ml kanamycin and identified through PCR Primer Premier software, ver. 5.0 (PREMIER Biosoft Int, and restriction enzyme digestion. Palo Alto, CA ). The PCR reactions were carried out in a 50-μl volume Preparation of dialyzed cell-free extracts containing 100 ng DNA template solution, 5 μl of 10× LA 2+ PCR Buffer II (Mg plus), 2.5 U of LA Taq Polymerase, Fresh single colony-forming units (CFUs) of each test 10 nmol of each deoxyribonucleoside triphosphate and 10 strain were inoculated into 10 ml of proper culture medium. pmol of each primer. The PCR reaction was performed After incubation at 37°C, total cell numbers were adjusted under following conditions: an initial denaturation step at to 10 CFU/ml, then cells of each strain were harvested by 94°C for 5 min followed by 30 cycles of denaturation at centrifugation at 5000 g for 10 min. Cell pellets were then 94°C for 1 min, annealing at 58°C for 40 s, and extension at quickly washed twice and resuspended in deionized water 72°C for 40 s, with a final elongation step at 72°C for followed by ultrasonic disruption (5 min, 200 W, 50% 10 min. All PCRs were carried out with Eppendorf working time) on a Sonicator JY96-II (Scientz Biotechnology, Mastercycler ep gradient S (Hamburg, Germany). The Ningbo, China) with constant cooling on ice, The supernatant 132 Ann Microbiol (2012) 62:129–137 Measurement of hydrogen peroxide scavenging activity Hydrogen peroxide scavenging activity was measured accordingtoPickand Keisari(1980) with minor modification. Briefly, 50 μlof5 mM H O solution and 50 μlof fresh 2 2 sample or distilled water (control) were mixed and incubated at room temperature for 20 min. Then, 100 μl of horseradish peroxidase (HRP)–phenol red solution (300 μg/ml HRP and 4.5 mM phenol red in 100 mM phosphate buffer) was added and after 10 min of incubation, the sample absorbance was monitored at 610 nm. The scavenging effect was calculated according to the following equation: Hydrogen peroxide scavenging activityðÞ % ¼ 1 A = A 100: Sample Control Expression, purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant protein A single colony of recombinant E. coli BLSod was inoculated in 2 ml LB medium containing 50 μg/ml kanamycin and cultivated at 37°C at 200 rpm. The overnight culture was Fig. 1 Construction of the pETsod plasmid. BamHI, NdeI Restriction diluted 100-fold into 50 ml of fresh LB medium containing enzymes, sod superoxide dismutase, kan kanamycin, r resistance 50 μg/ml kanamycin, grown to an OD of about 0.6 and then induced with 1 mM isopropylthiogalactoside (IPTG). (cell-free extract, CFE) was recovered and used immediately The culture was subsequently incubated to express the fusion in subsequent experiments. protein. After 9 h, wet cells were collected from 50 mL of bacteria culture by centrifugation and resuspended in 5 ml of Antioxidant activity assays Native Binding Buffer (50 mM NaH PO ,0.5M NaCl, pH 2 4 8.0); 8 mg lysozyme was then added and the mixture Measurement of superoxide radical scavenging activity incubated on ice for 30 min and subjected to ultrasonication. In order to determine whether the recombinant SOD protein Superoxide radical scavenging activity was measured by was expressed, or not, and if expressed whether it is in the the method of Nishikimi et al. (1972). Briefly, 1 ml of form of inclusion bodies, the bacteria cells were sonicated and nitroblue tetrazolium (NBT) solution (156 μMin 100 mM separated into soluble and insoluble fractions by centrifuga- phosphate buffer, pH 7.4), 1 ml of coenzyme I reduced tion at 10,000 g at 4°C for 10 min. Both the soluble and disodium salt (NADH) solution (468 μM in 100 mM insoluble fractions were analyzed by 12% sodium dodecyl phosphate buffer, pH 7.4) and 0.1 ml of fresh sample or sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) distilled water (control) were mixed. The reaction was according to the protocol of the manufacturer (Bio-Rad, startedbyadding100 μl of phenazine methyl sulfate Hercules, CA). The ultrasonicated supernatant (soluble frac- (PMS) solution (60 μM PMS in 100 mM phosphate buffer, tion) was then subjected to His-tag purification and purified pH 7.4) to the mixture. The mixture was then incubated at by the Ni-NTA Purification system (Invitrogen, Carlsbad, CA) 25°C for 5 min and the absorbance measured at 560 nm. according to the manufacturer’s instructions. Both the crude Decreased absorbance of the reaction mixture indicated and purified protein fractions were boiled for 5 min and increased superoxide anion scavenging activity. The analyzed by SDS–PAGE. Proteins were visualized by scavenging activity of superoxide radical was calculated Coomassie brilliant blue staining. as follows: Measurement of purified SOD activity Superoxide radical scavenging activityðÞ % Enzyme activity was measured as described above. One ¼ 1 A =A 100: Sample Control unit of SOD activity was defined as the amount of purified Ann Microbiol (2012) 62:129–137 133 enzyme that consumed 1 μmol of PMS from the substrate coli BL21(DE3) to form the recombinant strain E. coli per minute. The specific activity was defined as units of BLSod. activity per milligram of purified protein (purified SOD). The protein concentrations were determined by the Expression and purification of the recombinant protein Lowry method (Liong and Shah 2005) using bovine serum as the standard. Using the T7 promoter and the N-terminal His-tag of pETsod in recombinant E. coli BLSod, the expression of Amino acids sequence analysis the fusion protein was induced by incubation in the presence of 1 mM IPTG at 37°C for 9 h when the OD The deduced amino acid sequence of the sod gene was reached 0.6. SDS–PAGE analysis of the soluble and analyzed using BioEdit software ver. 7.0.1. insoluble fractions revealed that most of the induced protein was present in the soluble fraction. SDS–PAGE also Nucleotide sequence accession number revealed a distinct band of the expected size (about 24 kDa) in the recombinant E. coli BLSod lane following The sod gene nucleotide sequences and the 16S rDNA gene induction with 1 mM IPTG (Fig. 3, lane 4) and the absence of L. casei Lc18 and Lc2 has been deposited in the of this band in the controls, namely, BL21(DE3) (Fig. 3, GenBank database under accession numbers HM070825, lane 1), BL21(DE3) harboring pET-28a(+) (Fig. 3, lane 2) HM070024, HM070025. and BLSod without induction (Fig. 3, lane 3). These findings indicate that the SOD protein may be expressed normally in E. coli BLSod with the induction of IPTG. We Results then used the Ni-NTA purification system to confirm these results. Following nickel ion metal affinity chromatography, Construction and Identification of recombinants the final eluate (the purified target protein) produced a single distinct band (Fig. 3, lane 7) that had the same size as The 700-bp PCR-fragment cloned from L. casei Lc18 was the expected SOD (about 24 kDa) in lane 4 of Fig. 3; there identified as the sod gene by a BLAST search of the was no similar band in the other two controls, BL21(DE3) GenBank database. The cloned sod gene contained an 618-bp (Fig. 3, lane 5) and BL21(DE3) harboring pET-28a(+) open reading frame (ORF) that encoded a protein of 205 (Fig. 3, lane 6). An enzyme activity test of the purified amino acids with a predicted molecular weight of 23.3 kDa. SOD revealed that the enzyme activity of O scavenging The recombinant plasmid pETsod was demonstrated to be was 39.97 U/mg. An additional band of about 27 kDa was correct by restriction enzyme digestion (Fig. 2) and DNA visualized in both lanes 4 and 7, which was interpreted as sequencing (nucleotide sequence accession number an endogenous protein of BL21(DE3) containing several HM070825). Figure 2 shows that the recombinant plasmid seriate histidines that was induced by IPTG. pETsod was successfully constructed and transformed into E. Antioxidative activity of the tested strains Table 2 shows antioxidative activities, including superoxide radical scavenging activity and hydrogen peroxide scavenging activity, from both intact cells and CFEs of E. coli BL21 (DE3), E. coli BLSod, L. casei Lc18, L. casei Lc2, L. acidophilus LA11 and L. acidophilus LA12. The results suggested that both the intact cells and CFEs of all six strains had some antioxidative ability and that the abilities of the CFEs were generally higher than those of intact cells. The antioxidative abilities of both L. acidophilus are quite low compared to the other four strains. The superoxide radical scavenging activity of E. coli increased from 37.17 to 43.24% (intact cells) and from 44.31 to 55.20% (CFEs) after recombination with the sod gene; the hydrogen peroxide scavenging activity also Fig. 2 Identification of recombinant plasmid pETsod by restriction increased, from 52.08 to 55.37% (intact cells) and from enzyme digestion. Lanes: 1 5-kb DNA ladder, 2 digestion of pETsod. 51.04 to 58.15% (CFEs) after recombination with sod Arrows Two digestion products; upper band digested plasmid vector, lower band sod gene gene. 134 Ann Microbiol (2012) 62:129–137 Fig. 3 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis harboring pETsod] without induction of IPTG, 4 total protein of analysis of SOD heterologous expression in Escherichia coli BLSod. recombinant strain BLSod [E. coli BL21(DE3) harboring pETsod] Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the with induction of IPTG, 5 purified protein of control strain [E. coli growth medium at a concentration of 1 mM for SOD induction, and BL21(DE3)] by Ni-NTA purification system, 6 purified protein of the culture was then incubated at 37°C for 9 h prior to the analysis of control strain [E. coli BL21(DE3) harboring pET-28a(+)] by Ni-NTA cellular proteins. Lanes: M pre-stained protein molecular weight purification system, 7 purified recombinant SOD of strain BLSod with marker, 1 total protein of control strain [E. coli BL21(DE3)], 2 total IPTG induction by Ni-NTA purification system. Arrows Target SOD protein of control strain [E. coli BL21(DE3) harboring pET-28a(+)], proteins 3 total protein of recombinant strain BLSod [E. coli BL21(DE3) Sequence analysis and comparison gene of L. casei Lc18 is quite different from that of many other species, including humans, animals, plants and other The results of the antioxidant activity assays and recombinant bacteria. However, a homology comparison of the sequence protein analysis demonstrated that the sod gene was amplified of the L. casei Lc18 sod gene with other SOD protein successfully from the genome DNA of L. casei Lc18. A sequences revealed that the SOD protein sequence deduced BLAST search of the GenBank database revealed that the sod from the sod gene of L. casei Lc18 is more similar to the three Table 2 Antioxidative activities Strain O scavenging activity (%) H O scavenging activity (%) 2 2 2 of the tested strains Intact cells BL21(DE3) 37.17±0.47 52.08±1.73 BLSod 43.24±2.29 55.37±3.71 L. casei Lc18 49.37±2.58 17.40±1.37 L. casei Lc2 69.95±3.27 43.59±2.11 L. acidophilus LA11 10.42±2.86 5.48±1.50 L. acidophilus LA12 5.48±2.74 9.07±1.27 Cell-free extracts BL21(DE3) 44.31±1.09 51.04±2.94 BLSod 55.20±3.11 58.15±4.70 L. casei Lc18 58.91±2.63 21.74±3.37 L. casei Lc2 75.21±3.41 47.37±1.88 L. acidophilus LA11 7.14±1.24 3.44±0.29 Data are given as the mean ± standard deviation of three L. acidophilus LA12 9.00±2.01 6.74±0.98 independent experiments. Ann Microbiol (2012) 62:129–137 135 MnSOD protein sequences from Streptococcus thermophilus by catalytically scavenging O . During the past 20 years, (Bruno-Bárcena et al. 2004), L. lactis (Sanders et al. 1995)and many sod genes have been cloned from various organisms, E. coli (Roy et al. 1993)(Fig. 4)thantothetwoFeSODsfrom including E. coli (Roy et al. 1993), L. lactis (Sanders et al. Pseudomonas ovalis (Isobe et al. 1987)and Photobacterium 1995), P. ovalis (Isobe et al. 1987), S. thermophilus leiogathi (Barra et al. 1987). The iron- and manganese- (Bruno-Bárcena et al. 2004), Trichoderma harzianum containing SODs can be distinguished by analysis of their (Yang et al. 2009), Deschampsia antarctica (Sánchez- primary structures (Parker and Blake 1988). Thesizeofthe L. Venegas et al. 2009), and humans (Tibell et al. 1987). For casei Lc18 SOD (205 amino acid residues) is within the range some widely used probiotics, such as L. acidophilus, that of thesizeofSODs from thefiveaforementionedbacteria have no SOD and low antioxidative activities, heterologous (194–206 residues) (Fig. 4). The amino acids involved in the expression of the sod gene from L. casei or L. sakei is of binding of metal ligands are present in the L. casei Lc18 SOD critical importance in the construction of genetic engineering (His-27, His-82, Asp-174 and His-178; Fig. 4), as are the of Lactobacillus with higher SOD activity. The functional postulated active-site residues (Parker et al. 1987;Parkerand gene from L. casei is more suitable for expression in other Blake 1988). The L. casei Lc18 SOD contains the typical Lactobacillus because L. casei is a Gram-positive non-spore- residues (Gly-76, Gly-77, Phe-85, Gln-153, Asp-154; Fig. 4) forming organism belonging to the same group that has a of MnSODs and none of the typical residues (Gla-76, Gln-77, low GC content; as such the L. casei sod gene is close to that Thr-85, Gla-153, Gly-154; Fig. 4)ofFeSODs(Parkerand of other strains in the Lactobacillus group. In this study, the Blake 1988), which indicates that the L. casei Lc18 SOD sod gene from Lactobacillus was heterologously expressed belongs to the MnSODs. in another prokaryote for the first time. The antioxidative effects of every bacterial strain tested in this study were examined in intact cells and in Discussion intracellular extracts (CFEs). The results shown in Table 2 show that all of the tested bacterial strains had some The SODs are very important because they provide a antioxidative activity and that the antioxidative effects of cellular defense mechanism against the toxicity of oxygen disrupted cells were higher than those of intact cells. A Fig. 4 Aligned amino acid sequences of manganese and iron SODs. Invariant residues are boxed 136 Ann Microbiol (2012) 62:129–137 Brioukhanov AL, Netrusov AI (2004) Catalase and superoxide similar finding was previously reported by Saide and dismutase: distribution, properties, and physiological role in cells Gilliland (2005) who found that intracellular extracts of of strict anaerobes. Biochemistry 69(9):949–962 lactobacilli possessed higher antioxidative activity than Bruno-Bárcena JM, Andrus JM, Libby SL, Klaenhammer TR, Hassan intact cells. The higher antioxidative activity of the HM (2004) Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection intracellular extracts could be due to the better accessibility against hydrogen peroxide toxicity. Appl Environ Microbiol 70 of antioxidative enzymes or metal ions to the oxidant (8):4702–4710 substrates. Guarner F, Schaafsma GJ (1998) Probiotics. Int J Food Microbiol 39 Although the antioxidative activities of L. casei Lc2 (3):237–238 Hassan HM (1997) Cytotoxicity of oxyradicals and the evolution of were higher than those of L. casei Lc18, we failed to superoxide dismutases. In: Massaro D, Clerch L (eds) Oxygen, amplify the sod gene from L. casei Lc2 using the same gene expression and cellular function. Marcel Dekker, New York, PCR conditions employed for L. casei Lc18. The PCR pp 25–52 primers were designed based on the genome of L. casei Isobe T, Fang YI, Muno D, Okuyama T, Ohmori D, Yamakura F (1987) Amino acid sequence of iron-superoxide dismutase from ATCC 334. Therefore, the inability to amplify the sod gene Pseudomonas ovalis. FEBS Lett 223(1):92–96 from L. casei Lc2 may indicate that this strain was Keele BB Jr, McCord JM, Fridovich I (1970) Superoxide dismutase genetically altered during its evolution or did not contain from Escherichia coli B. A new manganese-containing enzyme. J a sod gene at all. However, this strain still displayed high Biol Chem 245(22):6176–6181 Kodali VP, Sen R (2008) Antioxidant and free radical scavenging superoxide radical scavenging activity and hydrogen activities of an exopolysaccharide from a probiotic bacterium. peroxide scavenging activity, which might have been due Biotechnol J 3(2):245–251 2+ to the high concentration of Mn in its cells or other Korpela R, Peuhkuri K, Laehteenmaeki T, Sievi E, Saxelin M, Vapaatalo unknown antioxidative mechanisms. H (1997) Lactobacillus rhamnosus GG shows antioxidative properties in vascular endothelial cell culture. Milchwissenschaft The O scavenging activity and H O scavenging 2 2 2 52(9):503–505 activity of the recombinant E. coli BLSod was slightly Kullisaar T, Zilmer M, Mikelsaar M, Vihalemm T, Annuk H, Kairane higher than that of E. coli BL21(DE3). The slight increase C, Kilk A (2002) Two antioxidative lactobacilli strains as in hydrogen peroxide scavenging activity may be explained promising probiotics. Int J Food Microbiol 72(3):215–224 Lin MY, Yen CL (1999) Antioxidative ability of lactic acid bacteria. J by the positive interaction between SOD and catalase. Agric Food Chem 47(4):1460–1466 Superoxide radicals can inactivate catalase; therefore, SOD Liong MT, Shah NP (2005) Bile salt deconjugation ability, bile salt can protect catalase due to its O scavenging activity hydrolase activity and cholesterol co-precipitation ability of (Brioukhanov and Netrusov 2004). lactobacilli strains. Int Dairy J 15(4):391–398 Meile L, Fischer K, Leisinger T (1995) Characterization of the The aim of this study was to clone the sod gene from L. superoxide dismutase gene and its upstream region from casei into E. coli BL21(DE3) for ectopic expression in order Methanobacterium thermoautotrophicum Marburg. FEMS to demonstrate that the SOD from Lactobacillus is able to Microbiol Lett 128(3):247–253 function in a heterologous prokaryote with considerable Nishikimi M, Appaji N, Yagi K (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and enzyme activity. Further work is required to clone the sod of molecular oxygen. Biochem Biophys Res Commun 46(2):849–854 L. casei into other species of Lactobacillus,such as L. Parker MW, Blake CC (1988) Iron- and manganese-containing acidophilus, which is widely used as a probiotic but lacks superoxide dismutases can be distinguished by analysis of their the SOD protein and has low antioxidative activity. primary structures. FEBS Lett 229(2):377–382 Parker MW, Blake CC, Barra D, Bossa F, Schinina ME, Bannister WH, Bannister JV (1987) Structural identity between the iron- and Acknowledgments This work was supported by the National Hi- manganese-containing superoxide dismutases. Protein Eng 1 Tech Research and Development Program of China (“863” Program, (5):393–397 grant 2007AA10Z355), the National Basic Research Program of Pick E, Keisari Y (1980) A simple colorimetric method for the China (“973” Program, grant 2007CB714301) and the Medical- measurement of hydrogen peroxide produced by cells in culture. Engineering Joint Research Foundation of Shanghai Jiao Tong J Immunol Methods 38(1–2):161–170 University (grant YG2009MS11). Roy DG, Klaenhammer TR, Hassan HM (1993) Cloning and expression of the manganese superoxide dismutase gene of Escherichia coli in Lactococcus lactis and Lactobacillus gasseri. Mol Gen Genet 239(1–2):33–40 References Sánchez-Venegas JR, Navarrete A, Dinamarca J et al (2009) Cloning and constitutive expression of Deschampsia antarctica Cu/Zn Adams MR, Marteau P (1995) On the safety of lactic acid bacteria superoxide dismutase in Pichia pastoris. BMC Res Notes 2:207 from food. 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Annals of Microbiology – Springer Journals
Published: Mar 3, 2011
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