Nonheme Catalase Research Articles

Potential antioxidant activity of Lactobacillus fermentum KF7 from virgin coconut oil products in Padang, West Sumatra, Indonesia

Abstract. Syukur S, Safrizayanti, Zulaiha S, Supriwardi E, Fahmi A, Nurfadilah KK, Purwati E. 2021. Potential antioxidant activity of Lactobacillus fermentum KF7 from virgin coconut oil products in Padang, West Sumatra, Indonesia. Biodiversitas 23: 1628-1634. The isolation and characterization of five commercial Virgin Coconut Oil (VCO) products circulating in the local market of Padang city were conducted. In this study, eighteen isolates of lactic acid bacteria (LAB) were isolated and characterized microscopically, and biochemically. VCO-LAB isolates were Gram-positive bacilli and cocci, catalase-negative, and homofermentative (except VD was heterofermentative). There were three isolates of VCO-LAB (VB.3, VD and VE.4) that could produce gamma-aminobutyric acid (GABA). The highest concentration of GABA (19.5 mg/mL) was produced by isolate VB.3 after 72 hours of incubation and the addition of 7% MSG. VB.3 isolate shows the highest antioxidant activity with the addition of Monosodium glutamate (MSG), 68.13% for 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ABTS and 88.02%. In addition, GABA-producing VCO-LAB could be grown in media containing 0.2-1.0 mM hydrogen peroxide, with the lowest optical density value of 0.82 nm. The antioxidant activity of VCO-LAB was affected by the GABA produced and by secondary metabolites such as peptides and antioxidant enzymes (NADH-oxidase, superoxide dismutase, NADH peroxidase and non-heme catalase). The highest antioxidant production was obtained in isolate VB.3 or Lactobacillus fermentum KF7.

Role of the Mn-Catalase in Aerobic Growth of Lactobacillus plantarum ATCC 14431

Lactobacilli are Gram-positive aerotolerant organisms that comprise the largest genus of Lactic Acid Bacteria (LAB). Most lactobacilli are devoid of the antioxidant enzymes, superoxide dismutases, and catalases, required for protection against superoxide radicals and hydrogen peroxide (H2O2), respectively. However, some lactobacilli can accumulate millimolar concentrations of intracellular manganese and spare the need for superoxide dismutase, while others possess non-heme catalases. L. plantarum is associated with plant materials and plays an important role in fermented foods and gut microbiomes. Therefore, understanding the effects of the environment on the growth and survival of this organism is essential for its success in relevant industrial applications. In this report, we investigated the physiological role of Mn-catalase (MnKat) in Lactobacillus plantarum ATCC 14431. To this end, we compared the physiological and morphological properties of a ΔMnkat mutant strain and its isogenic parental strain L. plantarum ATCC 14431. Our data showed that the MnKat is critical for the growth of L. plantarum ATCC 14431 in the presence of oxygen and resistance to H2O2. The aerobic growth of the mutant in presence or absence of H2O2 was improved in the Mn-rich medium (APT) as compared to the growth in MRS medium. Furthermore, under aerobic conditions the mutant strain possessed atypical cellular morphology (i.e., shorter, and fatter). In conclusion, the MnKat of L. plantarum ATCC 14431 is important for aerobic growth, protection against H2O2, and maintenance of the rod-shaped cell morphology under aerobic conditions.

Aerobic and respirative growth of heterofermentative lactic acid bacteria: A screening study

Heterofermentative lactic acid bacteria (76 strains) belonging to Lactobacillus, Leuconostoc and Weissella species which are important in fermentation, spoilage or as probiotics were screened in a factorial experiment for their ability to grow, produce catalase and consume oxygen in aerobiosis or in anaerobiosis, with or without supplementation with hemin and/or menaquinone in a medium containing glucose as a carbohydrate source. Aerobiosis improved growth with a few exceptions. The effect of supplementation with heme and/or menaquinone was strain specific and clear evidence of heme-boosted respiration was found in some cases. Heme-catalase was produced by strains of L. brevis, W. minor and Leuc. mesenteroides; some strains of the latter species produced non-heme catalase. Shaken flasks experiments showed that aerobic growth resulted in increased maximum growth rate and in a limited increase in biomass. Heme supplementation during aerobic growth resulted in a further increase in growth rate and final biomass only for a few strains; this was often related to catalase, which was also responsible for increased tolerance of H2O2. In both experiments we found evidence of heme toxicity, especially in anaerobiosis and in absence of menaquinone. Dose response curves for aerobic growth in the presence of combinations of hemin and menaquinone were non-monotonic, with growth stimulation at low doses of heme (<2.5 mg/l) and toxicity at higher doses. Menaquinone at 0.25–8 mg/l increased growth stimulation and partially reduced toxicity.

In-situ electrochemical immobilization of [Mn(bpy)2(H2O)2]2+ complex on MWCNT modified electrode and its electrocatalytic H2O2 oxidation and reduction reactions: A Mn-Pseudocatalase enzyme bio-mimicking electron-transfer functional model

Mn-Pseudocatalase is a non-heme catalases family enzyme produced by various bacteria that involves in a two-electron H2O2 catalytic cycle in a manner similar to that of the heme-based Catalase enzymes. Herein, we report a bio-mimicking functional model system prepared by in-situ electrochemical oxidation of Mn(bpy)2Cl2 precursor to a surface-confined [MnII(bpy)2(H2O)2]2+ complex, wherein, bpy = 2,2′-bipyridyl, on a carboxylic acid functionalized multiwalled‑carbon nanotube (f-MWCNT)/Nafion modified glassy carbon electrode for biomimicking H2O2 disproportionation reaction. The modified electrode showed a well-defined redox peaks at an apparent standard electrode potentials (Eo′), 0.65 ± 0.05 V and 0.2 V vs Ag/AgCl with surface-excess (ΓMn) values, 6.24 × 10−9 mol cm−2 and 0.43 × 10−9 mol cm−2 for the MnIV/III and MnIII/II sites of the Mn-complex in neutral pH solution respectively. Physico-chemical characterizations of the system by FTIR, UV–Vis and ESI-MS (ethanolic extract of the electrode) confirming the conversion of [Mn(bpy)2(H2O)2]2+ complex (m/z, 403.09). A strong π-π interaction between the f-MWCNT's graphitic sp2 carbons and the bpy's aromatic electrons, hydrogen bonding between the oxygen and water molecules and ionic interaction between the complex and sulphonic site of nafion favor the stability of the complex. The hybrid system showed selective current signals for mediated oxidation and reduction reactions of H2O2 (disproportional reaction) without any dissolved oxygen interference. As an independent study, selective electro-catalytic oxidation and amperometric detection of H2O2 was demonstrated.

Comparative proteomic analyses for elucidating metabolic changes during EPS production under different fermentation temperatures by Lactobacillus plantarum Q823

Exopolysaccharide (EPS)-producing bacteria are of growing interest in industrial processes, mainly concerning food. Lactic acid bacteria are widely appreciated for their GRAS (generally recognized as safe) status and their ascertained or putative probiotic features. Detailed investigation on what happens at metabolic level during EPS production is scarce in the literature. The facultative heterofermenter Lactobacillus plantarum Q823 was studied in order to compare growth and EPS production at 30°C and 37°C. A higher growth rate was observed at 37°C, whereas, a significantly higher (tenfold increase) EPS amount was produced at 30°C. To understand the molecular mechanisms leading to the different EPS production in the two conditions, a comparative proteomic experiment was performed. The results of the in-gel proteomics revealed that: i) at 37°C a higher abundance of proteins involved in carbon catabolism and nucleic acid biosynthesis together with a significant amount of stress proteins was observed; ii) at 30°C the production of an atypical manganese-containing non-heme catalase (pseudocatalase) was increased, in agreement with previous data reporting that growth-rates of catalase negative Lactobacillus plantarum strains were greater than that of catalase positive strains. Taken together, all these findings provide further insights about the metabolic pathways stimulated during EPS production, and the mechanism that triggers EPS biosynthesis.

Transcriptome Profiling and Functional Analysis ofAgrobacterium tumefaciensReveals a General Conserved Response to Acidic Conditions (pH 5.5) and a Complex Acid-Mediated Signaling Involved inAgrobacterium-Plant Interactions

Agrobacterium tumefaciens transferred DNA (T-DNA) transfer requires that the virulence genes (vir regulon) on the tumor-inducing (Ti) plasmid be induced by plant phenolic signals in an acidic environment. Using transcriptome analysis, we found that these acidic conditions elicit two distinct responses: (i) a general and conserved response through which Agrobacterium modulates gene expression patterns to adapt to environmental acidification and (ii) a highly specialized acid-mediated signaling response involved in Agrobacterium-plant interactions. Overall, 78 genes were induced and 74 genes were repressed significantly under acidic conditions (pH 5.5) compared to neutral conditions (pH 7.0). Microarray analysis not only confirmed previously identified acid-inducible genes but also uncovered many new acid-induced genes which may be directly involved in Agrobacterium-plant interactions. These genes include virE0, virE1, virH1, and virH2. Further, the chvG-chvI two-component system, previously shown to be critical for virulence, was also induced under acid conditions. Interestingly, acidic conditions induced a type VI secretion system and a putative nonheme catalase. We provide evidence suggesting that acid-induced gene expression was independent of the VirA-VirG two-component system. Our results, together with previous data, support the hypothesis that there is three-step sequential activation of the vir regulon. This process involves a cascade regulation and hierarchical signaling pathway featuring initial direct activation of the VirA-VirG system by the acid-activated ChvG-ChvI system. Our data strengthen the notion that Agrobacterium has evolved a mechanism to perceive and subvert the acidic conditions of the rhizosphere to an important signal that initiates and directs the early virulence program, culminating in T-DNA transfer.

Non-Heme Catalase Activity of Lactic Acid Bacteria

A number of 71 species of lactic acid bacteria (LAB) were investigated for non-heme and heme catalase activity and their potential to form hydrogen peroxide. Non-heme catalase activity was restricted to strains of Lactobacillus plantarum , Lactobacillus mali and Pediococcus pentosaceus whereas heme catalase activity was present in 21 species belonging to virtually all genera of LAB. P. pentosaceus LTH 416 exhibited maximum non-heme catalase activity when cultured aerobically at the stationary phase. The activity increased after glucose was exhausted. Transfer of cells from glucose minimal medium (0.05%) to media containing 0.2% and 1% glucose, respectively, caused reduction of non-heme catalase activity. The effect of glucose was also seen if it was added to cells growing under static pH conditions (pH 6.5). The non-heme catalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 were characterized with regard to environmental factors important in sausage fermentation such as pH, temperature, concentration of nitrate or nitrite and water activity. The results permit an estimation of the non-heme catalase activity in the two strains during the fermentation process.

Luminescence of a bacterial luciferase intermediate by reaction with H 2O 2: the evolutionary origin of luciferase and source of endogenous light emission

The bacterial luciferase reaction was shown to emit light in the presence of hydrogen peroxide (H 2O 2) instead of aldehyde. Although the light intensity was weaker than that with an aldehyde, the bioluminescence emission spectrum of the luciferase reaction ( λ max 495 nm) with H 2O 2 closely agreed with that using a long chain aliphatic aldehyde (decanal). It was also found that the chemiluminescence of reduced or unreduced FMN with H 2O 2 was enhanced by peroxidase. Light emission in the luciferase reaction with H 2O 2 is attributed to a catalase-like reaction, in which the abstraction of one atom of oxygen from intermediate II (luciferase-bound FMNH-OOH) by H 2O 2 leads to the formation of excited hydroxide (E-FMNH-OH∗), H 2O and O 2. Since H 2O 2 is formed in the spontaneous breakdown of intermediate II in the absence of aldehyde, the reaction of this H 2O 2 with intermediate II can explain the origin of endogenous (non aldehyde) light emission. In the presence of luciferase, intermediate II can be formed both from FMNH 2 and oxygen, and by the reaction of H 2O 2 with FMN. The functional origin of the bacterial luciferase is postulated to be a FMN-dependent non-heme catalase which scavenges H 2O 2, concomitant with weak light emission. Prior to the association of luciferase genes ( lux AB) with the aldehyde synthesis genes ( lux CDE), forming the lux operon in luminous bacteria, the genes may have been expressed independently such that the bacteria lacked a high level of light emission.

Oxygen metabolism of catalase-negative and catalase-positive strains of Lactobacillus plantarum.

Two catalase-negative strains of Lactobacillus plantarum and a strain producing the atypical, nonheme catalase were studied to determine if the ability to produce the atypical catalase conferred any growth advantage upon the producing strain. Both catalase-negative strains grew more rapidly than the catalase-positive strain under aerobic or anaerobic conditions in a glucose-containing, complex medium. Upon exhaustion of glucose from the medium, all three strains continued growth under aerobic but not under anaerobic conditions. The continued aerobic growth was accompanied by production of acetic acid in addition to the lactic acid produced during growth on glucose. Oxygen was taken up by exponential phase-cell suspensions grown on glucose when glucose or glycerol were used as substrates. Cells harvested from glucose-exhausted medium oxidized glucose, glycerol, and pyruvate. Oxygen utilization by a catalase-negative strain increased as did the specific activity of reduced nicotinamide adenine dinucleotide peroxidase during late growth in the glucose-exhausted medium. The catalase-positive strain and the catalase-negative strain tested both possessed low but readily detectable levels of superoxide dismutase throughout growth. The growth responses are discussed in terms of the presence of enzymes which would allow the cells to remove potentially damaging reduction products of O2.

ISOLATION AND CHARACTERIZATION OF THE CYANIDE-RESISTANT AND AZIDE-RESISTANT CATALASE OF LACTOBACILLUS PLANTARUM.

Johnston, M. A. (Cornell University, Ithaca, N.Y.), and E. A. Delwiche. Isolation and characterization of the cyanide-resistant and azide-resistant catalase of Lactobacillus plantarum. J. Bacteriol. 90:352-356. 1965.-Lactobacillus plantarum T-1403-5 has been shown to possess a very active cyanide- and azide-resistant catalase. By means of fractional ammonium sulfate precipitation, removal of nucleic acids with protamine sulfate, adsorption on calcium phosphate gel, and pH gradient chromatography on diethylaminoethyl cellulose, the catalase "activity" was purified approximately 14-fold. The purified enzyme preparation was insensitive to the heme poisons cyanide and azide, the metal chelating agents ethylenediaminetetraacetate and o-phenanthroline, and the sulfhydryl binding agent p-chloromercuribenzoate. The purified enzyme moved at a uniform rate in the electrophoretic field (isoelectric point, pH 4.7). The ultraviolet-light absorption spectrum was negative for heme-iron components, and fluorescence measurements yielded negative results with regard to flavin components. Acriflavin and Atabrine had no effect on enzyme activity. The nonheme catalase displayed a much broader pH range of activity than the heme-iron catalase of a control culture of Escherichia coli and the azide-sensitive catalase developed by L. plantarum NZ48 when grown in the presence of preformed hematin. The nonheme catalase was more resistant to heat inactivation. No retention of the enzyme on a chromatographic column could be obtained with Sephadex 200, nor could the enzyme be separated from crystalline beef-liver catalase by the gel filtration technique. Sedimentation was obtained in a centrifugal field of 144,000 x g for 12 hr.