Jacqueline Destain

Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis.

In this work, the potential of Bacillus subtilis strain M4 at protecting plants against fungal diseases was demonstrated in different pathosystems. We provide evidence for the role of secreted lipopeptides, and more particularly of fengycins, in the protective effect afforded by the strain against damping-off of bean seedlings caused by Pythium ultimum and against gray mold of apple in post-harvest disease. This role was demonstrated by the strong biocontrol activity of lipopeptide-enriched extracts and through the detection of inhibitory quantities of fengycins in infected tissues. Beside such a direct antagonism of the pathogen, we show that root pre-inoculation with M4 enabled the host plant to react more efficiently to subsequent pathogen infection on leaves. Fengycins could also be involved in this systemic resistance-eliciting effect of strain M4, as these molecules may induce the synthesis of plant phenolics involved in or derived from the defense-related phenylpropanoid metabolism. Much remains to be discovered about the mechanisms by which Bacillus spp suppress disease. Through this study on strain M4, we reinforce the interest in B. subtilis as a pathogen antagonist and plant defense-inducing agent. The secretion of cyclic fengycin-type lipopeptides may be tightly related to the expression of these two biocontrol traits.

Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by plackett-burman design

Bacillus subtilis S499 is well-known for its ability to produce two families of surfactant lipopeptides: Iturin A and Surfactin S1. Fermentation optimization for this strain was performed to amplify the surfactant production. Ten active variables were analyzed by two successive Plackett-Burman designs, consisting respectively of 12 and 16 experiments to give an optimized medium. The amount of biosurfactant lipopeptides in the supernatant of a culture carried out in this optimized medium was about five times higher than that obtained in nonoptimized rich medium. The analysis of the surfactant molecules produced in such optimized conditions has revealed the presence of a third family of lipopeptides: the fengycins.The time-dependent production of these three families of molecules in bioreactors showed that surfactin S1 is produced during the exponential phase and iturin A and fengycins during the stationary phase.

Improvement of lipase production from Yarrowia lipolytica

Extracellular lipase production by Yarrowia lipolytica was increased by mutant selection from 28 U/ml to 1000 U/ml. This activity was also reached in a 500 l bioreactor. The properties of the mutant lipase were the same of those of the wild type: M 38 kDa, optimum pH 7 and optimum temperature 37¡C.

Purification Of Antifungal Lipopeptides By Reversed-Phase High-Performance Liquid-Chromatography

A rapid procedure for the purification of antifungal lipopeptides from Bacillus subtilis, a potential agent for biocontrol of plant diseases, was tested. It consists of a solid-phase extraction on C18 gel followed by reversed-phase chromatography using a biocompatible PepRPC HR 5/5 column with a pharmacia fast protein liquid chromatographic system. This is a very effective method for isolating and fractionating iturin A and surfactin, two lipopeptides of different nature, co-produced by Bacillus subtilis strain S499. The presence of homologous lipopeptides was easily detected.

Biochemistry of lactone formation in yeast and fungi and its utilisation for the production of flavour and fragrance compounds

The consumers’ demand for natural flavour and fragrances rises. To be natural, compounds have to result from the extraction of natural materials and/or to be transformed by natural means such as the use of enzymes or whole cells. Fungi are able to transform some fatty acids into lactones that can thus be natural. Although some parts of this subject have been reviewed several times, the present article proposes to review the different pathways utilised, the metabolic engineering strategies and some current concerns on the reactor application of the transformation including scaling up data. The main enzymatic steps are hydroxylation and β-oxidation in the traditional way, and lactone desaturation or Baeyer–Villiger oxidation. Although the pathway to produce γ-decalactone is rather well known, metabolic engineering strategies may result in significant improvements in the productivity. For the production of other lactones, a key step is the hydroxylation of fatty acids. Beside the biotransformation, increasing the production of the various lactones requires from biotechnologists to solve two main problems which are the toxicity of lactones toward the producing cell and the aeration of the emulsified reactor as the biochemical pathway is very sensitive to the level of available oxygen. The strategies employed to resolve these problems will be presented.

Bacteriocin Activity By Lactobacillus Curvatus Cwbi-B28 To Inactivate Listeria Monocytogenes In Cold-Smoked Salmon During 4 Degrees C Storage

The inhibition effectiveness of a bacteriocin produced by Lactobacillus curvatus CWBI-B28 against Listeria monocytogenes was investigated in cold-smoked salmon during storage at 4 degrees C. Three bacteriocin-based strategies for the control of L. monocytogenes in foods (i.e., producing bacteriocin in situ, spraying with partially purified bacteriocin, and packaging in bacteriocin-coated plastic film), plus a newly developed method that uses cell-adsorbed bacteriocin (i.e., a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustments), were assessed. Although all the approaches inactivated L. monocytogenes in cold-smoked salmon, various efficacy levels were observed. The behavior of L. monocytogenes was similar in samples treated with either partially purified bacteriocin or in situ bacteriocin production. In both of these cases, the counts of the pathogen declined to below the detectable limit of 0.7 log CFU/cm2 within the first week, but a approximately 0.95- and 1.3-log increase, respectively, occurred after day 14. The bioactive packaging film resulted in a slower inactivation of the pathogen but prevented any subsequent increase in the CFU throughout 22 days of storage at 4 degrees C. Application of the cell-adsorbed bacteriocin was shown to be the most effective means, as it resulted in a complete inactivation of the pathogen within 3 days, and no increase in Listeria counts occurred up to 22 days.

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.

Investigation of the effect of different extracellular factors on the lipase production by Yarrowia lipolityca on the basis of a scale-down approach

The influence of three extracellular factors (namely, the methyl oleate dispersion in the broth, the dissolved oxygen variations, and the pH fluctuation) on the lipase production by Y. lipolytica in batch bioreactor has been investigated in different scale-down apparatus. These systems allow to reproduce the hydrodynamic phenomena encountered in large-scale equipments for the three specified factors. The effects of the extracellular factors have been observed at three distinct levels: the microbial growth, the extracellular lipase production, and the induction of the gene LIP2 encoding for the main lipase of Y. lipolytica. Among the set of environmental factors investigated, the dissolved oxygen fluctuations generated in a controlled scale-down reactor (C-SDR) have led to the more pronounced physiological effect by decreasing the LIP2 gene expression level. The other environmental factors observed in a partitioned scale-down reactor, i.e., the methyl oleate dispersion and the pH fluctuations, have led to a less severe stress traduced only by a decrease of the microbial yield and thus of the extracellular lipase specific production rate.

Identification and characterization of a new xylanase from Gram-positive bacteria isolated from termite gut (Reticulitermes santonensis)

Termites are world champions at digesting lignocellulosic compounds, thanks to cooperation between their own enzymes and exogenous enzymes from microorganisms. Prokaryotic cells are responsible for a large part of this lignocellulolytic activity. Bacterial enzyme activities have been demonstrated in the higher and the lower termite gut. From five clones of Gram-positive bacteria isolated and identified in a previous work, we constructed a genomic DNA library and performed functional screening for alpha-amylase, beta-glucosidase, and xylanase activities. One candidate, Xyl8B8, showed xylanase activity. Sequence analysis of the genomic insert revealed five complete ORFs on the cloned DNA (5746bp). Among the encoded proteins were a putative endo-1,4-beta-xylanase (XylB8) belonging to glycoside hydrolase family 11 (GH11). On the basis of sequence analyses, genomic DNA organization, and phylogenetic analysis, the insert was shown to come from an actinobacterium. The mature xylanase (mXylB8) was expressed in Escherichia coli and purified by affinity chromatography and detected by zymogram analysis after renaturing. It showed maximal xylanase activity in sodium acetate buffer, pH 5.0 at 55 °C. Its activity was increased by reducing agents and decreased by Cu(2+), some detergents, and chelating agents. Its substrate specificity appeared limited to xylan.

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate.

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.