Separation and identification of exo- and endoinulinase from Aspergillus ficuum

A new and convenient method was developed to separate and identify exo- and endoinulinase from Aspergillus ficuum by native polyacrylamide gel electrophoresis. Eight protein bands were obtained. Three bands were identified as exoinulinase, and two bands were endoinulinase, by using TLC and HPLC. The five bands were all detected as glycoproteins with the sensitive periodic acid-silver stain.     

Isolation and properties of recombinant inulinases from <Emphasis Type="Italic">Aspergillus</Emphasis> sp.

The genes inuA and inu1, encoding two inulinases (32nd glycosyl hydrolase family) from filamentous fungi Aspergillus niger and A. awamori, were cloned into Penicillium canescens recombinant strain. Using chromatographic techniques, endoinulinase InuA (56 kDa, pI 3) and exoinulinase Inu1 (60 kDa, pI 4.3) were purified to homogeneity from the enzymatic complexes of P. canescens new transformants. The properties, such as substrate specificity, pH- and T-optima of activity, stability at different temperatures, influence of cations and anions on the catalytic activity, etc., of both recombinant inulinases were studied.     

Cloning and Nucleotide Sequence of the Endoinulinase-Encoding Gene, inu2, from Aspergillus ficuum

A 2.3 kb DNA fragment that contains a gene encoding endoinulinase, inu2, from Aspergillus ficuum ATCC 16882 was isolated and analyzed. It includes an open reading frame of 1,551 bp, coding for a polypeptide with calculated molecular weight of 55,790 Da, including a putative signal peptide of 22 amino acids. Alignment of amino acid sequences revealed 73.3% identity and 93.9% similarity between A. ficuum and Penicillium purpurogenum endoinulinase.     

Growth Kinetics of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> ssp. <Emphasis Type="Italic">diacetylactis</Emphasis> Harboring Different Plasmid Content

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac⁺- and Prt⁺-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation. Received: 27 July 2002 / Accepted: 27 August 2002     

Endo‐ and exo‐inulinases: Enzyme‐substrate interaction and rational immobilization

Three‐dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoinulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase resolved so far. Docking and molecular dynamics simulations were performed to investigate the differences between the two inulinases in terms of substrate selectivity. The analysis of the structural differences between the two inulinases provided the basis for the explanation of their different regio‐selectivity and for the understanding of enzyme‐substrate interactions. Surface analysis was performed to point out structural features that can affect the efficiency of enzymes also after immobilization. The computational analysis of the three‐dimensional models proved to be an effective tool for acquiring information and allowed to formulate an optimal immobilized biocatalyst even more active that the native one, thus enabling the full exploitation of the catalytic potential of these enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Electrophoretic karyotype of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> and its variation among monokaryotic progenies

 The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0 Mb or more. Received: October 11, 2001 / Accepted: November 11, 2002 Acknowledgments We thank Professor T. Morinaga, Hiroshima Prefectural University, and Dr. T. Arima for their technical advice regarding CHEF gel electrophoresis. Correspondence to:E. Tanesaka     

Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

Identification and characterization of inulinases by bioinformatics analysis of bacterial glycoside hydrolases family 32 (GH32)

The glycoside hydrolase family contains enzymes that break the glycosidic bonds of carbohydrates by hydrolysis. Inulinase is one of the most important industrial enzymes in the family of Glycoside Hydrolases 32 (GH32). In this study, to identify and classify bacterial inulinases initially, 16,002 protein sequences belonging to the GH32 family were obtained using various databases. The inulin-effective enzymes (endoinulinase and exoinulinase) were identified. Eight endoinulinases (EC 3.2.1.7) and 4318 exoinulinases (EC 3.2.1.80) were found. Then, the localization of endoinulinase and exoinulinase enzymes in the cell was predicted. Among them, two extracellular endoinulinases and 1232 extracellular exoinulinases were found. The biochemical properties of 363 enzymes of the genus Arthrobacter, Bacillus, and Streptomyces (most abundant) showed that exoinulinases have an acid isoelectric point up to the neutral range due to their amino acid length. That is, the smaller the protein (336 aa), the more acidic the pI (4.39), and the larger the protein (1207 aa), the pI is in the neutral range (8.84). Also, a negative gravitational index indicates the hydrophilicity of exoinulinases. Finally, considering the biochemical properties affecting protein stability and post-translational changes studies, one enzyme for endoinulinase and 40 enzymes with desirable characteristics were selected to identify their enzyme production sources. To screen and isolate enzyme-containing strains, now with the expansion of databases and the development of bioinformatics tools, it is possible to classify, review and analyze a lot of data related to different enzyme-producing strains. Although, in laboratory studies, a maximum of 20 to 30 strains can be examined. Therefore, when more strains are examined, finally, strains with more stable and efficient enzymes were selected and introduced for laboratory activities. The findings of this study can help researchers to select the appropriate gene source from introduced strains for cloning and expression heterologous inulinase, or to extract native inulinase from introduced strains.     

Utilization of chicory roots for microbial endoinulinase production

AIMS: The optimal culture conditions for endoinulinase production using chicory roots were studied in shake-flask culture. METHODS AND RESULTS: Much higher enzyme production was achieved with Xanthomonas sp. (15 U ml(-1)) than with Pseudomonas sp. (3 U ml(-1)). Optimized culture conditions of Xanthomonas sp. for endoinulinase production in flask culture were: chicory powder, 5 g l(-1); temperature, 37 degrees C; pH, 7.0; agitation speed, 100 rev min(-1). CONCLUSION: Maximum bacterial growth and enzyme production were 6.2 g l(-1) and 20 U ml(-1) under optimal conditions, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Chicory roots could be used as a fermentation substrate for the production of enndoinulinase.     

Seasonal development of secondary xylem and phloem in <Emphasis Type="Italic">Schizolobium parahyba</Emphasis> (Vell.) Blake (Leguminosae: Caesalpinioideae)

The cambial activity and periodicity of secondary xylem and phloem formation have been less studied in tropical tree species than in temperate ones. This paper describes the relationship between seasonal cambial activity, xylem and phloem development, and phenology in Schizolobium parahyba, a fast growing semideciduous seasonal forest tree from southeastern Brazil. From 2002 to 2003, wood samples were collected periodically and phenology and climate were recorded monthly in the same period. S. parahyba forms annual growth increments in wood, delimited by narrow initial parenchyma bands. The reduction of the cambial activity to a minimum correlates to the dry season and leaf fall. The higher cambial activity correlates to the wet season and the presence of mature leaves. In phloem, a larger conductive region was observed in the wet season, when the trees were in full foliage. The secondary phloem did not exhibit any incremental zone marker; however, we found that the axial parenchyma tends to form irregular bands.     

Regulation of Extracellular Chitinases and Proteases in the Entomopathogen and Acaricide <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS–PAGE. Received: 8 May 2002 / Accepted: 8 June 2002     

Inter- and intra-specific variation in the facial colours of <Emphasis Type="Italic">Perisesarma eumolpe</Emphasis> and <Emphasis Type="Italic">Perisesarma indiarum</Emphasis> (Crustacea: Brachyura: Sesarmidae)

The colour of the characteristic facial bands in the mangrove crabs Perisesarma eumolpe (De Man) and Perisesarma indiarum (Tweedie) were investigated in relation to species, sex and size. Quantitative red/blue/green (RBG) values extracted from close-up digital photographs of 27 P. indiarum and 48 P. eumolpe specimens revealed significant differences between species and sexes. Males of both species possessed facial bands characterized by a greater intensity of blue, whereas green was more pronounced in female facial bands. It is postulated that these colour differences play a role in intra-specific sexual recognition. There existed a significant positive relationship between size and the intensity of blue in the facial bands of females of both species, suggesting these traits function as an indicator of their maturity (and possibly body condition). In large males, facial band colours contrast strongly against the surrounding mudflat and may play an important role in conspecific signalling during territorial disputes or contests over access to females. Handling editor: K. Martens     

Allergenic and mutagenic characterization of 14 <Emphasis Type="Italic">Penicillium</Emphasis> species

Extracts of 14 Penicillium species, P. aurantiogriseum, P. brevicompactum, P. citrinum, P. chrysogenum, P. expansum, P. glabrum, P. hirsutum, P. italicum, P. janthinellum, P. melini, P. oxalicum, P. purpurescens, P. simplicissimum, and P. viridicatum were investigated by total protein, specific enzyme determinations, isoelectric focusing (IEF), sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using pooled human atopic IgE. Considerable variation was observed between the Penicillium species with respect to protein yield and the number of distinct protein bands resolved in IEF. Using the Api-Zym system, the most common activities observed among the extracts included acid and alkaline phosphatase, phosphodiamidase and β-glucosaminidase. The number of discrete atopic IgE-reactive bands in immunoblots of Penicillium extracts ranged from 1 (P. chrysogenum) to 9 (P. viridicatum). Certain allergens showed potential for cross-reactivity between species, including 52 and 54 kDa proteins in P. citrinum, P. purpurescens, P. viridicatum and 40 kDa proteins in several species. The extracts were also nonmutagenic when tested with the Ames assay using Salmonella strains TA98 and TA100. The results tend to indicate that P. viridicatum, P. janthinellum, P. oxalicum, P. brevicompactum and P. italicum, which are highly immunogenic as well as allergenic, could possibly be good candidates for allergen cloning studies through the construction of cDNA libraries. The extracts were non-mutagenic and can be used safely for skin testing.*Presented in part at the 7th International meeting of Aerobiology, Montebello, August, 2002.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: occurrence in the air and clinical significance

Trichoderma harzianum, a filamentous fungus, is being widely used as a potential biopesticide. The potential of this fungus in causing skin sensitization, however, was poorly investigated as yet. The objective of this study was to monitor the occurrence of T. harzianum in the air and to explore its skin sensitizing potential. Seasonal periodicity of T. harzianum was studied for the years 2002–2004 by an Andersen air sampler. The skin sensitizing potential of T. harzianum extract was studied in 389 patients with suspected respiratory allergy by skin prick test (SPT) and specific IgE level was determined by ELISA. SDS–PAGE and immunoblotting were also performed. T. harzianum colony count varied from 3.69 to 134.88 CFU m⁻³ with the peak achieved in February. Relative humidity was found to be a significant (P < 0.05) factor predicting the occurrence of T. harzianum in the air. Positive skin reaction (wheal diameter ≥ 3 mm) was observed in 105 patients (26.99%). T. harzianum crude extract was resolved in 18 protein bands (12–72 kDa) on SDS–PAGE (12% gel) including two IgE-binding protein bands (21 and 32 kDa). T. harzianum can be considered an important inhalant allergen.     

Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml^?1, 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l^?1 inulin, an enzyme concentration of 40 U g^?1 substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.     

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus

Summary. Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, ulica Pasteura 3, 02-093 Warsaw, Poland.Received October 7, 2002; accepted December 2, 2002; published online August 26, 2003     

Identification and Characteristics of Nisin Z-Producing <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> Isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100°C for 1 h and at 121°C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or β-glucosidase. There were some differences in characteristics from those of nisins described previously. Received: 21 June 2002 / Accepted: 22 July 2002