Alginate-deriving oligosaccharide production by alginase from newly isolated Flavobacterium sp. LXA and its potential application in protection against pathogens

Aims:  To examine algino-oligosaccharide production by alginase from newly isolated Flavobacterium sp. LXA and its elicitor and antibacterial activity.
Methods and Results:  Algino-oligosaccharide production from alginate was carried out using alginase obtained from a newly isolated Flavobacterium sp. LXA. When alginase was partially purified by dual ammonium sulfate precipitation and used for alginate degradation, the viscosity loss correlated well with the release of reducing terminals. The optimal temperature and pH for alginate degradation was 40°C and pH 7·0, respectively. When alginate was added at an initial concentration of more than 0·8%, the maximal degradation rate of alginate was obtained. Under these optimal reaction conditions and with partially purified alginase, the average degrees of polymerization (DP) of alginate-degraded products was about 6·0, which favoured algino-oligosaccharide production. The algino-oligosaccharides showed an elicitor activity stimulating the accumulation of phytoalexin and inducing phenylalanine ammonia lyase in soybean cotyledon, and antimicrobial activity on Pseudomonas aeruginosa .
Conclusions:  Algino-oligosaccharide could be degraded from alginate by the partially purified alginase and its maximal bioactivity occurred on the oligosaccharide with average DP 6·8.
Significance and Impact of the Study:  Algino-oligosaccharide was first reported to have elicitor and antibacterial activity and have potential as a biological agent for protection against plant or human disease.     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Enzymatic degradation of alginate by marine fungi

A total of 72 pre-selected strains of 19 species of marine fungi were tested for their ability to decompose sodium alginate, calcium alginate or freshly prepared calcium alginate gel. Active alginate decomposition was evident in 18 strains (25% of total tested). These belong to only three different species: Asteromyces cruciatus, Corollospora intermedia, and Dendryphiella salina. In broth culture, decomposition of sodium alginate by the two deuteromycetes was followed by gravimetric, electrometric, viscometric, photometric and chromatographic methods in order to characterize the alginase enzyme system and its degradation products. The alginase enzyme complex consisted of at least two different enzyme components: the already known alginate lyase (eliminase) and a new endo-alginate hydrolase. In summary, a model is presented on the alginase-mediated structural and molecular decomposition of sodium alginate by marine fungi.     

Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: the enzyme participates in protoplast formation of Schizophyllum commune

Chitinase A of Streptomyces cyaneus SP-27 or chitinase I of Bacillus circulans KA-304 showed the protoplast-forming activity when combined with alpha-1,3-glucanase of B. circulans KA-304. The gene of chitinase A was cloned. It consisted of 903 nucleotides encoding 301 amino acid residues, including a putative signal peptide (35 amino acid residues). The deduced N-terminal moiety of chitinase A showed sequence homology with the chitin-binding domain of chitinase F from Streptomyces coelicolor and chitinase 30 from Streptomyces olivaceoviridisis. The C-terminal moiety also showed high sequence similarity to the catalytic domain of several Streptomyces family 19 chitinases as well as that of chitinase I of B. circulans KA-304. Recombinant chitinase A was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the recombinant enzyme were almost the same as those of chitinase A purified from a culture filtrate of S. cyaneus SP-27. The recombinant enzyme was superior to B. circulans KA-304 chitinase I not only in respect to protoplast forming activity in a mixture containing alpha-1,3-glucanase, but also to antifungal activity and powder chitin-hydrolyzing activity.     

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R

Background  

The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related α-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans.     

Influence of different sources of carbon and nitrogen on the biosynthesis of proteolytic complexes by Bacillus circulans 693, Bacillus sp. 27 and Yarrowia lipolytica 2061 strains

The influence of different factors on the biosynthesis of extracellular proteolytic complexes by strains-producers Bacillus circulans 693, Bacillus sp. 27 and Yarrowia lipolytica 2061 at submerged cultivation has been investigated. It has been shown that ammonium hydrocarbonate and gelatin with glucose were optimum carbon and nitrogen sources for synthesis of proteolytic activity of B. circulans strain 693, gelatin with arabinose--for Bacillus sp. 27, gelatin and glycine with sorbose--for Y. lipolytica 2061. It has been established that the cultivation of producers on optimal sources of carbon and nitrogen increased protease activity of cultural liquid of B. circulans 693 3.8 times, Bacillus sp. 27--2.7 times, Y. lipolytica 2061--3.4 times. It has been found that the usage of different protein substrates in cultural medium permitted to obtain the proteolytic enzymes with various specificity with respect to globular and fibrillar proteins.     

An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

Purification and some properties of a novel alpha-L-fucosidase capable of acting on alpha-(1----6)-L-fucosidic linkages from Bacillus circulans M28

Two types of alpha-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the alpha-(1----2), alpha-(1----3), and alpha-(1----4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl alpha-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved alpha-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl alpha-L-fucoside, but also 2-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the alpha-(1----6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma

O kagbue , R.N. & L ewis , M.J. 1985. Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma. Journal of Applied Bacteriology 59 , 243–255.
In mixed culture Bacillus circulans WL-12 hydrolysed cell walls of Phaffia rhodozyma and rendered astaxanthin extractable from the yeast. pH control was critical to survival and lytic activity of the bacillus; the optimum range was 6.2–6.8. The optimum range of temperature was 20–24C. Glucose (1–2%) was efficient in minimizing catabolite repression of the lytic enzyme complex of the bacillus. Slow-feeding of glucose improved ultimate yields of lytic enzyme but did not acclerate yeast cell wall modification. A relatively high inoculum level of B. circulans accelerated modification of P. rhodozyma in the mixed culture: when the bacterial inoculum was four times that of the yeast, over 80% of total astaxanthin was extractable in 48 h. High bacterial inoculum size also stimulated yeast autolysis and necessitated early harvest of the mixed culture. Results obtained in shake flasks were duplicated in 5-litre fermentors and suggest that the mixed culture has potential industrial value for producing a biomass containing biologically-available astaxanthin. Extractability of astaxanthin was also achieved when mixed culture filtrate was incubated with pure cultured Phaffia cells. When suitably fortified with nutrients, the filtrate also supported simultaneous yeast growth and modification of the yeast cell walls. A scheme incorporating mixed culture with B. circulans WL-12 and re-use of culture filtrate has been proposed for enzymatic processing of Phaffia rhodozyma for inclusion in animal diets.     

The aman6 gene encoding a yeast mannan backbone degrading 1,6-alpha-D-mannanase in Bacillus circulans: cloning, sequence analysis, and expression

A gene (aman6) encoding endo-1,6-alpha-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned. The putative aman6 was 1,767 base pairs long and encoded a mature 1,6-alpha-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1,6-alpha-D-mannanase from the Escherichia coli transformant showed 61-kDa protein, and N-terminal amino acid sequence and other general properties of the recombinant enzyme were identical to those of 1,6-alpha-D-mannanase from Bacillus circulans TN-31.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans, five were B. circulans and four were B. licheniformis. Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65 degrees C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70 degrees C for 30 min.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans , five were B. circulans and four were B. licheniformis . Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65°C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70°C for 30 min.     

Purification and characterization of a Bacillus cereus exochitinase

Five extracellular chitinases of Bacillus cereus 6E1 were detected by a novel in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant violet 5R (CM-chitin-RBV) as a substrate. The major chitinase activity was associated with a 36-kDa (Chi36) gel band. Chi36 was purified by a one-step, native gel purification procedure derived from the new in-gel chitinase assay. The purified Chi36 has optimal activity at pH 5.8 and retains some enzymatic activity between pH 2.5-8. The temperature optimum for Chi36 was 35 degrees C, but the enzyme was active between 4-70 degrees C. Based on its ability to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta-D-glucosaminide)(2), Chi36 is characterized as a chitobiosidase, a type of exochitinase. The N-terminal amino acid sequence of mature Chi36 was determined (25 amino acids). Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36. The N-terminal sequence of Chi36 demonstrated highest similarity with Bacillus circulans WL-12 chitinase D and significant similarity with several other bacterial chitinases.     

The crystal structure of beta-glucosidase from Bacillus circulans sp. alkalophilus: ability to form long polymeric assemblies

Family 1 of glycosyl hydrolases is a large and biologically important group of enzymes. A new three-dimensional structure of this family, beta-glucosidase from Bacillus circulans sp. alkalophilus is reported here. This is the first structure of beta-glucosidase from an alkaliphilic organism. The model was determined by the molecular replacement method and refined to a resolution of 2.7 A. The quaternary structure of B. circulans sp. alkalophilus beta-glucosidase is an octamer and subunits of the octamer show a similar (beta/alpha)(8) barrel fold to that previously reported for other family 1 enzymes. The crystal structure suggested that Cys169 in the active site is substituted. The Cys169 is located near the putative acid/base catalyst Glu166 and it may contribute to the high pH optimum of the enzyme. The crystal structure also revealed that the asymmetric unit contains two octamers which have a clear binding interaction with each other. The ability of the octamers to link with each other suggested that beta-glucosidase from Bacillus circulans sp. alkalophilus is able to form long polymeric assemblies, at least in the crystalline state.     

Regulation of dihydrodipicolinate synthase during growth and sporulation of Bacillus cereus.

A four- to sixfold increase in specific activity of dihydrodipicolinic acid synthase was observed during sporulation of Bacillus cereus. The enzyme from cells harvested before and after the increase in specific activity appeared to be very similar as judged by pH optima, heat denaturation kinetics, apparent Michaelis constants, chromatography on diethylaminoethyl-cellulose and Sephadex G-200, and polyacrylamide gel electrophoresis. Studies with various combinations of amino acids and one of the enzyme substrates, pyruvate, failed to give evidence for control of the enzyme by activation, inhibition, repression, induction, or stabilization. Omission of calcium from the sporulation medium had no significant effect on the specific activity pattern of the enzyme as a function of age of culture.     

Possible role of cAMP in the synthesis of β-glucanases and β-xylanases of Bacillus circulans WL-12

Abstract Bacillus circulans WL-12 secretes 1,4-β- d -xylanase and 1,3-β- d - and 1,6-β- d -glucanase activities. All of them are catabolites regulated by glucose and, while xylanase needs xylan as the inducer, the two latter enzyme activities are formed once glucose is depleted. Cyclic nucleotides such as adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5' monophosphate (cGMP) exhibit a negative effect on enzyme synthesis if added to the culture media. Based on the fact that only cAMP is found in cells growing in glucose-rich media we propose a model for B. circulans WL-12 in which cAMP acts as a negative effector for regulating the synthesis of these enzymes. The model is not, however, extrapolated to other Bacillus species and all B. circulans strains.     

Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans

A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.     

Purification and characterization of chitinase A of Streptomyces cyaneus SP-27: an enzyme participates in protoplast formation from Schizophyllum commune mycelia

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity. Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to alpha-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with alpha-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.