Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.     

Structure and glycosylation of lipoteichoic acids in Bacillus strains.

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.     

Cloning and strong expression of a Bacillus subtilis WL-3 mannanase gene in B. subtilis

A gene encoding the mannanase of Bacillus subtilis WL-3, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and the nucleotide sequence of a 2.7-kb DNA fragment containing the mannanase gene was subsequently determined. The mannanase gene, designated manA, consisted of 1,080 nucleotides encoding polypeptide of 360 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to glycosyl hydrolase family 26. The manA gene was strongly expressed in B. subtilis 168 by cloning the gene downstream of a strong B. subtilis promoter of plasmid pJ27Delta 88U. In flask cultures, the production of mannanase by recombinant B. subtilis 168 reached maximum levels of 300 units/ml and 450 units/ml in LB medium and LB medium containing 0.3% locust bean gum, respectively. Based on the zymogram of the mannanase, it was found that the mannanase produced by recombinant B. subtilis could be maintained stably without proteolytic degradation during the culture time.     

Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage

Liquid packaging boards and blanks were examined for microbial contaminants. A total of 218 strains were identified and representatives of the most frequent species were characterized for their potential for food spoilage. Contaminants found were aerobic spore-forming bacteria, mostly Bacillus megaterium, B. licheniformis, B. cereus group, B. pumilus, Paenibacillus macerans, P. polymyxa, P. pabuli and B. flexus. Production of amylolytic, proteolytic, lipolytic and phospholipolytic enzymes was common. Approximately 50% of the B. cereus group strains were positive in the diarrhoeal enterotoxin immunoassay test or in the enterotoxin reversed passive latex agglutination test. Strains capable of growth at 6°C were found among B. cereus group, P. pabuli, P. validus, B. megaterium and P. polymyxa. All B. licheniformis strains grew at 55°C. The spores of B. licheniformis were most resistant to hydrogen peroxide. The B. cereus group strains were recognizable by fatty acid components not present in any of the other paperboard strains, 11-methyldodecanoic acid (13:0 iso) and trans-9-hexadecenoic acid (16:1 ω 7 trans), each contributing 7% or more to the total cellular fatty acids.     

Production and properties of beta-mannanase by free and immobilized cells of Aspergillus oryzae NRRL 3488

Seven fungi were tested for production of mannanases. The highest mannanase activities were produced by Aspergillus oryzae NRRL 3488 after 7 days in static cultures. Mannanases were induced by gum locust bean (1.0%). The highest mannanase activity was produced when a mixture of peptone, urea and ammonium sulphate was used as nitrogen source. Zn2+ or Co2+ favoured enzyme production. The immobilized cells on Ca-alginate and agar were able to produce beta-mannanase for four runs with a slight decrease in the activity. The optimum temperature for enzyme reaction was 50-55 degrees C at pH 6.0. In the absence of substrate the enzyme was thermostable retaining 75% activity for 1 h at 50 degrees C, and 68% activity for 1 h at 60 degrees C.     

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.     

长江流域水稻根际芽杆菌属固氮菌株的分离与鉴定

Rice rhizosphere soil samples were colected from 10 sites of 7 provinces in the Yangzi River Valley, and from the soil samples 16 endospore-forming strains with ARA (Acetylene Reduction Activity) were isolated, the nitrogen fixing ability was tested by the method of 15N tracer and the atom 15N% excess are ranged from 0.0297% to 0.4714%. The strains were identified as Bacillus licheniformis, B. subtilis, B. azotoformans, B. cereus, B. pumilus, B. brevis, B. megaterium, B. firmu.     

Screening of mannanases in actinomycetes and their potential application in the biobleaching of pine kraft pulps

Fifty actinomycete strains were screened for the production of mannanase activity during growth in both liquid and solid media. Streptomyces scabies CECT 3340 and Streptomyces ipomoea CECT 3341 were selected for their ability to produce high levels of mannanase (294.3 U/l and 242.9 U/l, respectively) during growth in liquid culture. β-Mannosidase (15.3 U/l) and α-galactosidase (7.7 U/l) activities were also detected in culture filtrate from S. scabies CECT 3340. Highest levels of mannanase activity for S. scabies CECT 3340 were achieved in media containing locust bean gum and asparagine (4.8 U mg⁻¹ protein) whilst in S. ipomoea CECT 3341 greatest activity was detected in media containing locust bean gum and yeast extract (13.2 U mg⁻¹ protein). No carboxymethylcellulase activity was detected. In biobleaching experiments, enzyme treatment, carried out with mannanase activity produced by S. ipomoea CECT 3341, followed by alkaline extraction of pine kraft pulp resulted in the release of colour (A ₄₆₅, 0.69) and chromophoric material from the pulp (A ₂₃₇, 12.9; A ₂₅₄, 6.9 and A ₂₈₀, 6.7). The ability of this enzyme complex to improve the bleaching of pine kraft pulps was also shown by a pulp brightness increase (2.4 units ISO) and a reduction in kappa number (from 21.4 units to 20.1 units) with the absence of variations on the viscosity values. Received: 23 February 1999 / Received revision: 1 April 1999 / Accepted: 6 April 1999     

几种芽胞杆菌溶血素BL基因及其溶血素的检测

用PCR方法对几种芽胞杆菌溶血素BL基因进行了检测,结果表明7株蜡样芽胞杆菌含有溶血素BL基因hblA、hblC、hblD,其他枯草芽胞杆菌、多粘类芽胞杆菌、地衣芽胞杆菌检测到部分溶血素基因;通过血平板培养的方法检测结果表明只有含有溶血素全部基因的菌株才会产生溶血环,从而为筛选不产生溶血素的有益芽胞杆菌奠定一定基础。     

长江流域水稻根际芽孢杆菌属固氮菌株的分离与鉴定

生物固氮资源的研究、开发和利用有利于发展持续农业,水稻田的氮素肥力比一般旱地高得多,活跃的生物固氮被认为是主要因素之一.苏宝林、崔宗均等系统地研究了我国北方稻区的固氮菌资源,分离鉴定出8属18种共35株固氮菌.在此基础上,我们对长江流域水稻根际异养固氮菌资源进行了系统研究.本文报道芽孢杆菌属(Bacillus)固氮菌株分离与鉴定的结果.     

Bacteria in food packaging paper and board

The bacteria of food packaging paper and board were studied. Most of the aerobic strains were spore-formers; members of the genus Bacillus with B. cereus group ( B. cereus, B. mycoides, B. thuringiensis ), B. polymyxa group ( B. polymyxa, B. circulans, B. macerans, B. pabuli ), B. brevis and B. licheniformis predominated. The main source of spore-forming bacteria in paper and board was the broke (rejected paper of board, which is repulped and recycled into the process). Gram-negative bacteria were rare in paper and board in spite of their abundance in the stock. A strain of B. pumilus forming clumping, hairy spores may be of significance in aseptic packaging.     

Bacteria in food packaging paper and board

The bacteria of food packaging paper and board were studied. Most of the aerobic strains were spore-formers; members of the genus Bacillus with B. cereus group (B. cereus, B. mycoides, B. thuringiensis), B. polymyxa group (B. polymyxa, B. circulans, B. macerans, B. pabuli), B. brevis and B. licheniformis predominated. The main source of spore-forming bacteria in paper and board was the broke (rejected paper or board, which is repulped and recycled into the process). Gram-negative bacteria were rare in paper and board in spite of their abundance in the stock. A strain of B. pumilus forming clumping, hairy spores may be of significance in aseptic packaging.     

The antimicrobial effect of organic acids, sour dough and nisin against Bacillus subtilis and B. licheniformis isolated from wheat bread

Prevention of growth in wheat bread for more than 6 d of approximately 10⁶ rope-producing Bacillus subtilis spores per gram of dough was achieved by addition of propionic or acetic acids at levels of 0·10% v/w (based on flour weight), or by addition of 15% sour dough fermented with Lactobacillus plantarum C11, Lact. brevis L62, Lact. plantarum ('vege-start 60'), Lact. plantarum (ch 20), Lact. maltaromicus (ch 15), or the commercial sour dough starter culture, Lact. sanfrancisco L99. These cultures resulted in an amount of total titratable acids above 10 in the sour dough and a pH value below 4·8 in the final bread. Bacteriocin-producing lactic acid bacteria added as starter cultures in wheat dough and nisin (Nisaplin) at levels up to 100 p.p.m. g⁻¹ flour had no effect against B. subtilis and B. licheniformis strains, despite the fact that nisin-producing strains of Lactococcus lactis ssp. lactis among 186 strains of lactic acid bacteria had demonstrated inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay.     

地衣芽胞杆菌dif序列的功能鉴定

地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difB8序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difB8以。序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI’：：difGm,通过电击转化法将质粒pHY-XI’：：difGm导入B．1icheniformis ATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer／dif／f位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。     

Biosynthesis of inulinases by Bacillus bacteria

Biosynthesis of extracellular inulinase by bacteria Bacillus polymyxa 29, B. polymyxa 722, and B. subtilis 68 was studied. The optimal parameters for the producer growth were as follows: pH 7.0, 33-35 degrees C, the growth duration within 72 h. The presence of mineral reduced or of organic nitrogen was necessary for the enzyme biosynthesis. The inulinase biosynthesis was sharply activated in the presence of carbohydrates. B. polymyxa 722 and B. polymyxa 29 displayed the maximal activity on a starch-containing culture medium, the maximal activity of B. subtilis 68 was found in the presence of sucrose. Inulin did not induce the inulinase biosynthesis by the strains studied. The time course of bacteria growth and of the enzyme biosynthesis was studied.     

Identification of bacteria in pasteurized zucchini purées stored at different temperatures and comparison with those found in other pasteurized vegetable purées

One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25 degrees C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis. A phylogenetic analysis pointed to one new species of Bacillus and three new species of Paenibacillus among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as B. circulans (group 2) or B. circulans/B. macerans/B. polymyxa were assigned to a new Paenibacillus taxon phylogenetically related to P. azotofixans. Storage conditions at 4 degrees C favored the development of "B. macroides/B. maroccanus" and Paenibacillus spp. in zucchini purées and Paenibacillus spp. in other purées. Storage conditions at 20 to 25 degrees C favored the development of B. subtilis group (B. licheniformis and B. subtilis) and B. cereus group strains. At 10 degrees C, Paenibacillus spp. were always present at high frequencies, whereas the occurrence of B. macroides/B. maroccanus (in zucchini purées), B. cereus, and B. pumilus varied with the experiment.     

Microbiology of controlled rotting of egusi (Colocynthis citrullus L.) fruits for the harvesting of the seeds

Microorganisms isolated from naturally rotting egusi fruits which are used as a source of dietary seeds, were mainly bacteria: Bacillus subtilis, B. licheniformis, B. polymyxa, B. megaterium, B. pumilus, Lactobacillus plantarum, La. brevis, Leuconostoc mesenteroides, Enterobacter aerogenes, E. cloacae, Klebsiella aerogenes, K. pneumoniae, Staphylococcus epidermidis, S. aureus, Micrococcus and Candida spp. The rotting period of egusi fruits could be decreased from 120 to 72 h by using a mixture of B. subtilis and B. licheniformis.     

Numerical analysis and DNA base compositions of some thermophilic Bacillus species.

Morphological, physiological, and biochemical characteristics and the DNA base compositions of 133 thermophilic Bacillus strains were determined. A total of 54 of these strains were received as identified species (mainly Bacillus stearothermophilus, Bacillus coagulans, Bacillus brevis, and Bacillus licheniformis) from international culture collections, and 79 newly isolated strains, which were isolated mainly from sugar diffusion juices of Italian plants, were also examined. Numerical taxonomy techniques (simple matching coefficient and unweight pair grouping using the mathematical average) and DNA G + C values showed that the strains aggregated into nine clusters. Both B. licheniformis and B. brevis were well separated from the other organisms. B. stearothermophilus and B. coagulans were confirmed as separate clusters and exhibited greater heterogeneity than previously shown. The B. stearothermophilus strains clustered into four groups, three of which have been recognized previously by other authors; the members of the fourth group had distinctive characteristics, including considerable biochemical inertness, an inability to grow at temperatures greater than 60 degrees C, and a high G + C content. Within the B. coagulans cluster the strains with characteristics very similar to those of the new species Bacillus smithii clustered together. However, the remaining strains were still clearly separated into two groups; one of these groups was considered B. coagulans sensu stricto, and the other was distinguished by morphological and biochemical criteria, such as spores which do not swell the sporangia, utilization of citrate, a higher proteolytic activity, and acidification of some carbohydrates. Our results were confirmed by comparing them with distinctive characteristics of recently described thermophilic Bacillus species.     

Isolation and characterization of an active mannanase-producing anaerobic bacterium, Clostridium tertiumKT-5A, from lotus soil

Of 10 strains of mannanase-producing anaerobicbacteria isolated from soils and methanogenic sludges, Clostridium tertium KT-5A,which was isolated from lotus soil, produced high amounts of extracellular β-1,4-mannanase. The isolate was an aerotolerant anaerobe without quinon systems; the cell growthcultivated with no addition of reducing agents was also stable. High yields of mannanasewere obtained by inducing enzyme production with galactomannan guar gum and beef extract/peptone as carbon and nitrogen sources, respectively. Fermentation endproducts on galactomannan fermentation were formate, acetate, lactate, butyrate, carbondioxide and hydrogen. The extracellular mannanase displayed high activity ongalactomannans of locust bean gum galactose/mannose (G/M) ratio 1:4 and spinogum (G/M 1:3), but weak activity on guar gum galactomannan (G/M 1:2) and konjac glucomannan. As far as is known, this is the first report on the isolation of an activemannanase-producing anaerobic bacterium from natural environments.     

Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp

Aim:  To investigate the effects of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium in terms of toxin and growth of pathogenic Vibrio harveyi .
Methods and Results:  Three Bacillus probionts were isolated from probiotic BZT aquaculture and identified using a 16S rDNA sequence. Growth inhibition assay showed that supernatants from the 24-h culture of three Bacillus species were able to inhibit the growth of V. harveyi (LMG 4044); B. subtilis was the most effective based on the well diffusion method. Results of a liquid culture model showed that B. subtilis was also widely effective in inhibiting three strains of V. harveyi (isolated from Thailand, the Philippines and LMG 4044), and that both B. licheniformis and B. megaterium inhibit the growth of V. harveyi isolated from the Philippines. Moreover, a haemolytic activity assay demonstrated that V. harveyi (IFO 15634) was significantly decreased by the addition of B. licheniformis or B. megaterium supernatant.
Conclusions:  Bacillus subtilis inhibited Vibrio growth, and both B. licheniformis and B. megaterium suppressed haemolytic activity in Vibrio .
Significance and Impact of the Study:  The cell-free supernatants produced by Bacillus probionts inhibit Vibrio disease, and Bacillus probionts might have an influence on Vibrio cell-to-cell communications.