Acid Pullulanase from Bacillus polymyxa MIR-23

Within the frame of a screening program aimed at the isolation of amylolytic sporeformers, one strain with high amylolytic activity designated MIR-23 was selected. The microbial characterization was carried out by morphological and biochemical tests and, by means of statistical treatment, was identified asBacillus polymyxa. The organism could grow in acidic conditions (pH 5.0) on a starch medium and produce α-amylase, pullulanase, and α-glucosidase. Batch cultures showed the highest enzyme activities in the stationary phase. Pullulanase activity exhibited an optimal temperature of 52–57°C at pH 4.5–5.5. These properties would allow its use in the saccharification processes in the starch industries.     

多粘类芽孢杆菌BS04拮抗成分分离纯化及其特性

用改良的琼脂孔扩散实验进行生物活性跟踪,分离纯化了多粘类芽孢杆菌BS04的拮抗成分,将纯化样品溶解在水中,测定其对青枯假单胞菌的拮抗作用,结果显示BS04拮抗成分能耐受广泛的pH,并且热稳定性好,活性不受蛋白酶K和胰蛋白酶影响,这些说明细菌BS04具有作为生物防治制剂的潜力。同时,拮抗成分的薄层层析、红外光谱以及质谱结果暗示此活性成分可能为4个分子量相近的肽类物质。     

Evaluation of solid and submerged fermentations for the production of cyclodextrin glycosyltransferase by Paenibacillus campinasensis H69-3 and characterization of crude enzyme

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Cyclodextrins have been shown to have a number of applications in the food, cosmetic, pharmaceutical, and chemical industries. In the current study, the production of CGTase by Paenibacillus campinasensis strain H69-3 was examined in submerged and solid-state fermentations. P. campinasensis strain H69-3 was isolated from the soil, which grows at 45°C, and is a Gramvariable bacterium. Different substrate sources such as wheat bran, soybean bran, soybean extract, cassava solid residue, cassava starch, corn starch, and other combinations were used in the enzyme production. CGTase activity was highest in submerged fermentations with the greatest production observed at 48–72 h. The physical and chemical properties of CGTase were determined from the crude enzyme produced from submerged fermentations. The optimum temperature was found to be 70–75°C, and the activity was stable at 55°C for 1 h. The enzyme displayed two optimum pH values, 5.5 and 9.0 and was found to be stable between a pH of 4.5 and 11.0.     

Fusaricidins from Paenibacillus polymyxa M‐1, a family of lipohexapeptides of unusual complexity—a mass spectrometric study

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P . polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15‐guanidino‐3‐hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P . polymyxa strain M‐1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix‐assisted laser‐desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed‐phase (rp) HPLC. Their structure was determined by MALDI‐LIFT‐TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m /z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino‐3‐hydroxyhepta‐ and nonadecanoic acid as fatty acid constituents. Apparently, the end‐standing guanidine group is not modified as shown by direct infusion nano‐electrospray ionization mass spectrometry (nano‐ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd.     

Lessons from the first international proficiency test for the detection of spores from the honey bee pathogen Paenibacillus larvae

We report on the first proficiency test in the context of honey bee disease testing on a broad international scale. Honey samples were distributed to 12 participating laboratories for isolation and identification of spores of the etiological agent of American foulbrood, Paenibacillus larvae. Of the 11 laboratories responding to this proficiency testing event, only 6 (54%) provided data that were in full agreement with the previously determined status of the honey samples; eight (72%) laboratories fell within the threshold for qualification as defined a priori in this proficiency test (=level of agreement of at least 90%). Some lessons can now be drawn from the organizational point of view and will certainly improve new initiatives to be taken.     

Characterization of novel neopullulanase fromBacillus polymyxa

Bacillus polymyxa CECT 155 produces an extracellular neopullulanase activity that degrades pullulan to panose. This activity was stimulated by the presence of pullulan in the culture, and repressed by glucose. The apparent mol wt determined for the enzyme was 58 kDa. The optimum pH and temperature for neopullulanase activity were pH 6.0 and 50°C, respectively. The enzyme was stable in a pH range of 4.0–8.0, and temperatures up to 60°C. These properties make it suitable for the saccharification processes in the starch industries.     

从作物根际分离的多粘芽孢杆菌固氮作用的研究

用乙炔还原法，研究了从作物根际分离的５１株多粘芽孢杆菌的固氮作用，结果表明：２８株在有氧条件下培养能合成固氮酶并固氮，其中ＨＷ－１菌株固氮酶活性达７８．３×１０－６ｍｏｌＬ－１ｈ－１．该菌株固氮活性在对数生长后期（３６ｈ）最高，最适固氮培养条件是３５℃和ｐＨ８．０．该菌株在有５％氧气的条件下培养时，固氮活性最高，在初始培养和乙炔还原体系中分别注入５０％氧气仍能检测到乙炔还原活性．培养基中ＮＨ＋４阻遏该菌株固氮酶合成，但对已合成的固氮酶活性无影响．     

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography

American foulbrood disease (AFB) is the main devastating disease that affects honeybees’ brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin‐1 and Chymotrypsin‐1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.