Impacts of UV radiation on Bacillus biocontrol agents and their resistance mechanisms.

Bacillus biocontrol agent(s) BCA(s) such as Bacillus cereus, Bacillus thuringiensis and Bacillus subtilis have been widely applied to control insects' pests of plants and pathogenic microbes, improve plant growth, and facilitate their resistance to environmental stresses. In the last decade, researchers have shown that, the application of Bacillus biocontrol agent(s) BCA(s) optimized agricultural production yield, and reduced disease risks in some crops. However, these bacteria encountered various abiotic stresses, among which ultraviolet (UV) radiation severely decrease their efficiency. Researchers have identified several strategies by which Bacillus biocontrol agents resist the negative effects of UV radiation, including transcriptional response, UV mutagenesis, biochemical and artificial means (addition of protective agents). These strategies are governed by distinct pathways, triggered by UV radiation. Herein, the impact of UV radiation on Bacillus biocontrol agent(s) BCA(s) and their mechanisms of resistance were discussed.

Study of the isomeric Maillard degradants, glycosylamine and Amadori rearrangement products, and their differentiation via MS2 fingerprinting from collision-induced decomposition of protonated ions.

RATIONALE: The focus of this work was to study glycosylamine and Amadori rearrangement products (ARPs), the two major degradants in the Maillard reactions of pharmaceutical interest, and utilize their MS2 fingerprints by liquid chromatography/high-resolution tandem mass spectrometry (LC/HRMS2 ) to quickly distinguish the two isomeric degradants. These two types of degradants are frequently encountered in the compatibility and stability studies of drug products containing primary or secondary amine active pharmaceutical ingredients (APIs), which are formulated with excipients consisting of reducing sugar functionalities. METHODS: Vortioxetine was employed as the primary model compound to react with lactose to obtain the glycosylamine and ARP degradants of the Maillard reaction, and their MS2 spectra (MS2 fingerprints) were obtained by LC/MS2 . Subsequently, the two degradants were isolated via preparative HPLC and their structures were confirmed by one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) determination. RESULTS: The MS2 fingerprints of the two degradants display significantly different profiles, despite the fact that many common fragments are observed. Specifically, protonated glycosylamine shows a prominent characteristic fragment of [Mvort + C2 H3 O]+ at m/z 341 (Mvort is the vortioxetine core), while protonated ARP shows a prominent characteristic fragment of [Mvort + CH]+ at m/z 311. Further study of the Maillard reactions between several other structurally diverse primary/secondary amines and lactose produced similar patterns. CONCLUSIONS: The study suggests that the characteristic MS2 fragment peaks and their ratios may be used to differentiate the glycosylamine and ARP degradants, the two isomeric degradants of the Maillard reaction, which are commonly encountered in finished dosage forms of pharmaceutical products containing primary and secondary amine APIs.

Ecologically controlling insect and mite pests of tea plants with microbial pesticides: a review.

Insect and mite pests are damaging stressors that are threatening the cultivation of tea plants, which result in enormous crop loss. Over the years, the effectiveness of synthetic pesticides has allowed for its prominent application as a control strategy. However, the adverse effects of synthetic pesticides in terms of pesticide residue, environmental contamination and insect pest resistance have necessitated the need for alternative strategies. Meanwhile, microbial pesticides have been applied to tackle the damaging activities of the insect and mite pests of tea plants, and their performances were scientifically adjudged appreciable and environmental friendly. Herein, entomopathogenic microbes that were effective against tea geometrid (Ectropis obliqua Prout), tea green leafhopper (Empoasca onukii Matsuda), paraguay tea ampul (Gyropsylla spegazziniana), tea mosquito bug (Helopeltis theivora Waterhouse) and red spider mite (Oligonychus coffea Nietner) have been reviewed. The current findings revealed that microbial pesticides were effective and showed promising performances against these pests. Overall, this review has provided the basic and integrative information on the integrated pest management (IPM) tool(s) that can be utilized towards successful control of the aforementioned insect and mite pests.

Identification and partial purification of thuricin 4AJ1 produced by Bacillus thuringiensis.

Thuricin 4AJ1, produced by Bacillus thuringiensis strain 4AJ1, showed inhibition activity against Bacillus cereus 0938 and ATCC 10987. It began to appear in the stationary phase and reached its maximum activity level of 209.958 U at 18 h against B. cereus 0938 and 285.689 U at 24 h against B. cereus ATCC 10987. Tricine-SDS-PAGE results showed that the partly purified thuricin 4AJ1 was about 6.5 kDa. The molecular weights of the known B. thuringiensis bacteriocins and the ones obtained by the two mainstream websites for predicting bacteriocins were inconsistent with the size of the thuricin 4AJ1, indicating that the bacteriocin obtained in this study may have a novel structure. Based on the biochemical properties, the thuricin 4AJ1 activities increased after treatment with proteinase K and lipase II, and were not affected by a-amylase, catalase, α-chymotrypsin VII and α-chymotrypsin II. It was heat tolerant, being active up to 90º C. In the pH 3-10 range, it maintained most of its activity. Finally, the sensitivity of the strain 4AJ1 to commonly used antibiotics was tested. In view of its stability and antibacterial activity, thuricin 4AJ1 may be applied as a food biopreservative.

Structure Elucidation and Mechanistic Study of a New Dimeric Degradant in Ropinirole Hydrochloride Extended-Release Tablets.

PURPOSE: The goal of the study was to elucidate the structure of a new degradant (1,3'-Dimer), generated in the stability testing of ropinirole extended-release tablets, and the formation mechanism of 1,3'-Dimer and its isomer (3,3'-Dimer). METHODS: The strategy of combining LC-PDA/UV-MSn (n = 1, 2) and NMR in conjunction with mechanism-based forced degradation study was employed to identify the structure of the unknown degradant and the formation mechanism of this dimeric degradant as well as its isomer, 3,3'-Dimer. The forced degradation was conducted by treating ropinirole API with formaldehyde under alkaline catalysis. A compatibility study between ropinirole and lactose was also performed. RESULTS: The degradant was isolated from the forced degradation sample and characterized by LC-PDA/UV-MSn as well as NMR measurement. The impurity was identified as a new dimeric degradant of ropinirole connected by a methylene bridge via the 1- and 3'-position of each ropinirole unit (i.e., 1,3'-Dimer of ropinirole), which is an isomer of a known dimeric degradant of ropinirole, namely 3,3'-Dimer. CONCLUSIONS: The newly occurred unknown degradant in ropinirole extended-release tablets was elucidated as the methylene-bridged 1,3'-Dimer of ropinirole. Based on the mechanistic study, 1,3'-Dimer and its isomer (3,3'-Dimer) were both formed by the reaction of ropinirole with residual formaldehyde present or formed in lactose, a main excipient of the formulation.

For: Pesticide biochemistry and physiology recG is involved with the resistance of Bt to UV.

As an ATP-dependent DNA helicase, RecG can repair DNA replication forks in many organisms. However, knowledge of recG in Bacillus thuringiensis (Bt) is limited. In our previous study, recG was found damaged in Bt LLP29-M19, which was more resistant to ultraviolet light (UV) after exposing Bt LLP29 to UV for 19 generations. To further understand the function of recG in the mechanism of Bt UV resistance, recG was knocked out and recovered with homologous recombination technology in Bt LLP29. Comparing the resistance of the different mutants to UVB, Bt ∆recG-LLP29 lacking recG was found more sensitive to UVB, hydroxyurea (HU) and H2O2 than LLP29 and the complementation strain. To compare the expression level of recG in the Bt strains under different UV treatments, Quantitative Real-time PCR (RT-qPCR) of recG was performed in the tested Bt strains, which showed that the expression level of recG in Bt ∆recG-LLP29 was substantially lower than that in the original strain and complementation strain. Interestingly, when exposed to UV for 20 min, RecG expression in both Bt LLP29 and Bt recG-R was the highest. The unwinding activity of recG in Bt LLP29 and the complementation strain were also found higher than that of the recG knockout strain, Bt ∆recG-LLP29. These results demonstrate that recG is involved with the resistance of Bt to UV. These findings not only enhance the understanding of the Bt UV resistance mechanism, but also provide an important theoretical basis for the application of Bt.

Heterologous expression and purification of BtCspB, a novel cold-shock protein-like bacteriocin from Bacillus thuringiensis BRC-ZYR2.

A novel Bacillus thuringiensis (Bt) bacteriocin BtCspB, active against a food-borne pathogen Bacillus cereus, was identified and purified by a traditional four-step chromatographic process with low yield (44.5 µg/L) in our lab previously. The aim of this study was to dramatically increase its yield by heterologous expression of BtCspB. The BtCspB gene from Bt BRC-ZYR2 was successfully heterologously expressed in Escherichia coli BL21 (DE3). Affinity chromatography was used to obtain the pure BtCspB up to 20 mg/L. The purified BtCspB showed a MIC value of 12.5 µg/mL and a MBC value of 50.0 µg/mL against Bacillus cereus ATCC 10987. The bacteriocin activity of BtCspB against B. cereus ATCC 10987 was further directly detected in a gel-overlay assay. The anti-B. cereus activity, however, was lower than the bacteriocin purified by the traditional four-step chromatographic process probably because of structural modifications. Compared with the traditional method, the yield of the bacteriocin by heterologous expression increased by 449 times, and the purification step was dramatically simplified, which laying a foundation for the industrial production of this novel cold-shock protein-like bacteriocin BtCspB active against B. cereus.

Nematicidal Activity of Cry1Ea11 from Bacillus thuringiensis BRC-XQ12 Against the Pine Wood Nematode (Bursaphelenchus xylophilus).

The nematicidal activity of 92 Bacillus thuringiensis strains against the pine wood nematode Bursaphelenchus xylophilus, one of the world's top 10 plant-parasitic nematodes, was determined. The insecticidal crystal proteins (ICPs) from Bacillus thuringiensis BRC-XQ12 were the most toxic to Bursaphelenchus xylophilus, with a lethal concentration 50 (LC50) of 32.13 µg/ml. Because the ICPs expressed by Bacillus thuringiensis BRC-XQ12 were closest to Cry1Ea6 and B. thuringiensis BRC-XQ12 contained four kinds of cry1 subgenes (cry1Aa, cry1Cb, cry1Ea, and cry1Ia), Cry1Ea was most likely to be the key active component against the nematode. The 3,516-bp cry1Ea11 gene from BRC-XQ12, as designated by the B. thuringiensis δ-endotoxin nomenclature committee, was expressed in Escherichia coli. Purified Cry1Ea11 showed an LC50 of 32.53 and 23.23 µg/ml at 24 and 48 h, with corresponding virulence equations of Y = 32.15X + 1.38 (R2 = 0.9951) and Y = 34.29X + 3.16 (R2 = 0.9792), respectively. In order to detect the pathway of B. thuringiensis Cry1Ea11 into Bursaphelenchus xylophilus, the nematode was fed with NHS-rhodamine-labeled GST-Cry1Ea11. The results of confocal laser-scanning microscopy showed that the 159-kDa GST-Cry1Ea11 could be detected in the stylet and the esophageal lumen of the pine wood nematode, indicating that GST-Cry1Ea11 could enter into the nematode through the stylet. As far as we know, no Cry1 proteins have been shown to have activity against plant-parasitic nematodes before. These results demonstrate that Cry1Ea11 is a promising nematicidal protein for controlling pine wilt disease rendered by B. xylophilus, further dramatically broadening the spectrum of Bacillus thuringiensis ICPs.

Keggin-type polyoxometalate-based ionic liquid gels.

A series of reversible phase transformation ammonium- and phosphonium-based polyoxometalate ionic liquid (POM-IL) gels were synthesized and studied with a focus on the correlation between their physicochemical properties and their chemical structure. The products were successfully characterized by IR, UV, XRD and TG-DTA, and their ionic conductivities were measured. The Keggin-type heteropolyanion clusters decorated with long alkyl chains demonstrated a tendency to exhibit a gel state at room temperature, while all the gels transformed into liquids after heating and then recovered after cooling. With a decrease in the alkyl chain length, a significant improvement in the thermal stability and conductivity of the ammonium-based POM-IL gels can be achieved. Moreover, compared with the corresponding ammonium compound, phosphonium-based POM-IL gel was found to be more stable at high temperature and exhibited better conductivity.

RAP-PCR fingerprinting reveals time-dependent expression of development-related genes following differentiation process of Bacillus thuringiensis.

Gene expression profiles are important data to reveal the functions of genes putatively involved in crucial biological processes. RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and specifically primed reverse transcription polymerase chain reaction (RT-PCR) were combined to screen differentially expressed genes following development of a commercial Bacillus thuringiensis subsp. kurstaki strain 8010 (serotype 3a3b). Six differentially expressed transcripts (RAP1 to RAP6) were obtained. RAP1 encoded a putative triple helix repeat-containing collagen or an exosporium protein H related to spore pathogenicity. RAP2 was homologous to a ClpX protease and an ATP-dependent protease La (LonB), which likely acted as virulence factors. RAP3 was homologous to a beta subunit of propionyl-CoA carboxylase required for the development of Myxococcus xanthus. RAP4 had homology to a quinone oxidoreductase involved in electron transport and ATP formation. RAP5 showed significant homology to a uridine kinase that mediates phosphorylation of uridine and azauridine. RAP6 shared high sequence identity with 3-methyl-2-oxobutanoate-hydroxymethyltransferase (also known as ketopantoate hydroxymethyltransferase or PanB) involved in the operation of the tricarboxylic acid cycle. The findings described here would help to elucidate the molecular mechanisms underlying the differentiation process of B. thuringiensis and unravel novel pathogenic genes.

Development of a Safe and Effective Bacillus thuringiensis-Based Nanobiopesticide for Controlling Tea Pests.

Mg(OH)2 was used as the nanocarrier of the Bacillus thuringiensis (Bt) Cry1Ac protein, and the synthesized Cry1Ac-Mg(OH)2 composites were regular and uniform nanosheets. Nano-Mg(OH)2 could effectively improve the insecticidal effect of the Cry1Ac protein toward Ectropis obliqua. It could enhance the damage degree of the Cry1Ac protein to intestinal epithelial cells and microvilli, induce and enrich the production of reactive oxygen species (ROS) in the midgut, and enhance the degradation of the Cry1Ac protein into active fragments. Furthermore, an anti-rinsing assay showed that the Cry1Ac-Mg(OH)2 composites were bound to the notch structure of the tea leaf surface. The retention of the Cry1Ac protein increased by 11.45%, and sprayed nano-Mg(OH)2 was rapidly absorbed by different tissues of tea plants. Moreover, nano-Mg(OH)2 and composites did not significantly affect non-target organisms. These results show that nano-Mg(OH)2 can serve as a safe and effective biopesticide carrier, which provides a new approach for stable and efficient Bt preparation.

Different toxicity of the novel Bacillus thuringiensis (Bacillales: Bacillaceae) strain LLP29 against Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae).

Bacillus thuringiensis (Bt) (Berliner) strain LLP29 produces a crystal protein Cyt1Aa6 toxic to mosquito vectors of human diseases. However, the susceptibility of Culex quinquefasciatus (Say) in the current study was 8.25 times higher than that of Aedes albopictus (Skuse) with this single protein Cyt1Aa6 purified from LLP29. To understand the mechanism of the novel mosquitocidal protein, the binding characteristic of brush border membrane vesicles from the two tested mosquitoes was investigated. Enzyme-linked immunosorbent assay showed that Cyt1Aa6 bound to the two mosquitoes' brush border membrane vesicles. However, the titer of Ae. albopictus was a little higher than that of Cx. quinquefasciatus, with 3.21 and 2.91, respectively. Ligand Western blot analysis showed Cyt1Aa6 toxin specifically bound to the same three proteins (i.e., 68, 54, and 26 kDa) in the two mosquitoes, but one another protein, approximately to 37 kDa, could just be detected in Cx. quinquefasciatus. However, little difference was found in the test of immunohistochemistry. Cyt1Aa6 was detected in the midguts of both mosquitoes with histopathological changes. It would of great importance to the knowledge of the novel toxin against to Cx. quinquefasciatus and Ae. albopictus.

Towards Sustainable Green Adjuvants for Microbial Pesticides: Recent Progress, Upcoming Challenges, and Future Perspectives.

Microbial pesticides can be significantly improved by adjuvants. At present, microbial pesticide formulations are mainly wettable powders and suspension concentrations, which are usually produced with adjuvants such as surfactants, carriers, protective agents, and nutritional adjuvants. Surfactants can improve the tension between liquid pesticides and crop surfaces, resulting in stronger permeability and wettability of the formulations. Carriers are inert components of loaded or diluted pesticides, which can control the release of active components at appropriate times. Protective agents are able to help microorganisms to resist in adverse environments. Nutritional adjuvants are used to provide nutrients for microorganisms in microbial pesticides. Most of the adjuvants used in microbial pesticides still refer to those of chemical pesticides. However, some adjuvants may have harmful effects on non-target organisms and ecological environments. Herein, in order to promote research and improvement of microbial pesticides, the types of microbial pesticide formulations were briefly reviewed, and research progress of adjuvants and their applications in microbial pesticides were highlighted, the challenges and the future perspectives towards sustainable green adjuvants of microbial pesticides were also discussed in this review.

Microbial ecology and association of Bacillus thuringiensis in chicken feces originating from feed.

To explain the association of Bacillus thuringiensis (Bt) with animal feces, an ecological analysis in chickens was conducted by introducing a cry(-) strain marked by production of green fluorescent protein (GFP). After feeding with the tagged Bt strains, the feces of the tested chickens were collected at different times, isolated, and the morphology of Bt was observed. It was shown that Bt strain HD-73GFP in spore form could be isolated from feces of chickens for a period of 13 d, and then it disappeared thereafter. Bt could be detected only up to day 4 (but not thereafter), when chickens were fed with vegetative cells of HD-73GFP. To confirm the source of newly isolated strains, the gfp gene was examined by polymerase chain reaction (PCR), which showed that all the isolated strains harbored the marker gene. Recent data from isolation and PCR had suggested that fecal Bt strains had originated from food. Chicken tissues were thus dissected to isolate Bt strains and to investigate whether Bt could be located in vivo. Bt was located within the duodenum in spore form. Compared to the morphology of the isolated strains at different growth times, the growth rates of all the tested Bt had little changes when passing through the digestive system to the feces. Dissection of the chickens confirmed that Bt was safe for the tested animal.

A differentially displayed mRNA related to resistance to Bacillus thuringiensis israelensis of Aedes albopictus selected in vitro-activated Cyt1Aa6.

We compared the differential display of Aedes albopictus cells, both resistant and susceptible to Bacillus thuringiensis israelensis (Bti), using differentially displayed reverse transcription polymerase chain reaction. We found 1 band about 200 base pairs long. After cloning and sequencing, the differentially expressed gene was similar to some partial messenger ribonucleic acid of Ae. aegypti, Culex quinquefasciatus, and Anopheles gambiae, rather than Ae. albopictus. This will be of some value for clarifying the mechanism of mosquito resistance to Bti products.

Characterization of Microorganisms from Protaetia brevitarsis Larva Frass.

Decomposers play an important role in the biogeochemical cycle. Protaetia brevitarsis larvae (PBLs) can transform wastes into frass rich in humic acid (HA) and microorganisms, which may increase the disease resistance of plants and promote plant growth. Beyond HA, the microorganisms may also contribute to the biostimulant activity. To address this hypothesis, we investigated the potential microbial community in the PBL frass samples and elucidated their functions of disease resistance and plant growth promotion. High-throughput sequencing analysis of four PBL-relevant samples showed that their frass can influence the microbial community of the surrounding environment. Further analysis showed that there were many microorganisms beneficial to agriculture, such as Bacillus. Therefore, culturable Bacillus microbes were isolated from frass, and 16S rDNA gene analysis showed that Bacillus subtilis was the dominant species. In addition, some Bacillus microorganisms isolated from the PBL frass had antibacterial activities against pathogenic fungi. The plant growth promotion pot experiment also proved that some strains promote plant growth. In conclusion, this study demonstrated that the microorganisms in the PBL frass are conducive to colonizing the surrounding organic matrix, which will help beneficial microbes to increase the disease resistance of plants and promote plant growth.

Plant Polysaccharides Modulate Biofilm Formation and Insecticidal Activities of Bacillus thuringiensis Strains.

After the biological pesticide Bacillus thuringiensis (Bt) is applied to the field, it has to remain on the surface of plants to have the insecticidal activities against insect pests. Bt can form biofilms on the surface of vegetable leaves, which were rich in polysaccharides. However, the relationship between polysaccharides of the leaves and the biofilm formation as well as the insecticidal activities of Bt is still unknown. Herein, this study focused on the effects of plant polysaccharides pectin and xylan on biofilm formation and the insecticidal activities of Bt strains. By adding pectin, there were 88 Bt strains with strong biofilm formation, 69 strains with weak biofilm formation, and 13 strains without biofilm formation. When xylan was added, 13 Bt strains formed strong biofilms, 98 strains formed weak biofilms, and 59 strains did not form biofilms. This indicated that two plant polysaccharides, especially pectin, modulate the biofilm formation of Bt strains. The ability of pectin to induce biofilm formation was not related to Bt serotypes. Pectin promoted the biofilms formed by Bt cells in the logarithmic growth phase and lysis phase at the air-liquid interface, while it inhibited the biofilms formed by Bt cells in the sporangial phase at the air-liquid interface. The dosage of pectin was positively correlated with the yield of biofilms formed by Bt cells in the logarithmic growth phase or lysis phase at the solid-liquid interfaces. Pectin did not change the free-living growth and the cell motility of Bt strains. Pectin can improve the biocontrol activities of the spore-insecticidal crystal protein mixture of Bt and BtK commercial insecticides, as well as the biofilms formed by the logarithmic growth phase or lysis phase of Bt cells. Our findings confirmed that plant polysaccharides modulate biofilm formation and insecticidal activities of Bt strains and built a foundation for the construction of biofilm-type Bt biopesticides.

Beyond Risk: Bacterial Biofilms and Their Regulating Approaches.

Bacterial biofilms are complex surface attached communities of bacteria held together by self-produced polymer matrixs mainly composed of polysaccharides, secreted proteins, and extracellular DNAs. Bacterial biofilm formation is a complex process and can be described in five main phases: (i) reversible attachment phase, where bacteria non-specifically attach to surfaces; (ii) irreversible attachment phase, which involves interaction between bacterial cells and a surface using bacterial adhesins such as fimbriae and lipopolysaccharide (LPS); (iii) production of extracellular polymeric substances (EPS) by the resident bacterial cells; (iv) biofilm maturation phase, in which bacterial cells synthesize and release signaling molecules to sense the presence of each other, conducing to the formation of microcolony and maturation of biofilms; and (v) dispersal/detachment phase, where the bacterial cells depart biofilms and comeback to independent planktonic lifestyle. Biofilm formation is detrimental in healthcare, drinking water distribution systems, food, and marine industries, etc. As a result, current studies have been focused toward control and prevention of biofilms. In an effort to get rid of harmful biofilms, various techniques and approaches have been employed that interfere with bacterial attachment, bacterial communication systems (quorum sensing, QS), and biofilm matrixs. Biofilms, however, also offer beneficial roles in a variety of fields including applications in plant protection, bioremediation, wastewater treatment, and corrosion inhibition amongst others. Development of beneficial biofilms can be promoted through manipulation of adhesion surfaces, QS and environmental conditions. This review describes the events involved in bacterial biofilm formation, lists the negative and positive aspects associated with bacterial biofilms, elaborates the main strategies currently used to regulate establishment of harmful bacterial biofilms as well as certain strategies employed to encourage formation of beneficial bacterial biofilms, and highlights the future perspectives of bacterial biofilms.

Solution degradant of mirabegron extended release tablets resulting from a Strecker-like reaction between mirabegron, minute amounts of hydrogen cyanide in acetonitrile, and formaldehyde in PEG during sample preparation.

During the related substances testing of mirabegron extended release tablets, an unknown peak was observed in HPLC chromatograms in a level exceeding the identification threshold. By using a strategy that combines LC-PDA/UV-MSn with mechanism-based stress studies, the unknown peak was rapidly identified as cyanomethyl mirabegron, a solution degradant that is caused by a Strecker-like reaction between the API, formaldehyde (an impurity in PEG), and HCN (an impurity in HPLC grade acetonitrile). The mechanism of the solution degradation chemistry was verified by stressing mirabegron with formaldehyde and trimethylsilyl cyanide (TMSCN, a synthetic reagent that generates HCN upon contact with water), in which the secondary amine group of mirabegron first reacts with formaldehyde to form the iminium ion intermediate; the latter then undergoes a nucleophilic attack by cyanide to yield the cyanomethyl mirabegron. The structure of the impurity was further confirmed through the synthesis of the impurity and subsequent structure characterization by 1D and 2D NMR. Due to the ubiquitous presence of formaldehyde in pharmaceutical excipients (e.g., PEG and polysorbate) and trace amount of HCN in HPLC grade acetonitrile, this type of solution degradation would likely occur in sample preparations of pharmaceutical finished products containing APIs with primary and secondary amine moieties. In a GMP environment, such an event may trigger undesirable out-of-specification (OOS) investigations; the results of this paper should help resolve such OOS investigations or even prevent these events from happening in the first place.

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

AHL-lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specifically hydrolyzes N-acyl-homoserine lactones (AHLs) secreted by Gram-negative bacteria and thereby attenuates the symptoms caused by plant pathogens. In this study, an aiiA gene was cloned from Bacillus subtilis BS-1 by PCR with a pair of degenerate primers. The deduced 250 amino acid sequence contained two small conserved regions, 103SHLHFDH109 and 166TPGHTPGH173, which are characteristic of the metallo-beta-lactamase family. Homology comparison revealed that the deduced amino acid sequence had a high degree of similarity with those of the known AiiA proteins in the B. cereus group. Additionally, the aiiA gene was expressed in Escherichia coli BL21 (DE3) pLysS and the expressed AiiA protein could attenuate the soft rot symptoms caused by Erwinia carotovora var. carotovora.