生淀粉酶生产菌株Paenibacillus sp.的筛选和酶的纯化及酶学性质

分离得到1株产生淀粉酶的菌株,通过扩增和测定16S rDNA序列并进行比对,发现是Paenibacillus属的细菌。液体摇瓶发酵结束后,其产生的生淀粉酶比酶活达108.5U/mL。通过饱和(NH4)2SO4沉淀、Sephacryl S-300层析的方法对其所产的生淀粉酶进行分离纯化,得到纯化的酶蛋白比酶活为5112.04U/mg,纯化倍数为14.1,相对分子质量约为1.0×105。该酶以木薯生淀粉为底物时,最适pH5.6,最适温度50℃。金属离子Ca2+、Mg2+对该酶具有激活作用,Zn2+、Cu2+、Fe2+、Ni2+、Mn2+、Co2+和EDTA2-对该酶均具有抑制作用。     

Paenibacillus sp. Strain SB-6 Induces Weathering of Ca-montmorillonite: Illitization and Formation of Calcite

Investigation of the weathering of silicate minerals is helpful to understand the process of soil development, cycling of nutrient elements, and potential applications in fixation of carbon dioxide from the atmosphere through carbonate precipitation. In this study, weathering experiments of calcium-montmorillonite were conducted using Paenibacillus sp. strain SB-6 for 70 days. The results indicated that the Si⁴⁺, Al³⁺, Ca²⁺ and Na⁺ concentrations in the medium of the biotic experiments were evidently higher than those of the abiotic experiments, and that Paenibacillus sp. could help the transformation of partial montmorillonite into an illite–montmorillonite mixed-layer. In the process of illitization, K⁺ went into the interlayer of montmorillonite and hydrated Ca²⁺ and Na⁺ released from it. In the late stage of the experiments, the Ca²⁺ released from montmorillonite combined with carbonate ions generated by the bacterial metabolism, forming calcite.     

Weathering of Basalts by Aspergillus sp. FS-4 Strain: Glass Compositions are Prone to Weathering

Abstract

Mechanisms underlying microbe-induced dissolution of basalt and its effect on glass composition remain unclear. Two powdered basalts, with a glass composition of 27?vol% (Basalt-T) and 18?vol% (Basalt-F), respectively, were inoculated with Aspergillus sp. FS-4 in Czapek medium. Changes in pH, siderophores and main dissolved elements were examined. Basalt-F led to a greater decrease in pH and higher production of siderophores than Basalt-T mainly due to the fungi activity. Element dissolution was 2 to 3 fold higher in Basalt-T than in Basalt-F, and the dissolution was non-stoichiometric for both basalts. FS-4 appears important for basalt weathering. Composition of geologic material had a considerable impact on weathering. Materials with a higher glass percentage are prone to weathering. Further quantitative evaluation is required.     

Complete genome sequence of Paenibacillus sp. strain JDR-2

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from sweetgum (Liquidambar styraciflua) wood, is able to efficiently depolymerize, assimilate and metabolize 4-O-methylglucuronoxylan, the predominant structural component of hardwood hemicelluloses. A basis for this capability was first supported by the identification of genes and characterization of encoded enzymes and has been further defined by the sequencing and annotation of the complete genome, which we describe. In addition to genes implicated in the utilization of β-1,4-xylan, genes have also been identified for the utilization of other hemicellulosic polysaccharides. The genome of Paenibacillus sp. JDR-2 contains 7,184,930 bp in a single replicon with 6,288 protein-coding and 122 RNA genes. Uniquely prominent are 874 genes encoding proteins involved in carbohydrate transport and metabolism. The prevalence and organization of these genes support a metabolic potential for bioprocessing of hemicellulose fractions derived from lignocellulosic resources.     

Non-contiguous finished genome sequence and description of Paenibacillus senegalensis sp. nov.

Paenibacillus senegalensis strain JC66^T, is the type strain of Paenibacillus senegalensis sp. nov., a new species within the genus Paenibacillus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. P. senegalensis strain JC66^T is a facultative Gram-negative anaerobic rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,581,254 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 48.2% and contains 5,008 protein-coding and 51 RNA genes, including 9 rRNA genes.     

Paenibacillus sp. strain HC1 xylanases responsible for degradation of rice bran hemicellulose

Paenibacillus sp. strain HC1 is the first bacterium capable of growing on rice bran hemicellulose as a sole carbon source. Two xylanases (Xyl-I and -II) were purified from the bacterial culture fluid and enzymatically characterized. Xyl-I and -II showed monomer forms with molecular masses of 30 and 18 kDa, respectively, and were most active at around pH 5.0 and 45 °C. Xylooligosaccharides were degraded to xylobiose and xylose by Xyl-I, but not by Xyl-II, suggesting that Xyl-I plays an important role in complete depolymerization of xylan. Both enzymes acted endolytically on rice bran hemicellulose, indicating that Xyl-I and -II contribute to the structure determination and practical use of the polysaccharide, an unutilized biomass in technology.     

Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach

Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.     

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.     

Non-contiguous finished genome sequence and description of Paenibacillus gorillae sp. nov.

Strain G1^T sp. nov. is the type strain of Paenibacillus gorillae a newly proposed species within the genus Paenibacillus. This strain, whose genome is described here, was isolated in France from the fecal sample of a wild western lowland gorilla from Cameroon. P. gorillae is a facultative anaerobic, Gram-negative, rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,257,967 bp long genome (one chromosome but no plasmid) contains 5,856 protein-coding and 62 RNAs genes, including 60 tRNA genes.     

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39

Aims:  The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.
Methods and Results:  Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60°C and pH 6·5. Addition of 1 mmol l⁻¹ of Ca²⁺ markedly enhanced both CMCase and avicelase activities of the enzyme.
Conclusions:  We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities.
Significance and Impact of the Study:  Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.     

Optimization of medium constituents for improved chitinase production by Paenibacillus sp. D1 using statistical approach

Aims:  Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1.
Methods and Results:  Urea, K₂HPO₄, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett–Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l⁻¹): urea, 0·33; K₂HPO₄, 1·17; MgSO₄, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml⁻¹ of chitinase.
Conclusions:  The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K₂HPO₄, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production.
Significance and Impact of the Study:  The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.     

一株类芽孢杆菌的分离鉴定及其抗革兰氏阴性菌活性测定

【背景】随着碳青霉烯类和多粘菌素类可转移耐药基因的发现及扩散,多重耐药革兰氏阴性细菌感染更加难以治疗。【目的】筛选有效拮抗革兰氏阴性菌的菌株,为新型抗生素的发掘奠定基础。【方法】利用胰蛋白胨大豆琼脂培养基筛选土壤源细菌,通过16S rRNA基因序列鉴定其种属;通过全基因组测序,antiSMASH比对分析菌株产抗生素潜能,双层琼脂平板法验证其抗菌活性;通过甲醇萃取其次级代谢产物,高效液相色谱串联质谱(HPLC-MS/MS)进行次级代谢产物分析。【结果】从北京周边土壤样品中分离到一株类芽孢杆菌CAU136 (Paenibacillus pabuli CAU136),经过生物信息学分析和antiSMASH比对,表明该菌株有较强合成次级代谢产物的潜能,双层琼脂平板法验证其能抑制多株革兰氏阴性菌生长,HPLC-MS/MS检测结果显示其可能分泌多粘菌素E。【结论】类芽孢杆菌CAU136可能分泌多粘菌素E,能有效拮抗革兰氏阴性菌。     

Isolation of an antifungal Paenibacillus strain HT16 from locusts and purification of its medium-dependent antagonistic component

Aims:  To isolate an antagonist for use in the biological control of the phytopathogenic fungus Penicillium expansum and purify the antifungal component produced by the antagonist.
Methods and Results:  An antifungal strain HT16 was isolated from locusts, showing strong inhibition to Pen. expansum . Based on its in vitro effectiveness, HT16 was characterized as a strain of Paenibacillus polymyxa by phenotypic tests and 16S rDNA sequence analysis. It was found that the antifungal component HT16 secreted was only induced by Poria cocos sclerotium (PCS), and it remained active after sterilization at 121°C for 15 min. The protein was purified by ammonium sulfate precipitation, heating process, and ultrafiltration using a 10 kDa cut-off membrane. The molecular weight of the purified antifungal protein, which was determined by mass spectrometry, was 4517 Da.
Conclusions:  A novel bacterial strain HT16 antagonistic to Pen. expansum was isolated from locusts and identified as Pae. polymyxa . The antifungal protein of 4517 Da was purified, and its production needed the inducer PCS in the fermentation medium.
Significance and Impact of the Study:  The antagonistic protein from Pae. polymyxa showed strong antifungal activity against phytopathogenic fungus Pen. expansum . This strain HT16 and the antifungal metabolite are therefore strong candidates for the biocontrol of phytopathogens in agriculture.     

Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic

This research was initiated to search for novel antimicrobial compounds produced by food or environmental microorganisms. A new bacterial strain, designated OSY-SE, which produces a unique and potent antimicrobial agent was isolated from soil. The isolate was identified as a Paenibacillus sp. through cultural, biochemical, and genetic analyses. An antimicrobial compound was extracted from Paenibacillus OSY-SE with acetonitrile and purified using liquid chromatography. After analyses by mass spectrometry (MS) and nuclear magnetic resonance (NMR), the antimicrobial compound was determined to be a cyclic lipopeptide consisting of a C(15) fatty acyl (FA) chain and 13 amino acids. The deduced sequence is FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-Val-Lys-Ile. The carboxyl-terminal Ile is connected to Thr by ester linkage. The new compound, designated paenibacterin, showed antagonistic activities against most Gram-positive and Gram-negative bacteria tested, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium. Paenibacterin is resistant to trypsin, lipase, α-glucosidase, and lysozyme. Its antimicrobial activity was lost after digestion by pronase and polymyxin acylase. Paenibacterin is readily soluble in water and fairly stable to exposure to heat and a wide range of pH values. The new isolate and its antimicrobial agent are being investigated for usefulness in food and medical applications.     

Demonstration of the carbon-sulfur bond targeted desulfurization of benzothiophene by thermophilic Paenibacillus sp. strain A11-2 capable of desulfurizing dibenzothiophene

Paenibacillus sp. strain A11-2, which had been primarily isolated as a bacterial strain capable of desulfurizing dibenzothiophene to produce 2-hydroxybiphenyl at high temperatures, was found to desulfurize benzothiophene more efficiently than dibenzothiophene. The desulfurized product was identified as o-hydroxystyrene by GC-MS and 1H-NMR analysis. Benzothiophene was assumed to be degraded in a way analogous to the 4S pathway, which has been well-known as a mode of dibenzothiophene degradation. These results suggest that benzothiophene desulfurization may share at least partially the reaction mechanism with dibenzothiophene desulfurization.     

Efficient Reduction of Iron Oxides by Paenibacillus spp. Strains Isolated from Tropical Soils

The genus Paenibacillus was hardly described as a Fe(III)-reducing agent, only limited to reduce soluble forms or Fe inserted in poorly crystallized structures. In this study, three Paenibacillus strains capable of reducing manganese oxides in addition to iron oxides were isolated from Cameroonian and Brazilian soils. These strains reduced iron minerals from poorly crystallized 2-line ferrihydrite to well-crystallized Al-substituted and pure goethite with a significant production of soluble ferrous iron. These Paenibacillus strains, inhabitants from ferralitic temporarily waterlogged soils, could play an important role in the bioweathering of minerals and metal mobility in soils.     

一株硅酸盐细菌的分离及解钾活性研究

目的利用硅酸盐细菌分离培养基,从昆明白泥山土壤样品中分离获得一株硅酸盐细菌——BN1。方法对分离获得的硅酸盐细菌——BM进行革兰染色、生理生化特征和16SrDNA测序分析,并对其解钾活性进行了初步研究。结果BN1初步鉴定为类芽胞杆菌属的菌株。结论该菌株——BN1对云母具有较强的解钾活性,为空白对照组的1．79倍。     

一株抗真菌内生多粘芽孢杆菌的分离鉴定及对水稻恶苗病菌的抑制作用

目的：从番茄叶片中筛选具广谱抑真菌活性的拮抗内生细菌,研究其对水稻恶苗病菌的抑制作用。方法：采用对峙培养法筛选拮抗内生细菌,根据菌株形态、生理生化特性结合16S rRNA基因序列分析鉴定菌株;采用硫酸铵沉淀法提取抗菌粗蛋白,研究其对水稻恶苗病菌菌丝生长和孢子萌发的影响。结果：从番茄叶片中筛选到一株抗真菌内生多粘芽孢杆菌（Paenibacillus polymyxa）SD-6,该菌株具有广谱抑菌活性,对供试的13种植物病原真菌均具较强的抑制作用;该菌株产生的抗菌粗蛋白能够显著抑制水稻恶苗病菌菌丝生长和孢子萌发,并能导致萌发孢子畸形和破裂。结论：从番茄叶片中分离到一株能产生抗真菌蛋白并具有广谱高效抑真菌作用的内生多粘芽孢杆菌,该菌株及其抗菌蛋白具有防治水稻恶苗病的潜力。