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Applied Microbiology and Biotechnology (v.84, #2)

Bioremediation of the organochlorine pesticides, dieldrin and endrin, and their occurrence in the environment by Emiko Matsumoto; Youhei Kawanaka; Sun-Ja Yun; Hiroshi Oyaizu (pp. 205-216).

Dieldrin and endrin are persistent organic pollutants that cause serious environmental problems. Although these compounds have been prohibited over the past decades in most countries around the world, they are still routinely found in the environment, especially in the soil in agricultural fields. Bioremediation, including phytoremediation and rhizoremediation, is expected to be a useful cleanup method for this soil contamination. This review provides an overview of the environmental contamination by dieldrin and endrin, along with a summary of our current understanding and recent advances in bioremediation and phytoremediation of these pollutants. In particular, this review focuses on the types and abilities of plants and microorganisms available for accumulating and degrading dieldrin and endrin.

Keywords: Bioremediation; Phytoremediation; Dieldrin; Endrin; Persistent organic pollutants

Microbiological production of tocopherols: current state and prospects by James C. Ogbonna (pp. 217-225).

Tocopherols are antioxidants that prevent various diseases caused by oxidative stress. Tocochromanols comprise four isoforms of tocopherols and four isoforms of tocotrienols but α-tocopherol is the most abundant and active isoform in human and animal tissues. Tocopherols are used as dietary supplements for human, as food preservatives, in manufacture of cosmetics, and for fortification of animal feed. Only photosynthetic cells are known to accumulate detectable concentrations of tocopherols. Tocopherols can be extracted and purified or concentrated from vegetable oils and other higher plant materials. However, the concentrations in these higher plant materials are very low and there are high proportions of the less-active homologues of tocopherols. Among the many strains of photosynthetic microorganisms known to accumulate tocopherols, Euglena gracilis is promising for commercial production of α-tocopherol. The growth rate and α-tocopherol contents are relatively high and α-tocopherol comprise more than 97% of all the tocopherols accumulated by Euglena gracilis. Although a lot of work has been done to increase the contents and composition of tocopherols in higher plants through genetic and metabolic engineering, work on genetic modification of microorganisms for increased tocopherol accumulation is scarce. Many cultivation systems have been investigated for efficient production of tocopherol by Euglena gracilis. However, those that involve heterotrophic metabolism are more promising. Bubble columns and flat-plate photobioreactors are more suitable for commercial production of tocopherols, than the tubular, internally illuminated, and open-air photobioreactors.

Keywords: Tocopherols; Antioxidants; Microbial production; Euglena gracilis ; Cultivation systems; Photobioreactors

Biochemistry of microbial polyvinyl alcohol degradation by Fusako Kawai; Xiaoping Hu (pp. 227-237).

Effect of minor chemical structures such as 1,2-diol content, ethylene content, tacticity, a degree of polymerization, and a degree of saponification of the main chain on biodegradability of polyvinyl alcohol (PVA) is summarized. Most PVA-degraders are Gram-negative bacteria belonging to the Pseudomonads and Sphingomonads, but Gram-positive bacteria also have PVA-degrading abilities. Several examples show symbiotic degradation of PVA by different mechanisms. Penicillium sp. is the only reported eukaryotic degrader. A vinyl alcohol oligomer-utilizing fungus, Geotrichum fermentans WF9101, has also been reported. Lignolytic fungi have displayed non-specific degradation of PVA. Extensive published studies have established a two-step process for the biodegradation of PVA. Some bacteria excrete extracellular PVA oxidase to yield oxidized PVA, which is partly under spontaneous depolymerization and is further metabolized by the second step enzyme (hydrolase). On the other hand, PVA (whole and depolymerized to some extent) must be taken up into the periplasmic space of some Gram-negative bacteria, where PVA is oxidized by PVA dehydrogenase, coupled to a respiratory chain. The complete pva operon was identified in Sphingopyxis sp. 113P3. Anaerobic biodegradability of PVA has also been suggested.

Keywords: Polyvinyl alcohol (PVA); PVA metabolism; Non-metabolic degradation of PVA; Two-step degradation of PVA; Anaerobic PVA degradation; pva operon

Biotechnological production of enantiopure epoxides by enzymatic kinetic resolution by Won Jae Choi (pp. 239-247).

Enantiopure epoxides are high value-added synthons for the production of pharmaceuticals, agrochemicals, as well as versatile fine chemicals and have broad scope of market demand for their applications. A major challenge in conventional organic synthesis is to generate such compounds in high enantiopurity with reasonable yield. Among possible chemical and biological technologies for enantiopure epoxide preparation, enzymatic kinetic resolution has been paid much attention with respect to its high enantioselectivity. Epoxide hydrolase (EH) has shown promising characteristics for the preparation of enantiopure epoxides and vicinal diols during enantioselective hydrolysis of racemic epoxides. EH is readily available from microbial resources thus it is being employed for biohydrolysis of a variety of epoxides. Recent technical progress in EH-catalyzed enantioselective hydrolysis is summarized in terms of exploration of novel EH, its functional improvement, high throughput assay, and preparative scale resolution process.

Keywords: Enantiopure epoxides; Kinetic resolution; Epoxide hydrolase; Enantioselective hydrolysis

Structural and physicochemical properties of polar lipids from thermophilic archaea by Nataša Poklar Ulrih; Dejan Gmajner; Peter Raspor (pp. 249-260).

The essential general features required for lipid membranes of extremophilic archaea to fulfill biological functions are that they are in the liquid crystalline phase and have extremely low permeability of solutes that is much less temperature sensitive due to a lack of lipid-phase transition and highly branched isoprenoid chains. Many accumulated data indicate that the organism’s response to extremely low pH is the opposite of that to high temperature. The high temperature adaptation does not require the tetraether lipids, while the adaptation of thermophiles to acidic environment requires the tetraether polar lipids. The presence of cyclopentane rings and the role of polar heads are not so straightforward regarding the correlations between fluidity and permeability of the lipid membrane. Due to the unique lipid structures and properties of archaeal lipids, they are a valuable resource in the development of novel biotechnological processes. This microreview focuses primarily on structural and physicochemical properties of polar lipids of (hyper)thermophilic archaea.

Keywords: Thermophiles; Archaeal polar lipids; Archaeosomes; Physicochemical properties; Biotechnological applications

Repeated-batch fermentation using flocculent hybrid, Saccharomyces cerevisiae CHFY0321 for efficient production of bioethanol by Gi-Wook Choi; Hyun-Woo Kang; Se-Kwon Moon (pp. 261-269).

In this study, the repeated-batch fermentation of liquefied cassava medium using the flocculent hybrid Saccharomyces cerevisiae CHFY0321 was investigated for semicontinuous, high-throughput production of bioethanol. Cassava medium was selected due to the industrial requirement for a cheap starchy substrate. Fermentations were performed by simultaneous saccharification and fermentation (SSF) with a set of ten batches successfully completing a series within the repeated fermentation process. In addition, pH of the culture medium was not controlled to simplify ethanol production for future use in industry. Optimal recycling volume was found to be 5%. Volumetric productivity, final ethanol concentration, and ethanol yield were measured at 3.34 g l⁻¹ h⁻¹, 84.5 g l⁻¹, and 90.7%, respectively. Cell recycling (24.5 g DCW l⁻¹) resulted in 1.8-fold decrease in fermentation time (24 h) and 1.8-fold increase in volumetric productivity compared with the ordinary batch fermentation. Therefore, repeated-batch SSF using flocculent CHFY0321 demonstrates the possibility of cost-effective bioethanol production by eliminating additional saccharification and inoculation steps.

Keywords: Bioethanol; Cassava; Repeated batch; SSF; Cell recycle

Optimizing emulsan production of A. venetianus RAG-1 using response surface methodology by Wen-Ta Su; Wen-Jang Chen; Yi-Fan Lin (pp. 271-279).

Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L ethanol, 8.2 g/L KH₂PO₄, 23.32 g/L K₂HPO₄, 5.77 g/L (NH₄)₂SO₄ and 0.354 g/L MgSO₄•7H₂O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production.

Keywords: Optimization; Emulsan; Acinetobacter venetianus RAG-1; Ethanol; Response surface methodology

Microalga Scenedesmus obliquus as a potential source for biodiesel production by Shovon Mandal; Nirupama Mallick (pp. 281-291).

Biodiesel from microalgae seems to be the only renewable biofuel that has the potential to completely replace the petroleum-derived transport fuels. Therefore, improving lipid content of microalgal strains could be a cost-effective second generation feedstock for biodiesel production. Lipid accumulation in Scenedesmus obliquus was studied under various culture conditions. The most significant increase in lipid reached 43% of dry cell weight (dcw), which was recorded under N-deficiency (against 12.7% under control condition). Under P-deficiency and thiosulphate supplementation the lipid content also increased up to 30% (dcw). Application of response surface methodology in combination with central composite rotary design (CCRD) resulted in a lipid yield of 61.3% (against 58.3% obtained experimentally) at 0.04, 0.03, and 1.0 g l⁻¹ of nitrate, phosphate, and sodium thiosulphate, respectively for time culture of 8 days. Scenedesmus cells pre-grown in glucose (1.5%)-supplemented N 11 medium when subjected to the above optimized condition, the lipid accumulation was boosted up to 2.16 g l⁻¹, the value ~40-fold higher with respect to the control condition. The presence of palmitate and oleate as the major constituents makes S. obliquus biomass a suitable feedstock for biodiesel production.

Keywords: Biodiesel; CCRD; N-limitation; P-limitation; RSM; Scenedesmus

Production of pigment-free pullulan by swollen cell in Aureobasidium pullulans NG which cell differentiation was affected by pH and nutrition by Bing-xue Li; Ning Zhang; Qing Peng; Tie Yin; Fei-fei Guan; Gui-li Wang; Ying Li (pp. 293-300).

A black yeast strain “NG” was isolated from strawberry fruit and identified as Aureobasidium pullulans. Strain NG displayed yeast-like cell (YL), swollen cell (SC), septate swollen cell (SSC), meristematic structure (MS), and chlamydospore (CH) morphologies. pH was the key factor regulating cell morphogenesis of strain NG. Differentiation of YL controlled by extracellular pH had no relationship with nutrition level. YL was maintained at pH >6.0, but was transformed into SC at pH ∼4.5. SC, a stable cell type of A. pullulans, could bud, septate, or transform into MS or CH, in response to nutrition level and low pH. SC produced swollen cell blastospores (SCB) at pH 2.1 with abundant nutrition, and could transform into MS at lower pH (1.5). SC was induced to form CH by low level nutrition and pH <3, and this transition was suppressed by adjusting pH to ∼4.5. Crude polysaccharides without pigment (melanin) were produced by SC of strain NG. Pullulan content of the polysaccharides was very high (98.37%). Fourier-transform infrared spectroscopy confirmed that chemical structures of the polysaccharides and standard pullulan were identical. Swollen cells produced 2.08 mg/ml non-pigmented polysaccharides at 96 h in YPD medium. Controlling pH of fermentation is an effective and convenient method to harvest SC for melanin-free pullulan production.

Keywords: Aureobasidium pullulans ; Swollen cell; pH; Pullulan; Melanin

Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27 by Lei Huang; Susanna Su Jan Leong; Rongrong Jiang (pp. 301-308).

This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l⁻¹, respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae.

Keywords: Antimicrobial activity; Antimicrobial peptide; Human β-defensin-26; Human β-defensin-27

New proteins orthologous to cerato-platanin in various Ceratocystis species and the purification and characterization of cerato-populin from Ceratocystis populicola by Cecilia Comparini; Lara Carresi; Eleonora Pagni; Francesca Sbrana; Federico Sebastiani; Nicola Luchi; Alberto Santini; Paolo Capretti; Bruno Tiribilli; Luigia Pazzagli; Gianni Cappugi; Aniello Scala (pp. 309-322).

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured α/β protein with a different percentage of the α-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth.

Keywords: Orthologous genes; Populus sp.; MALDI-TOF; Circular dichroism; PAMP activity; Induction of plant resistance

Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system by Guohua Yin; Zhaonan Sun; Nan Liu; Lin Zhang; Yunzhi Song; Changxiang Zhu; Fujiang Wen (pp. 323-333).

In many species, the introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference (RNAi). RNAi is the process of sequence-specific, posttranscriptional gene silencing (PTGS) in animals and plants, mediated by dsRNA homologous to the silenced genes. In plants, PTGS is part of a defense mechanism against virus infection, and dsRNA is the pivotal factor that induces gene silencing. Here, we report an efficient method that can produce dsRNA using a bacterial prokaryotic expression system. Using the bacteriophage λ-dependent Red recombination system, we knocked out the rnc genes of two different Escherichia coli strains and constructed three different vectors that could produce dsRNAs. This work explores the best vector/host combinations for high output of dsRNA. In the end, we found that strain M-JM109 or the M-JM109lacY mutant strain and the vector pGEM-CP480 are the best choices for producing great quantities of dsRNA. Resistance analyses and Northern blot showed that Tobacco mosaic virus infection could be inhibited by dsRNA, and the resistance was an RNA-mediated virus resistance. Our findings indicate that exogenous dsRNA could form the basis for an effective and environmentally friendly biotechnological tool that protects plants from virus infections.

Keywords: Double-stranded RNA; RNase III; TMV; RNA-mediated virus resistance

Increased PCP removal by Amylomyces rouxii transformants with heterologous Phanerochaete chrysosporium peroxidases supplementing their natural degradative pathway by A. M. Montiel-González; F. J. Fernández; N. Keer; A. Tomasini (pp. 335-340).

Fungal peroxidases and phenoloxidases are widely used in aromatic toxic compounds degradation. Peroxidases, such as lignin peroxidase and manganese peroxidase, as well as laccases are mainly produced by basidiomycetes and to a lower extent by other fungi, such as ascomycetes. Peroxidase-encoding genes have been described and homologous expression has been achieved in basidiomycetes. Heterologous expression has also been achieved in some non-producing peroxidase ascomycetes, like Penicillium and Aspergillus. In this work, heterologous expression of peroxidase-encoding genes, lignin peroxidase, and manganese peroxidase was achieved in a zygomycete producing only phenoloxidases (Amylomyces rouxii), aimed at coupling two different pathways used in nature for PCP removal in only one microbial strain. The ability of PCP removal was assayed with one of the obtained transformants, resulting in increased activity with respect to the ability of the parental strain cultured free of the inducer tyrosine (95% and 45%, respectively, of the initial PCP (12.5 mg L⁻¹) in 120 h, or 100% and 49%, respectively, of the initial PCP after 144 h of liquid culture).

Keywords: PCP removal; Peroxidases; Tyrosinase; Zygomycetes

Effects of Lactobacillus plantarum MA2 isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol diet by Yanping Wang; Nv Xu; Aodeng Xi; Zaheer Ahmed; Bin Zhang; Xiaojia Bai (pp. 341-347).

The objective of this study was to evaluate the effects of Lactobacillus plantarum MA2, an isolate from Chinese traditional Tibet kefir, on cholesterol-lowering and microflora of rat in vivo. Rats were fed on cholesterol-enriched experimental diet, supplemented with lyophilized L. plantarum MA2 powder, with a dose of 10¹¹ cells/day per mice. The results showed that L. plantarum MA2 feeding significantly lowered serum total cholesterol, low-density lipoprotein cholesterol, and triglycerides level, while there was no change in high-density lipoprotein cholesterol. In addition, liver total cholesterol and triglycerides was also decreased. However, fecal cholesterol and triglycerides was increased significantly (P < 0.05) in comparison with the control. Also, L. plantarum MA2 increased the population of lactic acid bacteria and bifidobacteria in the fecal, but it did not change the number of Escherichia coli as compared to control. Moreover, pH, moisture, and organic acids in the fecal were also measured. The present results indicate the probiotic potential of the L. plantarum MA2 strain in hypocholesterolemic effect and also increasing the probiotic count in the intestine.

Keywords: Lactobacillus plantarum MA2; Kefir; Cholesterol-lowering effect

Characterization of extracellular DNA production and flocculation of the marine photosynthetic bacterium Rhodovulum sulfidophilum by Hiromichi Suzuki; Masahide Daimon; Tomoyuki Awano; So Umekage; Terumichi Tanaka; Yo Kikuchi (pp. 349-356).

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids involved in its flocculation. Previously, we showed that the RNA fraction of these extracellular nucleic acids released into the culture medium contains mainly non-aminoacylated fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Here, we report the characterization of extracellular DNA itself and its production during cultivation. No differences were detected in nucleotide sequence between the intracellular DNA and extracellular soluble DNA on Southern blotting. Whole intracellular DNA seemed to be released from the cell. The bacterial floc was degraded by deoxyribonuclease or ribonuclease treatment, indicating that at least the extracellular DNA and RNAs in the floc are involved in the maintenance of the floc. When cultivated in nutritionally rich medium, the bacteria formed small flocs and produced large amounts of extracellular DNA, which were solubilized in the medium. In nutritionally poor medium, however, huge flocs of cells appeared and almost no extracellular soluble DNA was observed in the medium. As the floc was degraded by deoxyribonuclease treatment, it seems likely that the extracellular soluble DNA observed in the rich medium may be incorporated into the large floc and play a role in floc maintenance in poor medium. Addition of an inhibitor of quorum sensing, α-cyclodextrin, inhibited huge floc maintenance in the nutritionally poor medium. In the presence of α-cyclodextrin, the floc was rapidly degraded and extracellular soluble DNA production increased.

Keywords: α-cyclodextrin; Extracellular DNA; Extracellular RNA; Floc; Photosynthetic bacteria; Quorum sensing

Screening and degradation performances of dominant strains in high-salinity landfill leachate by Li Guo; Min Ji; Huiyu Dong; Yanjie Wei (pp. 357-364).

In order to enhance the removal efficiency of chemical oxygen demand (COD) in the high-salinity landfill leachate, the dominant strains were isolated from high-salinity landfill leachate. The dominant strains and bacteria consortium were screened for COD treatment potential using an aerobic COD concentration decrease test. Ten strains, TJ01–TJ10, were isolated, of which six strains TJ02, TJ03, TJ05, TJ06, TJ07, and TJ09 were found to have higher COD removal when the single bacteria were added, all more than 20%. The most effective combination was TJ06 + TJ09; the COD removal efficiency reached 45.57%. 16S rDNA gene sequence analysis revealed that TJ06 and TJ09 belonged to the genus Bacillus. The effects of the dominant bacteria consortium on the high-salinity landfill leachate varied with pH value and the volume fraction of leachate. The COD removal efficiencies maintained higher when the pH value was 6–8 and the volume fractions of leachate were less than 80%. The result also suggested that there is little effect on the growth of TJ06 and TJ09 when the range of Cl⁻ concentration is 0–30,000 mg/L.

Keywords: Landfill leachate; Dominant strains; Salinity; Chemical oxygen demand (COD)

Impact of dibenzofuran/dibenzo-p-dioxin amendment on bacterial community from forest soil and ring-hydroxylating dioxygenase gene populations by Nobutada Kimura; Yoichi Kamagata (pp. 365-373).

The impact of dibenzofuran (DF) and dibenzo-p-dioxin (DD) on the changes in bacterial community structure and the transition of catabolic genes were studied using forest soil. The bacterial community structure of soil suspensions amended with 1 µg/g of either DF or DD was analyzed by 16S rRNA and functional gene sequencing. To analyze the functional genes in the communities, we targeted a gene sequence that functions as the binding site of Rieske iron sulfur center common to ring-hydroxylating dioxygenases (RHDs) for monocyclic, bicyclic, and tricyclic aromatic compounds. The gene fragments were polymerase chain reaction-amplified from DNAs extracted from soil suspensions spiked with either DF or DD, cloned, and sequenced (70 clones). Bacterial community analysis based on 16S rRNA genes revealed that specific 16S rRNA gene sequences, in particular, phylotypes within alpha-Proteobacteria, increased in the soil suspension amended with DF or DD. RHD gene-based functional community analysis showed that, in addition to two groups of RHD genes that were also detected in unamended soil suspensions, another two groups of RHD genes, each of which is specific to DF- and DD-amended soil, respectively, emerged to a great extent. The DD-specific genotype is phylogenetically distant from any known RHDs. These results strongly suggest that soil microbial community potentially harbors a wide array of organisms having diverse RHDs including those previously unknown, and that they could quickly respond to an impact of contamination of hazardous chemicals by changing the microbial community and gene diversity.

Keywords: Ring-hydroxylating dioxygenase; Dibenzofuran; Dibenzo-p-dioxin

Development of an enzyme activity screening system for β-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting by Yan Zhou; Shin’ichiro Kajiyama; Kouichi Itoh; Takanori Tanino; Nobuo Fukuda; Tsutomu Tanaka; Akihiko Kondo; Kiichi Fukui (pp. 375-382).

Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying β-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.

Keywords: Yeast cell surface display; β-glucosidase; Screening system; Calcium alginate beads

Production of a cytotoxic cationic antibacterial peptide in Escherichia coli using SUMO fusion partner by Jian Feng Li; Jie Zhang; Ren Song; Jia Xin Zhang; Yang Shen; Shuang Quan Zhang (pp. 383-388).

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest was obtained per liter of fermentation culture. After the SUMO–CM4 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus, the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant cytotoxic peptides.

Keywords: Antibacterial peptide; ABP-CM4; SUMO; Escherichia coli

A method for obtaining DNA from compost by Liang Wu; Fenge Li; Changyan Deng; Dequan Xu; Siwen Jiang; Yuanzhu Xiong (pp. 389-395).

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical–chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.

Keywords: Compost; Cell lysis; DNA extraction; 16S rRNA

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Applied Microbiology and Biotechnology (v.84, #2)