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Applied Microbiology and Biotechnology (v.74, #1)

Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications by Baki Hazer; Alexander Steinbüchel (pp. 1-12).

A wide range of diverse polyhydroxyalkanoates, PHAs, is currently available due to the low substrate specificity of PHA synthases and subsequent modifications by chemical reactions. These polymers are promising materials for a number of different applications due to their biocompatibility and biodegradability. This review summarizes the large variability of PHAs regarding chemical structure and material properties that can be currently produced. In the first part, in vivo and in vitro biosynthesis processes for production of a large variety of different PHAs will be summarized with regard to obtaining saturated and unsaturated copolyesters and side chain functionalized polyesters, including brominated, hydroxylated, methyl-branched polyesters, and phenyl derivatives of polyesters. In the second part, established chemical modifications of PHAs will be summarized as that by means of grafting reactions and graft/block copolymerizations, as well as by chlorination, cross-linking, epoxidation, hydroxylation, and carboxylation, reactions yield further functionalized PHAs.

Keywords: Chemical modifications of PHAs; Copolyesters; Microbial polyesters; Poly(3-hydroxyalkanoate)s; PHA; PHA constituents

Alkane hydroxylases involved in microbial alkane degradation by Jan B. van Beilen; Enrico G. Funhoff (pp. 13-21).

This review focuses on the role and distribution in the environment of alkane hydroxylases and their (potential) applications in bioremediation and biocatalysis. Alkane hydroxylases play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Environmental studies demonstrate the abundance of alkane degraders and have lead to the identification of many new species, including some that are (near)-obligate alkanotrophs. The availability of a growing collection of alkane hydroxylase gene sequences now allows estimations of the relative abundance of the different enzyme systems and the distribution of the host organisms.

Bacillus methanolicus: a candidate for industrial production of amino acids from methanol at 50°C by Trygve Brautaset; Øyvind M. Jakobsen; Kjell D. Josefsen; Michael C. Flickinger; Trond E. Ellingsen (pp. 22-34).

Amino acids are among the major products in biotechnology in both volume and value, and the global market is growing. Microbial fermentation is the dominant method used for industrial production, and today the most important microorganisms used are Corynebacteria utilizing sugars. For low-prize bulk amino acids, the possibility of using alternative substrates such as methanol has gained considerable interest. In this mini review, we highlight the unique genetics and favorable physiological traits of thermotolerant methylotroph Bacillus methanolicus, which makes it an interesting candidate for overproduction of amino acids from methanol. B. methanolicus genes involved in methanol consumption are plasmid-encoded and this bacterium has a high methanol conversion rate. Wild-type strains can secrete 58 g/l of l-glutamate in fed-batch cultures at 50°C and classical mutants secreting 37 g/l of l-lysine have been selected. The relative high growth temperature is an advantage with respect to both reactor cooling requirements and low contamination risks. Key genes in l-lysine and l-glutamate production have been cloned, high-cell density methanol fermentation technology established, and recently a gene delivery method was developed for this organism. We discuss how this new knowledge and technology may lead to the construction of improved l-lysine and l-glutamate producing strains by metabolic engineering.

Applicability of pectate-entrapped Lactobacillus casei cells for l(+) lactic acid production from whey by P. S. Panesar; J. F. Kennedy; C. J. Knill; M. R. Kosseva (pp. 35-42).

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.

Keywords: Whey; Lactic acid; Lactose utilization; Immobilization; Pectate gel; L. casei

Microbial transformation of benzene to cis-3,5-cyclohexadien-1,2-diols by recombinant bacteria harboring toluene dioxygenase gene tod by Shao-Ping Ouyang; Shang-Yu Sun; Qian Liu; Jinchun Chen; Guo-Qiang Chen (pp. 43-49).

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylation of aromatic compounds. To achieve high efficient biotransformation of benzene to benzene cis-diols, Pseudomonas putida KT2442, Pseudomonas stutzeri 1317, and Aeromonas hydrophila 4AK4 were used as hosts to express TDO gene tod. Plasmid pSPM01, a derivative of broad-host plasmid pBBR1MCS-2 harboring tod from plasmid pKST11, was constructed and introduced into the above three strains. Their abilities to catalyze the biotransformation of benzene to benzene cis-diols, namely, cis-3,5-cyclohexadien-1,2-diols abbreviated as DHCD, were examined. In shake-flask cultivation under optimized culture media and growth condition, benzene cis-diols production by recombinant P. putida KT2442 (pSPM01), P. stutzeri 1317 (pSPM01), and A. hydrophila 4AK4 (pSPM01) were 2.68, 2.13, and 1.17 g/l, respectively. In comparison, Escherichia coli JM109 (pSPM01) and E. coli JM109 (pKST11) produced 0.45 and 0.53 g/l of DHCD, respectively. When biotransformation was run in a 6-l fermenter, DHCD production in P. putida KT2442 (pSPM01) was approximately 60 g/l; this is the highest DHCD production yield reported so far.

Keywords: Pseudomonas putida ; Pseudomonas stutzeri ; Aeromonas hydrophila ; Toluene dioxygenase; Biotransformation; Benzene cis-diols; cis-3,5-Cyclohexadien-1,2-diols

Enhanced ethanol fermentation of brewery wastewater using the genetically modified strain E. coli KO11 by Kripa Rao; Vaibhav Chaudhari; Sasidhar Varanasi; Dong-Shik Kim (pp. 50-60).

We have used liquid waste obtained from a beer brewery process to produce ethanol. To increase the productivity, genetically modified organism, Escherichia coli KO11, was used for ethanol fermentation. Yeast was also used to produce ethanol from the same feed stock, and the ethanol production rates and resulting concentrations of sugars and ethanol were compared with those of KO11. In the experiments, first the raw wastewater was directly fermented using two strains with no saccharification enzymes added. Then, commercial enzymes, α-amylase, pectinase, or a combination of both, were used for simultaneous saccharification and fermentation, and the results were compared with those of the no-enzyme experiments for KO11 and yeast. Under the given conditions with or without the enzymes, yeast produced ethanol more rapidly than E. coli KO11, but the final ethanol concentrations were almost the same. For both yeast and KO11, the enzymes were observed to enhance the ethanol yields by 61–84% as compared to the fermentation without enzymes. The combination of the two enzymes increased ethanol production the most for the both strains. The advantages of using KO11 were not demonstrated clearly as compared to the yeast fermentation results.

Keywords: Ethanol fermentation; E. coli KO11; Simultaneous saccharification and fermentation; Brewery wastes; Yeast; Pectinase

Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate by Z. J. Xiao; P. H. Liu; J. Y. Qin; P. Xu (pp. 61-68).

The nutritional requirements for acetoin production by Bacillus subtilis CICC 10025 were optimized statistically in shake flask experiments using indigenous agroindustrial by-products. The medium components considered for initial screening in a Plackett–Burman design comprised a-molasses (molasses submitted to acidification pretreatment), soybean meal hydrolysate (SMH), KH₂PO₄·3H₂O, sodium acetate, MgSO₄·7H₂O, FeCl₂, and MnCl₂, in which the first two were identified as significantly (at the 99% significant level) influencing acetoin production. Response surface methodology was applied to determine the mutual interactions between these two components and optimal levels for acetoin production. In flask fermentations, 37.9 g l⁻¹ acetoin was repeatedly achieved using the optimized concentrations of a-molasses and SMH [22.0% (v/v) and 27.8% (v/v), respectively]. a-Molasses and SMH were demonstrated to be more productive than pure sucrose and yeast extract plus peptone, respectively, in acetoin fermentation. In a 5-l fermenter, 35.4 g l⁻¹ of acetoin could be obtained after 56.4 h of cultivation. To our knowledge, these results, i.e., acetoin yields in flask or fermenter fermentations, were new records on acetoin fermentation by B. subtilis.

Keywords: Acetoin fermentation; Statistical optimization; Molasses; Soybean meal hydrolysate

Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by Pseudomonas putida KT2440 by Zhiyong Sun; Juliana A. Ramsay; Martin Guay; Bruce A. Ramsay (pp. 69-77).

Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h⁻¹ but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (μ = 0.15 h⁻¹) produced 70 g l⁻¹ biomass containing 75% PHA. At a higher exponential feed rate (μ = 0.25 h⁻¹), the overall productivity was increased but less biomass (56 g l⁻¹) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.

Keywords: Pseudomonas putida ; Fed-batch; High-density; Fermentation; PHA

Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by Agaricus blazei by Gao-Qiang Liu; Xiao-Ling Wang (pp. 78-83).

Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.

Keywords: Agaricus blazei ; Submerged culture; Response surface methodology; Box–Behnken design; Extracellular polysaccharide

Biochemical characterization of a novel 2-Cys peroxiredoxin from Antrodia camphorata by Jenq-Kuen Huang; Chuian-Fu Ken; Hui-Ming Huang; Chi-Tsai Lin (pp. 84-92).

Peroxiredoxins (Prxs) play important roles in antioxidation and cell signaling. A gene encoding a novel 2-Cys Prx was identified based on sequence homology in an expressed sequence tag database of the Antrodia camphorata, a medicinal mushroom found only in Taiwan. The 2-Cys Prx cDNA (940 bp) encodes a protein of 188 amino acid residues with calculated molecular mass of 20,965 Da and a pI of 5.89. The coding region was subcloned into pAVD10, transformed into Escherichia coli, and expressed as a His-tagged fusion protein. The purified enzyme was characterized under various conditions. The Prx retained 68% activity after being heated at 60°C for 2 min. It was stable under a broad pH range from 5 to 11. The enzyme activity was slightly decreased in the presence of 1% sodium dodecyl sulfate. The enzyme was somewhat susceptible to chymotrypsin treatment but resistant to digestion by trypsin.

Keywords: Mushroom; Antrodia camphorata ; Expression; 2-Cys Peroxiredoxin (2-Cys Prx)

A new approach to the production of the recombinant SOD protein by methylotrophic Pichia pastoris by Ping Yu (pp. 93-98).

The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity.

Keywords: Copper, zinc–superoxide dismutase gene; PCR amplification; Similarity comparison; Pichia pastoris

Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme by Zhiqiang Liu; Yin Li; Yingying Xu; Lifeng Ping; Yuguo Zheng (pp. 99-106).

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.

Keywords: Cloning; Prokaryotic expression; Epoxide hydrolase; Rhodococcus opacus ML-0004; Characterization

Characterization of thermostable native alkaline phosphatase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1 by Is Helianti; Takako Okubo; Yasutaka Morita; Eiichi Tamiya (pp. 107-112).

This paper reports the characterization of an alkaline phosphatase (AP) from an aerobic hyperthermophilic Archaeon Aeropyrum pernix K1. The native AP was purified into homogeneity. The enzyme is predicted as a homodimeric structure with a native molecular mass of about 75 kDa and monomer of about 40 kDa. Apparent optimum pH and temperature were estimated at 10.0 and above 95°C, respectively. Magnesium ion increased both the stability and the activity of the enzyme. A. pernix AP has been demonstrated as a very thermostable AP, retaining about 76% of its activity after being incubated at 90°C for 5.5 h and 67% of its activity after being incubated at 100°C for 2.5 h, respectively, under the presence of Mg(II). Enzyme activity was increased in addition of exogenous Mg(II), Ca(II), Zn(II), and Co(II).

Keywords: Hyperthermophilic; Aeropyrum pernix ; Thermostable; Alkaline phosphatase

A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut by Nisha Palackal; Christopher S. Lyon; Seema Zaidi; Peter Luginbühl; Paul Dupree; Florence Goubet; John L. Macomber; Jay M. Short; Geoffrey P. Hazlewood; Dan E. Robertson; Brian A. Steer (pp. 113-124).

A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different β-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes β-1,4-linked mannan substrates, while the second catalytic site hydrolyzes β-1,4-linked xylan and β-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing.

Keywords: Metagenomics; Environmental DNA; Glucanase; Mannanase; Xylanase; Multidomain

Isolation and characterization of an antifungal peptide with antiproliferative activity from seeds of Phaseolus vulgaris cv. ‘Spotted Bean’ by H. X. Wang; T. B. Ng (pp. 125-130).

A 7.3-kDa antifungal peptide with an N-terminal sequence exhibiting remarkable homology to defensins from other leguminous plants was isolated from Phaseolus vulgaris cv. ‘Spotted Bean’. The isolation procedure involved ion exchange chromatography on O-diethylaminoethyl (DEAE) cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and SP-Sepharose. It exerted an antifungal action on Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited mycelial growth in F. oxysporum with an IC₅₀ value of 1.8 μM. It suppressed [methyl-³H]-thymidine incorporation by leukemia L1210 cells and MBL2 cells with an IC₅₀ value of 4.0 and 9.0 μM, respectively. There was no effect on HIV-1 reverse transcriptase activity when the peptide was tested up to 0.1 mM.

Engineering the lycopene synthetic pathway in E. coli by comparison of the carotenoid genes of Pantoea agglomerans and Pantoea ananatis by Sang-Hwal Yoon; Ju-Eun Kim; Sook-Hee Lee; Hye-Min Park; Myung-Suk Choi; Jae-Yean Kim; Si-Hyoung Lee; Yong-Chul Shin; Jay D. Keasling; Seon-Won Kim (pp. 131-139).

The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-d-thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli.

Strain-typing of Lentinula edodes in China with inter simple sequence repeat markers by Ruiying Zhang; Chenyang Huang; Suyue Zheng; Jinxia Zhang; Tzi Bun Ng; Ruibo Jiang; Xuemei Zuo; Hexiang Wang (pp. 140-145).

To validate strain typing by inter simple sequence repeat (ISSR) analysis in Lentinula edodes cultivars, 17 Chinese L. edodes strains including 15 cultivated strains cultivated on a large scale and two wild strains were analyzed with the ISSR technique. With the use of two ISSR primers, a total of 32 DNA products were detected, of which, 31 DNA products (96.9% of the detected products) were polymorphic between two or more strains. The profiles of those two primers could be employed to differentiate all of the tested strains. A cluster analysis based on ISSR data revealed that the 17 strains could be classified into two distinct groups. One group consisted of eight strains in which the cultivated strains were H (high-temperature)-type or B (broad-temperature)-type, and the other group comprised cultivated strains that were of the L (low-temperature)-type or M (medium-temperature)-type. In contrast to the two wild strains, the genetic diversity of 15 cultivated strains was very rich based on a similarity coefficient analysis.

Keywords: Lentinula edodes ; ISSR

Bacterial expression of a Trichosanthes kirilowii defensin (TDEF1) and its antifungal activity on Fusarium oxysporum by Li Da-Hui; Jian Gui-Liang; Zhang Ying-Tao; Ai Tie-Min (pp. 146-151).

The gene encoding Trichosanthes kirilowii defensin (TDEF1) was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). The newly discovered TDEF1 cDNA contains 231 bp (Genbank accession number DQ526373) and encodes a 76-amino acid protein, which consists of a 29-amino acid signal peptide and a 47-amino acid mature peptide. The partial cDNA, corresponding to the mature peptide coding region of TDEF1, was inserted into bacterial expression vector pET32a(+). Subsequent expression showed that TDEF1 was produced as a 26-kDa fusion protein in the form of inclusion body in Escherichia coli BL21 (DE3). After protein refolding and purification, the fusion TDEF1 displayed an inhibitive activity against the fungal pathogen, Fusarium oxysporum, with EC₅₀ of 247 μg/ml by means of fungal growth inhibition method.

Keywords: Defensin; Expression; Fusarium oxysporum ; RT-PCR; Trichosanthes kirilowii

Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin by Sung Hyun Kim; Gyun Min Lee (pp. 152-159).

Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75–89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54–87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45–79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.

Keywords: Chinese hamster ovary (CHO) cells; Lactate dehydrogenase-A (LDH-A); Small interfering RNA (siRNA); Specific lactate production rate

High efficiency and throughput system in directed evolution in vitro of reporter gene by Ai-Sheng Xiong; Ri-He Peng; Jin-Ge Liu; Jing Zhuang; Yu-Shan Qiao; Fang Xu; Bing Cai; Zhen Zhang; Jian-Min Chen; Quan-Hong Yao (pp. 160-168).

In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli β-glucuronidase (gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50–100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100°C for 30 min.

Keywords: GUS reporter gene; DNA directed evolution; Gene shuffling; β-Glucuronidase; Thermostability

Analysis of the 7.6-kb cryptic plasmid pNC500 from Rhodococcus rhodochrous B-276 and construction of Rhodococcus–E. coli shuttle vector by Toru Matsui; Hisashi Saeki; Naoya Shinzato; Hitoshi Matsuda (pp. 169-175).

Four circular cryptic plasmids were detected from propene-degrading Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 and the smallest 7.6-kb plasmid, named pNC500 was used to construct Rhodococcus–E. coli shuttle vector, pNC5403. Sequence analysis of pNC500 revealed that the plasmid contains eight potential ORFs, namely 1 through 8. The deduced amino acid sequences for ORFs 3, 4, 6, and 7 show homology with those of Rep A, Rep B, DNA methyl-transferase (M.XamI), and restriction nuclease (R.XamI), respectively. The region responsible for replication in the potent oil-desulfurization bacterium, Rhodococcus opacus T09 was determined as 3.7 kb-XbaI/BalI fragment which contains ORFs 3 and 4, while no transformants were obtained when ORF 4 was partially deleted, suggesting that both are required for its replication. Alignment of the predicted amino acid sequences revealed that ORFs 3 and 4 were DNA binding protein and DNA primase, respectively. A compatibility test with pAL5000-related plasmid vector, pRHK1, which contains pRC4, revealed that pNC5403 was compatible with pRHK1 suggesting that each replication origin would be different. ORFs 3 and 4 containing a pNC5403 derivative, pN5DXB, was stably maintained for over 80 generations in the absence of antibiotic selective conditions.

Keywords: Rhodococcus ; Shuttle vector; Nocardioform; Replication; Plasmid

Toxicogenomic analysis of sodium hypochlorite antimicrobial mechanisms in Pseudomonas aeruginosa by David A. Small; Wook Chang; Freshteh Toghrol; William E. Bentley (pp. 176-185).

Sodium hypochlorite (bleach) is routinely used in hospitals and health care facilities for surface sterilization; however, the mechanism of action by which this disinfectant kills and the extent to which Pseudomonas aeruginosa is resistant to sodium hypochlorite have not been elucidated. Consequently, nosocomial infections from P. aeruginosa result in considerable casualties and economic hardship. We report the genome-wide transcriptome response of P. aeruginosa to sodium hypochlorite-induced oxidative stress via the use of DNA microarrays. In addition to a general oxidative stress response, our data revealed a downregulation of virtually all genes related to oxidative phosphorylation and electron transport and an upregulation of many organic sulfur transport and metabolism genes.

Keywords: Antimicrobial; Toxicogenomic; Microarray

Identification of genetic markers to distinguish the virulent and avirulent subspecies of Pantoea stewartii by comparative proteomics and genetic analysis by Qiong Wu; Zide Jiang; Jinliang Liao; Zhinan Chen; Huaping Li; Mantong Mei; Lian-Hui Zhang (pp. 186-193).

Pantoea stewartii subsp. stewartii (Pnss), the causal agent of Stewart’s bacterial wilt and leaf blight of maize and sweet corn, is one of the quarantine pathogens in many countries and regions. In contrast, P. stewartii subsp. indologenes (Pnsi), the closely related subspecies of Pnss, is avirulent on these plants. In this study, the protein expression profiles of these two subspecies were compared using two-dimensional gel electrophoresis analysis. Twenty-one unique protein spots consistently detected in Pnss but not in Pnsi were analyzed by mass spectrometry. Some of these Pnss-specific proteins are known to be essential for virulence and survival in host, such as FoxR and HrcJ, which are the key components of iron uptake and Type III secretion systems, respectively. For further genetic analysis, six Pnss-specific proteins were characterized by peptide sequencing. Southern and Northern blot analyses revealed that the differences in protein expression profiles of the two subspecies were either due to the discrepancy at genome level or because of the variations in transcriptional expression. The results provide novel genetic markers to distinguish the two closely related subspecies and may also serve as useful clues for investigation of the genetic basis accounting for their sharp difference in virulence.

Keywords: Genetic marker; Quarantine; Pantoea stewartii ; Proteomics; Genetic variation

Effects of environmental settings on MTBE removal for a mixed culture and its monoculture isolation by Chi-Wen Lin; Shen-Long Tsai; Sung-Nan Hou (pp. 194-201).

A mixed culture was utilized to evaluate methyl tert-butyl ether (MTBE) removal under various conditions and to isolate a MTBE-degrading pure culture. The results showed that high MTBE removal efficiencies can be reached even in the presence of other substrates. The biodegradation sequence of the target compounds by the mixed culture, in order of removal rate, was toluene, ethyl benzene, p-xylene, benzene, MTBE, ethyl ether, tert-amyl methyl ether, and ethyl tert-butyl ether. In addition, preincubation of the mixed cultures with benzene and toluene showed no negative effect on MTBE removal; on the contrary, it could even increase the degradation rate of MTBE. The kinetic behavior showed that the maximum specific growth rate and the saturation constant of the mixed culture degrading MTBE are 0.000778 h⁻¹ and 0.029 mg l⁻¹, respectively. However, a high MTBE concentration (60 mg l⁻¹) was slightly inhibiting to the growth of the mixed culture. The pure culture isolated from the enrichments in the bubble-air bioreactor showed better efficiency in MTBE removal than the mixed culture; whereas, tert-butyl alcohol was formed as a metabolic intermediate during the breakdown of MTBE.

Keywords: Biodegradation; Kinetics; Methyl tert-butyl ether; Oxygenates

Adsorption of bisphenol A by lactic acid bacteria, Lactococcus, strains by Yasushi Endo; Nobuhiro Kimura; Ikuo Ikeda; Kenshiro Fujimoto; Hiromi Kimoto (pp. 202-207).

Ten strains of the genus Lactococcus were examined for their ability to remove bisphenol A [2, 2-bis(4-hydroxyphenyl)propane; BPA], which is known as an endocrine disrupter. Nine strains of the lactococci tested could remove BPA from media during growth, although the removal ratio was below 9%. When BPA was incubated with lyophilized cells of lactococci for 1 h, the concentration of BPA in the media was decreased by 9–62%. Especially, the highest removal ratio of BPA was observed for Lactococcus lactis subsp. lactis 712. The lactococci could adsorb BPA but not degrade it, because the lactococci maintained the ability to remove BPA from the medium after autoclaving. When the lyophilized cells of L. lactis subsp. lactis 712 were also incubated with six analogues of BPA, they effectively adsorbed hydrophobic compounds such as 2, 2′-diphenylpropane and bisphenol A dimethylether. The BPA-adsorbing ability of lactococci could be due to the hydrophobic binding effect. The removal ratio of BPA by L. lactis subsp. lactis 712 was increased after treatment with sodium dodecyl sulfate and decreased after digestion with trypsin. These results suggest that the hydrophobic proteins on cell surface may be involved in the BPA-adsorbing ability of lactococci.

Keywords: Bisphenol A; Lactococcus ; Lactic acid

Formation of extracellular polymeric substances from acidogenic sludge in H₂-producing process by Guo-Ping Sheng; Han-Qing Yu (pp. 208-214).

In this study, the formation of extracellular polymeric substances (EPS) and surface characteristics of an acidogenic sludge in anaerobic H₂-producing process was investigated. Results show that carbohydrates, proteins, and humic substances were the dominant components in bound EPS (BEPS), while in soluble EPS (SEPS), carbohydrates were the main component. The total content of BEPS initially increased but then kept almost unchanged during fermentation from 25 to 35 h; after that, it slightly decreased. The total content of SEPS increased to 172.5 ± 0.05 mg C g⁻¹ volatile suspended solid with the time that increased to 23.5 h, and then rapidly decreased until 43 h; thereafter, it kept almost unchanged. The SEPS had good correlations with the specific H₂ production rate, substrate degradation rate, and specific aqueous products formation rate, but the BEPS seemed to have no such correlations with these specific rates. Results also confirm that part of EPS could be utilized by the H₂-producing sludge. As the substrate was in short supply, the EPS would be hydrolyzed to sever as carbon and energy source.

Keywords: Extracellular polymeric substances (EPS); Hydrogen production; Acidogenesis; Surface characteristics; Sludge

Biofiltration of toluene-contaminated air using an agro by-product-based filter bed by B. Krishnakumar; A. M. Hima; Ajit Haridas (pp. 215-220).

An innovative, coir-pith-based, filter bed for degrading vapor phase toluene in a gas biofilter over 160 days without any external nutrient supply is reported in this study. Indigenous microflora present in the coir pith as well as in the aerobic sludge added at the start-up stage metabolized the toluene, and correspondingly, CO₂ was produced in the biofilter. Inlet toluene concentration in the range of 0.75 to 2.63 g/m³ was supplied to the biofilter in short acclimation periods. The maximum elimination capacity achieved was 96.75 g/m³·h at 120.72 g/m³·h loading where around 60% was recovered as CO₂. The filter bed maintained a stable low-pressure drop (0–4 mm H₂O), neutral pH range (6.5–7.5), and moisture content of 60–80% (w/w) throughout the period. In addition to toluene-degrading microbial community, a grazing fauna including rotifer, bacteriovoric nematode, tardigrade, and fly larvae were also present in the filter bed. The overall performance of the biofilter bed in pollutant removal and sustainability was analyzed in this study.

Keywords: Biofiltration; Coir pith; Toluene degradation

Denitrifying degradation of dimethyl phthalate by Da-Wei Liang; Tong Zhang; Herbert H. P. Fang (pp. 221-229).

Results of batch experiments on the denitrifying degradation of dimethyl phthalate (DMP) was most favorable at pH 7–9 and 30–35°C. DMP was first degraded to monomethyl phthalate (MMP), which was in turn degraded to phthalate before complete mineralization. There was no fatty acid residue in the mixed liquor throughout the experiments. The maximum specific degradation rates were 0.32 mM/(gVSS·h) for DMP, 0.19 mM/(gVSS·h) for MMP, and 0.14 mM/(gVSS·h) for phthalate. About 86% of available electron in DMP was utilized for denitrification; the remaining 14% was presumable conserved in the new biomass with an estimated yield of 0.17 mg/mg DMP. Based on 16S rDNA analysis, the denitrifying sludge was mainly composed of β-subdivision and α-subdivision of Proteobacteria (33 and 5 clones out of a total of 43 clones, respectively), plus some Acidobacteria. Using a primer set specifically designed to amplify the denitrification nirK gene, 10 operational taxonomy units (OTUs) were recovered from the clone library. They clustered into a group in the α-subdivision of Proteobacteria most closely related to denitrifier Bradyrhizobium japonicum USDA110 and several environmental clones.

Keywords: Phthalic esters; Denitrification; 16S rDNA; Dimethyl phthalate; nirK gene; Phylogenetic analysis

Effects of electron donors and acceptors on anaerobic reduction of azo dyes by Shewanella decolorationis S12 by Yiguo Hong; Xingjuan Chen; Jun Guo; Zhicheng Xu; Meiying Xu; Guoping Sun (pp. 230-238).

Shewanella decolorationis S12 was able to reduce various azo dyes in a defined medium with formate, lactate, and pyruvate or H₂ as electron donors under anaerobic conditions. Purified membranous, periplasmic, and cytoplasmic fractions from strain S12 analyzed, respectively, only membranous fraction was capable of reducing azo dye in the presence of electron donor, indicating that the enzyme system for anaerobic azoreduction was located on cellular membrane. Respiratory inhibitor Cu²⁺, dicumarol, stigmatellin, and metyrapone inhibited anaerobic azoreduction by purified membrane fraction, suggesting that the bacterial anaerobic azoreduction by strain S12 was a biochemical process that oxidizes the electron donors and transfers the electrons to the acceptors through a multicompound system related to electron transport chain. Dehydrogenases, cytochromes, and menaquinones were essential electron transport components for the azoreduction. The electron transport process for azoreduction was almost fully inhibited by O₂, 6 mM of $${ ext{NO}}^{ - }_{3} $$ , and 0.9 mM of $${ ext{NO}}^{ - }_{2} $$ , but not by 10 mM of Fe³⁺. The inhibition may be a result from the competition for electrons from electron donors. These findings impact on the understanding of the mechanism of bacterial anaerobic azoreduction and have implication for improving treatment methods of wastewater contaminated by azo dyes.

Decolorization of a dye industry effluent by Aspergillus fumigatus XC6 by Xian-Chun Jin; Gao-Qiang Liu; Zheng-Hong Xu; Wen-Yi Tao (pp. 239-243).

The strain Aspergillus fumigatus XC6 isolated from mildewing rice straw was evaluated for its ability to decolorize a dye industry effluent. The strain was capable of decolorizing dyes effluent over a pH range 3.0–8.0 with the dyes as sole carbon and nitrogen sources. The optimum pH was 3.0; however, supplemented with either appropriate nitrogen sources (0.2% NH₄Cl or (NH₄)₂SO₄ ) or carbon sources (1.0% sucrose or potato starch), the strain decolorized the effluent completely at the original pH of the dyes effluent. Therefore, A. fumigatus XC6 is an efficient strain for the decolorization of reactive textile dyes effluents, and it might be a practical alternative in dyeing wastewater treatment.

Keywords: Aspergillus fumigatus ; Biosorption; Decolorization; Dye; Effluent

A simple method for extracting C-phycocyanin from Spirulina platensis using Klebsiella pneumoniae by Y. Zhu; X. B. Chen; K. B. Wang; Y. X. Li; K. Z. Bai; T. Y. Kuang; H. B. Ji (pp. 244-248).

C-phycocyanin (C-PC) was extracted from fresh Spirulina platensis by deploying a species of non-pathogenic nitrogen-fixing bacteria, namely, Klebsiella pneumoniae. The algal slurry was neither washed nor centrifuged; the bacterial culture was poured into the slurry, the vessel sealed, and crude C-PC extracted after about 24 h. The extraction was clean and efficient, and the purity and concentration of C-PC proved to be of adequate quality.

Keywords: C-phycocyanin; Klebsiella pneumoniae ; Extraction; Spirulina platensis

A simple preparation method of crystals of soybean hull peroxidase by Junhong Liu; Haizhou Liu; Yuanyuan Zhang; Longhui Qiu; Feng Su; Fengmei Li; Zhongliang Su; Junfeng Li (pp. 249-255).

Soybean hull peroxidase (SHP) was crystallised from an enzyme solution with low purity by a simple method. The enzyme solution was purified by cooperation salting out of acetone and ammonium sulphate, and lumpy crystals were obtained with the size of about 40 × 30 μm when ammonium sulphate was quickly added to the enzyme solution. The crystal was examined and confirmed to be an SHP crystal by the method of activity test. The result shows that, though the purity of the enzyme solution was not high, crystals could be formed when the enzyme solution rapidly reached to a degree of supersaturation, which was different from the traditional methods of protein crystallisation. Additionally, a purification method of acetone and ammonium sulphate fractional salting out was also studied, in which the procedure was simplified, and a satisfactory purification effect was obtained.

Keywords: Soybean hull peroxidase; Purification; Cooperation salting out; Crystallisation

A screening system for active and enantioselective amidase based on its acyl transfer activity by Ren-Chao Zheng; Yu-Guo Zheng; Yin-Chu Shen (pp. 256-262).

A novel enantioselective amidase screening system was developed and proved to be efficient and accurate. This screening system employed acyl transfer activity of amidase in the presence of hydroxylamine, leading to the formation of hydroxamic acids, followed by spectrophotometric quantification of hydroxamic acid/iron(III) complexes. The enantioselectivities of amidase were evaluated by employing (R, S)-2, 2-dimethyl cyclopropanecarboxamide (1), (S)-2, 2-dimethyl cyclopropanecarboxamide and their mixture as substrates concurrently under the same conditions. To prove the accuracy of the screening system, enantioselectivity of acyl transfer reaction (E _T) and that of hydrolytic reaction (E _H) was compared. With this method, we obtained eight microorganism strains with enantioselective amidase from 523 isolates, two of which showed R-stereospecific avtivity for (R, S)-1.

Keywords: High-throughput screening; Amidase; Acyl transfer reaction; Hydroxamic acid; 2, 2-Dimethyl cyclopropanecarboxamide

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Applied Microbiology and Biotechnology (v.74, #1)

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Applied Microbiology and Biotechnology (v.74, #1)