Expression of Arginine Deiminase from <Emphasis Type="Italic">Pseudomonas plecoglossicida</Emphasis> CGMCC2039 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and Its Anti-Tumor Activity

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent in clinical trials for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. The arcA gene encoding ADI was cloned from a recently isolated strain Pseudomonas plecoglossicida CGMCC2039. The nucleotide sequence of ADI comprises an ORF of 1,254 bp encoding 417 amino acids. The deduced ADI protein sequence has a calculated molecular weight of 46.5 kDa and shows 97% and 85% identity to ADIs from P. putida and P. aeruginosa, respectively. The arcA from P. plecoglossicida CGMCC2039 was expressed in Escherichia coli BL21 with a N-terminal His₆-tag, and purified to homogeneity. A molecular mass of approximate 49 kDa was confirmed by SDS-PAGE analysis and specific activity was determined to be 4.76 U/mg (pH 6.0 and 37°C). In vivo activity study showed that the rADI could effectively inhibit H22 tumor growth at a total dose of 5 U/mouse over a 2-week dosing period.     

假单胞菌NyZ12基因组的可塑多变特征

【背景】假单胞菌是广泛存在于土壤、水体环境的微生物,其中Pseudomonas plecoglossicida NyZ12是一株能够以环己胺为唯一碳源和氮源生长的革兰氏阴性菌,其基因组达到7.0Mb左右。【目的】研究假单胞菌NyZ12的基因组是否具有可塑性和多变特征。【方法】以环己胺为唯一碳源和氮源生长的P. plecoglossicida NyZ12为研究对象,以琥珀酸或者代谢中间产物环己酮为碳源连续传代让其自然发生突变,然后筛选在以环己胺为唯一碳源和氮源的无机盐培养基上不能生长的突变体。将获得的突变体进行全基因组测序,并与野生型假单胞菌NyZ12的全基因组进行比对。【结果】以琥珀酸和环己酮为碳源分别筛选到一株突变体T1和T2,测序比对后发现假单胞菌突变体T1、T2的基因组发生大量的缺失和突变。对基因丢失的原因进行了分析,丢失的2个大片段中存在大量的重复序列、转座酶、转座子和原噬菌体。【结论】假单胞菌NyZ12的基因组具有可塑多变的特征。其可能的机制为进一步揭示微生物的适应和进化提供了参考。     

Squamocin mode of action to stimulate biofilm formation of Pseudomonas plecoglossicida J26, a PAHs degrading bacterium

Squamocin, an annonaceous acetogenin (ACG) extracted from Annona cherimolia (Annonaceae), has been shown to increase biofilm production of Pseudomonas plecoglossicida J26 (closely related to P. plecoglossicida), a polycyclic aromatic hydrocarbon degrading bacterium. PAHs have become priority pollutants for bioremediation due to their carcinogenicity and toxicity. The effect of various stressful stimuli (naphthalene, octanol, HCl, and NaCl) on cell growth, biofilm formation and autoinducer production of P. plecoglossicida were evaluated and compared with the effect of squamocin to establish its mode of action on biofilm formation. All stressors that inhibited growth stimulated autoinducer production while squamocin was growth stimulant at concentrations above 2.5 μg ml⁻¹. Despite structural similarities, squamocin is not an autoinducer agonist. It indirectly stimulates autoinducer production and increases P. plecoglossicida J26 cell growth. This is the first report on an ACG mode of action in the formation of biofilm in a naphthalene-degrading strain.     

Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Summary Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the␣L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of␣L.␣plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L.␣plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.     

精氨酸脱亚胺酶在大肠杆菌中的分泌型表达

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arc A克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢB中,经鉴定得到重组质粒pBAD-ADI。将重组质粒转化大肠杆菌TOP10F'后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002%(w/v),25℃下诱导5 h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同)。采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL。SDS-PAGE分析显示,重组蛋白大小约为46 kD。     

Isolation and characterization of arsenite-oxidizing bacteria from arsenic-contaminated soils in Thailand

Two hundred and eighty-eight arsenic-resistant bacteria were isolated by an enrichment culture method from a total of 69 arsenic-contaminated soil-samples collected from Dantchaeng district in Suphanburi province (47 samples), and from Ron Phiboon district in Nakhon Sri Thammarat province (22 samples), in Central and Southern Thailand, respectively. Twenty-four of the 288 isolated arsenic-resistant bacteria were found to be arsenite-oxidizing bacteria. On the basis of their morphological, cultural, physiological, biochemical and chemotaxonomic characteristics, and supported by phylogenetic analysis based upon their 16S rRNA gene sequences, they were divided into five groups, within the genera Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium and Sphingomonas, respectively. Within genera, phylogenetic analysis using the 16S rRNA gene sequences suggested that they were comprised of at least ten species, five isolates being closely related to known bacteria (Acinetobacter calcoaceticus NCCB 22016^T, Pseudomonas plecoglossicida FPC951^T, Ps. knackmussii B13^T, Sinorhizobium morelense Lc04^T, and Sphingomonas subterranea IFO16086^T). The other five proposed species are likely to be new species closely related to Flavobacterium johnsoniae, Sinorhizobium morelense, Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, but this awaits further characterization for confirmation of the taxonomic status. No overlap in isolated species or strains was observed between the two sites. The strain distribution and characterization are described.     

Degradation of cyclohexylamine by a new isolate of <Emphasis Type="Italic">Pseudomonas plecoglossicida</Emphasis>

A strain utilizing cyclohexylamine as the sole source of carbon and nitrogen, designated NyZ12, was isolated from soil and identified by 16S rDNA sequencing as Pseudomonas plecoglossicida. This bacterium released ammonia into the medium when grown on cyclohexylamine, and also grows readily on cyclohexanone as the sole carbon source, suggesting that degradation involves an initial deamination step.     

Rapid evolution of arginine deiminase for improved anti-tumor activity

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K _m value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100–120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime–thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K _m under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K _m value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers.     

Degradation of perfluorooctanyl sulfonate by strain <Emphasis Type="Italic">Pseudomonas plecoglossicida</Emphasis> 2.4-D

A bacterial strain has been isolated from soil contaminated by waste from petrochemical production. Based on its cultural-morphological, physiological-biochemical properties and an analysis of the nucleotide sequence of the 16S rRNA gene and phylogenetic analysis data, the strain was defined as Pseudomonas plecoglossicida 2.4-D. The unique ability of this bacterium to use perfluorooctanyl sulfonate (PFOS) as the only source of carbon and energy is shown. When cultivated in a liquid medium, the strain completely utilizes this substance in 6 days. It is proved that P. plecoglossicida 2.4-D transforms PFOS to perfluoroheptanoic (perfluoroenanthic) acid, while free fluorine ions are released into the medium. Upon introduction into the soil, the strain was capable of PFOS degradation by 75%. The strain P. plecoglossicida 2.4-D is recommended for environmental protection. It can be used in the development of biotechnologies for the transformation (utilization) of fluoroorganic compounds.     

嗜盐四联球菌arc operon多拷贝基因在精氨酸代谢中的作用

【背景】嗜盐四联球菌(Tetragenococcus halophilus)是一类存在于发酵食品中的耐盐乳酸菌,研究其精氨酸(arginine,Arg)代谢对解析食品发酵过程中氨基甲酸乙酯(ethyl carbamate,EC)前体积累机制和保障食品安全具有重要意义。【目的】研究酱醪来源嗜盐四联球菌精氨酸脱亚氨基(arginine deiminase,ADI)途径的基因构成,揭示这些基因对菌株精氨酸代谢和氨基甲酸乙酯前体瓜氨酸(citrulline,Cit)利用与积累的影响。【方法】采用PCR扩增与测序分析不同菌株的ADI途径基因组成,通过比较ADI途径关键基因转录水平和关键酶活性,探究环境因素对嗜盐四联球菌代谢氨基酸能力的影响及各拷贝基因参与氨基酸代谢的功能。【结果】酱醪来源嗜盐四联球菌基因组中ADI途径基因类型主要有两大类：以菌株R23为代表含有完整arc操纵子(operon)基因且具有最多基因拷贝数;以菌株C3为代表缺失arcA和arcB但含有多拷贝arcB和arcC。基因组中有arcA的菌株才具有利用精氨酸能力,并通过利用精氨酸生成瓜氨酸。体系中精氨酸含量和乙醇与脂肪酸的存在均可影响嗜盐四联球菌利用精氨酸积累中间产物瓜氨酸。当精氨酸含量大于5 g/L或体系中含有乙醇与脂肪酸时,嗜盐四联球菌会利用精氨酸积累中间产物瓜氨酸。脂肪酸和乙醇对ADI途径的3个关键酶均有显著抑制作用,可使精氨酸脱亚氨基酶(arginine deiminase,ADI)、鸟氨酸氨甲酰基转移酶(ornithine transcarbamylase,OTC)和氨甲酰磷酸激酶(carbamate kinase,CK)的活性分别降低41.0%、46.4%和60.0%。嗜盐四联球菌中arcB转录水平分别是其拷贝arcB₁和arcB₂的10.5倍和29.8倍,arcC的转录水平分别是arcC₁、arcC₂、arcC₃的17.6、20.3、23.9倍,说明arcB和arcC在瓜氨酸代谢中起主要作用。【结论】精氨酸含量和乙醇加脂肪酸是影响嗜盐四联球菌代谢精氨酸能否积累瓜氨酸的关键环境因素。嗜盐四联球菌arc operon的多拷贝基因中,arcB和arcC基因在瓜氨酸代谢中起主要作用。     

Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonasaeruginosa, P. plecoglossicida and P. mosselii

Of 80 fluorescent pseudomonad strains screened for phosphate solubilization, three strains (BFPB9, FP12 and FP13) showed the ability to solubilize tri-calcium phosphate (Ca₃(PO₄)₂). During mineral phosphate solubilization, decrease of pH in the culture medium due to the production of organic acids by the strains was observed. These phosphate solubilizing strains produced indole-3-acetic acid (IAA) and protease as well as exhibited a broad-spectrum antifungal activity against phytopathogenic fungi. When tested in PCR using the gene-specific primers, strain BFPB9 showed the presence of hcnBC genes that encode hydrogen cyanide. On the basis of phenotypic traits, 16S rRNA sequence homology and subsequent phylogenetic analysis, strains BFPB9, FP12 and FP13 were designated as Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii, respectively. Present investigation reports the phosphate solubilization potential and biocontrol ability of new strains that belong to P. plecoglossicida and P. mosselii. Because of the innate potential of phosphate solubilization, production of siderophore, IAA, protease, cellulase and HCN strains reported in this study can be used as biofertilizers as well as biocontrol agents.     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate) with flexible 3-hydroxyhexanoate content in<Emphasis Type="Italic"> Aeromonas hydrophila</Emphasis> CGMCC 0911

Aeromonas hydrophila CGMCC 0911 isolated from lake water was found to be able to synthesize a polyhydroxyalkanoate (PHA) copolymer (PHBHHx) consisting of 3-hydroxybutyrate (HB) and 4–6 mol% 3-hydroxyhexanoate (HHx). The wild-type bacterium accumulated 49% PHBHHx containing 6 mol% HHx in terms of cell dry weight (CDW) when grown on lauric acid for 48 h. When A. hydrophila CGMCC 0911 expressed the Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli, the recombinant strain could accumulate 47% PHBHHx, while the HHx content reached 17.4 mol%. The presence of changing glucose concentration in the culture changed the HHx content both in wild type and recombinant A. hydrophila CGMCC 0911. When 5 g l^–1 glucose was added to a culture containing 5 g l^–1 lauric acid as co-substrate, 45% PHBHHx/CDW consisting of 8.8 mol% HHx was produced by wild-type A. hydrophila CGMCC 0911 compared with only 5% in the absence of glucose. When the recombinant A. hydrophila CGMCC 0911 was grown on a mixed substrate containing lauric acid and 8–10 g l^–1 glucose, the HHx content could be further increased to 35.6 mol%. When the glucose concentration exceeded 10 g l^–1, cell growth, PHA content and mole percentages of HHx in PHBHHx were significantly reduced.     

Penicillium Mycobiota in Arctic Subglacial Ice

Fungi have been only rarely isolated from glacial ice in extremely cold polar regions and were in these cases considered as random, long-term preserved Aeolian deposits. Fungal presence has so far not been investigated in polar subglacial ice, a recently discovered extreme habitat reported to be inhabited exclusively by heterotrophic bacteria. In this study we report on the very high occurrence (up to 9000 CFU L⁻¹) and diversity of filamentous Penicillium spp. in the sediment-rich subglacial ice of three different polythermal Arctic glaciers (Svalbard, Norway). The dominant species was P. crustosum, representing on the average half of all isolated strains from all three glaciers. The other most frequently isolated species were P. bialowiezense, P. chrysogenum, P. thomii, P. solitum, P. palitans, P. echinulatum, P. polonicum, P. commune, P. discolor, P. expansum, and new Penicillium species (sp. 1). Twelve more Penicillium species were occasionally isolated. The fungi isolated produced consistent profiles of secondary metabolites, not different from the same Penicillium species from other habitats. This is the first report on the presence of large populations of Penicillium spp. in subglacial sediment-rich ice.     

Inactivation of type I polyhydroxyalkanoate synthase in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> resulted in discovery of another potential PHA synthase

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC _mcl) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC _mcl gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.     

Isolation and characterization of three new PGPR and their effects on the growth of Arabidopsis and Datura plants

This study characterizes three bacterial strains isolated from plant rhizospheres and evaluates their performance as plant-growth-promoting rhizobacteria. Pseudomonas plecoglossicida strain Pp20 was isolated from the rhizosphere of a date palm in Bechar (Algerian Sahara), Bacillus spec. strain Bt04 isolated from the rhizosphere of pear in Ghardaia (Algerian Sahara) and Lysinibacillus fusiformis strain Lf89 was isolated from the rhizosphere of tomato in Ain Defla (northern Algeria). Their effects on plant growth and development were analyzed in different in vitro cultures: an Arabidopsis thaliana plate assay and two hydroponic systems for Datura stramonium and Datura tatula. Our results show that all strains significantly improve plant growth of the plant species tested and some strains produce a shift in the C/N ratio in A. thaliana. Inoculation had no effect on alkaloid production per gram leaf dry weight in D. stramonium, but specific plant-growth-promoting rhizobacteria interactions may alter the alkaloid composition in the shoot.     

Isolation, identification and characterization of algicidal bacteria against Stephanodiscus hantzschii and Peridinium bipes for the control of freshwater winter algal blooms

Five strains (HYY0510-SK04, HYY0511-SK09, HYK0512-SK12, HYK0512-PK04 and HYY0512-PK05) of algicidal bacteria against the harmful bloom forming diatom Stephanodiscus hantzschii and dinoflagellate Peridinium bipes, were isolated. Among these strains, HYY0510-SK04, HYY0511-SK09 and HYK0512-SK12 have an effective algicidal activity for S. hantzschii, while HYK0512-PK04 and HYY0512-PK05 have an algicidal effect against P. bipes. Sequence analysis of 16S rDNA showed that HYY0510-SK04 and HYY0511-SK09 were closely related to Acidovorax delafieldii ATCC 17505^T. HYK0512-SK12, HYK0512-PK04 and HYY0512-PK05 showed high homology with Variovorax paradoxus IAM 12373^T (98.9%), Hydrogenophaga palleronii ATCC 49743^T (98.8%) and Pseudomonas plecoglossicida ATCC 700383^T (98.3%), respectively. HYY0510-SK04, HYY0511-SK09 and HYK0512-SK12 degraded S. hantzschii cells within two weeks when those bacteria were inoculated at densities of ≥10⁷cells mL⁻¹ to the lag or logarithmic growth phase of the algal culture. HYK0512-PK04 and HYY0512-PK05 degraded more than 90% of P. bipes cells within 14 and 8 days, respectively, when these bacteria were inoculated at densities of ≥10⁷cells mL⁻¹. Among the five bacterial strains, HYK0512-SK12 and HYY0512-PK05 showed the most effective growth inhibition of all the algae and cyanobacteria tested. Biochemical assays revealed that the main algicidal substance from all isolates were likely to be extracellular substances. These results indicate that the bacterial strains isolated for this study are potential agents for the control of harmful algal blooms in eutrophic reservoirs.     

A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, its application as anticancer drug is hampered by its poor stability under physiological conditions in the bloodstream. Commonly, random PEGylation is being used for increasing the stability of ADI and in turn the improved half-life. However, the traditional random PEGylation usually leads to poor PEGylation efficiency due to the limited number of Lys on the protein surface. To boost the PEGylation efficiency and enhance the stability of ADI further, surface engineering of PpADI (an ADI from Pseudomonas plecoglossicida) to increase the suitable PEGylation sites was carried out. A new in silico approach for increasing the PEGylation sites was developed. The validation of this approach was performed on previously identified PpADI variant M31 to increase potential PEGylation sites. Four Arg residues on the surface of PpADI M31 were selected through three criteria and subsequently substituted to Lys, aiming for providing primary amines for PEGylation. Two out of the four substitutions (R299K and R382K) enhanced the stability of PEGylated PpADI in human serum. The average numbers of PEGylation sites were increased from ~12 (tetrameric PpADI M31, starting point) to ~20 (tetrameric PpADI M36, final variant). Importantly, the PEGylated PpADI M36 after PEGylation exhibited significantly improved T_m values (M31: 40°C; M36: 40°C; polyethylene glycol [PEG]-M31: 54°C; PEG-M36: 64°C) and half-life in human serum (M31: 1.9 days; M36: 2.0 days; PEG-M31: 3.2 days; PEG-M36: 4.8 days). These proved that surface engineering is an effective approach to increase the PEGylation efficiency which therefore enhances the stability of therapeutic enzymes. Furthermore, the PEGylated PpADI M36 represents a highly attractive candidate for the treatment of arginine-auxotrophic tumors.     

Gene cloning,expression, and characterization of a novel trehalose synthase from <Emphasis Type="Italic">Arthrobacter aurescens</Emphasis>

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 °C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg²⁺, Mn²⁺, and Ca²⁺ while strongly inhibited by 5 mM Cu²⁺ and SDS.