Optimisation of biodegradation conditions for waste canola oil by cold-adapted Rhodococcus sp. AQ5-07 from Antarctica

BACKGROUND: The potential waste canola oil-degrading ability of the cold-adapted Antarctic bacterial strain Rhodococcus sp. AQ5-07 was evaluated. Globally, increasing waste from food industries generates serious anthropogenic environmental risks that can threaten terrestrial and aquatic organisms and communities. The removal of oils such as canola oil from the environment and wastewater using biological approaches is desirable as the thermal process of oil degradation is expensive and ineffective. RESULTS: Rhodococcus sp. AQ5-07 was found to have high canola oil-degrading ability. Physico-cultural conditions influencing its activity were studied using one-factor-at-a-time (OFAT) and statistical optimisation approaches. Considerable degradation (78.60%) of 3% oil was achieved by this bacterium when incubated with 1.0 g/L ammonium sulphate, 0.3 g/L yeast extract, pH 7.5 and 10% inoculum at 10°C over a 72-h incubation period. Optimisation of the medium conditions using response surface methodology (RSM) resulted in a 9.01% increase in oil degradation (87.61%) when supplemented with 3.5% canola oil, 1.05 g/L ammonium sulphate, 0.28g/L yeast extract, pH 7.5 and 10% inoculum at 12.5°C over the same incubation period. The bacterium was able to tolerate an oil concentration of up to 4.0%, after which decreased bacterial growth and oil degradation were observed. CONCLUSIONS: These features make this strain worthy of examination for practical bioremediation of lipid-rich contaminated sites. This is the first report of any waste catering oil degradation by bacteria originating from Antarctica.

Expression and characterization of a novel cold-adapted lipase from psychrotolerant bacteria Psychrobacter sp. S1B

@#Aim: This study aimed to isolate, express and characterize the lipase derived Psychrobacter sp. S1B in Escherichia coli expression system. Methodology and results: Exploration towards S1B lipase characteristic was conducted where shotgun cloning method was applied to obtain lipase encoded gene and E. coli expression system through pET28a was used to overexpress S1B lipase. Lipase activity was measured by using p-nitrophenol method. The S1B lipase gene is 1005 bp in length with molecular weight of 46 kDa, optimum pH was 10.0, showed hydrolytic activity preference toward p-nitrophenyl caprylate (C8) and p-nitrophenyl hexanoate (C6) substrates (C6 < C8). The best temperature for S1B lipase activity was at 30 °C while exhibited high activity at lower temperature (10-25 °C) with above 90% of maximum activity, therefore it is classified as cold adaptive lipase. In addition, S1B lipase showed stability against various metal ions, including Cu2+ and Zn2+ which commonly act as inhibitors of lipases derived from Psychrobacter species. Moreover, S1B lipase exhibited great tolerance against up to 50% (v/v) hexane and some non-ionic detergents such as 1% (v/v) DMSO and 1% (v/v) Triton X-100. Conclusion, significance and impact of study: The study proposes a novel cold-adapted lipase which has potential as a biocatalyst for synthesis caprylic acid ester

Biodiversity of psychrotrophic microbes and their biotechnological applications

The extreme cold environments harbor novel psychrotrophic microbes. The psychrotrophic microbes have been reportedas plant growth promoters and biocontrol agents for sustainable agriculture, in industry as cold-adapted hydrolyticenzymes and in medicine as secondary metabolites and pharmaceutical important bioactive compounds. Inoculationwith psychrotrophic/psychrotolerant strains significantly enhanced root/shoot biomass and nutrients uptake as comparedto non-bacterized control. The psychrotrophic microbes play important role in alleviation of cold stress in plant growingat high hill and low temperature and conditions. The psychrotrophic microbes have been reported from worldwidefrom cold habitats and belong to all three domain archaea, bacteria, and eukarya including different phylum such asActinobacteria, Ascomycota, Bacteroidetes, Basidiomycota, Chloroflexi, Chlamydiae, Planctomycetes, Cyanobacteria,Euryarchaeota, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Mucoromycota, Proteobacteria, Spirochaetes,Thaumarchaeota and Nitrospirae. The most dominant genera belong to Arthrobacter, Bacillus, Exiguobacterium,Paenibacillus, Providencia, Pseudomonas, and Serratia have been reported from the cold habitats. The Psychrotrophicmicrobes have biotechnological applications in agriculture, medicine, industry, food, and allied sectors

Produção de L-asparaginase pela levedura Leucosporidium muscorum CRM 1648 isolada de sedimento marinho coletado na Península Antártica / L-asparaginase production by the yeast Leucosporidium muscorum CRM 1648 isolated from marine sediments collected in Antarctic Peninsula

L-asparaginase (L-ASNase) é uma enzima com propriedades interessantes para a indústria médica, farmacêutica e de alimentos, que tem recebido atenção especial, inclusive no Brasil, por fazer parte do protocolo de tratamento de distúrbios linfoproliferativos, como a leucemia linfoblástica aguda (LLA). No mercado desde a década de 1970, as enzimas de origem bacteriana enfrentam algumas limitações por provocarem reações adversas graves em quase 80% dos pacientes em tratamento. Nesse contexto, L-ASNases provenientes de leveduras se destacam como alternativa, por serem mais próximas às congêneres humanas. A Antártica ainda é um ambiente pouco explorado, com grande diversidade de microrganismos com potencial para a produção de moléculas biológicas de interesse industrial. Nesse contexto, 150 leveduras isoladas de amostras de sedimento marinho coletadas na Península Antártica como parte do projeto MICROSFERA (PROANTAR/CNPq) foram avaliadas para a produção de L-ASNase. A triagem resultou em 9 isolados produtores, dos quais 7 pertencem ao gênero Leucosporidium. A linhagem L. muscorum CRM 1648 foi a que produziu mais enzima (540 U.L-1), com maior produtividade (5,6 U.L-1.h-1) e, por isso, foi alvo deste estudo. A análise univariada de fontes de carbono e nitrogênio indicou maior crescimento desse microrganismo e produção de L-ASNase em meio CD com extrato de levedura, prolina e sacarose. Ureia, cloreto de amônio e sulfato de amônio resultaram em baixa ou nenhuma produção da enzima, sugerindo que a metabolização de fontes de nitrogênio por essa linhagem está sob a influência do fenômeno de repressão catabólica pelo nitrogênio (RCN). Dois delineamentos experimentais do tipo fatorial completo resultaram em um aumento de 10 vezes na produção e produtividade da enzima (4582,5 U.L-1 e 63,6 U.L-1.h-1, respectivamente). A análise univariada da concentração inicial de inóculo (X0), pH inicial do meio, temperatura e adição de água do mar mostrou que a melhor condição para a produção foi: pH = 5,5 ou 6,5, cultivo a 15°C com adição de água do mar (25-50% m/v). A variável X0 não foi significativa nas concentrações avaliadas. Cultivos em biorreator (batelada) foram conduzidos em quatro diferentes níveis de oxigênio dissolvido (OD): (1) OD não controlado e abaixo de 20%, (2) OD não controlado e acima de 20%, (3) OD controlado em 80% e (4) OD controlado em 20%. Os resultados mostraram que OD é fator limitante para o crescimento de L. muscorum CRM 1648 e produção de L-ASNase por essa levedura e deve ser mantido acima de 35% para maior produção da enzima.Neste trabalho, a composição do meio e condições de cultivo foram estabelecidas para favorecer a produção de uma nova L-ASNase livre de atividade glutaminásica por levedura adaptada ao frio, abrindo espaço para novos estudos acerca de seu potencial antileucêmico e possível uso como alternativa às enzimas já existentes no mercado no tratamento de LLA

L-asparaginase (L-ASNase) is an enzyme with interesting properties for medical, pharmaceutical and food industry, which has received special consideration, especially in Brazil, for being part of lymphoproliferative disorders treatment, such as acute lymphoblastic leukemia (ALL). Bacterial enzymes are on the market since the 1970s and face some limitations related to theirserious adverse reactions that reach almost 80% of all patients in treatment. In this context, L-ASNases from yeasts are highlighted as important alternative to bacterial enzymes, due to the closerphylogeny to human congeners. Antarctic environment has much to be explored, with a vast diversity of microorganisms with potential to produce biomolecules with industrial interest. A total of 150 yeasts isolated from Antarctic marine sediments as part of MICROSFERA project (PROANTAR/CNPq) were evaluated for L-ASNase production. The screening resulted in 9 producers, 7 species from the genus Leucosporidium. L. muscorum CRM 1648 was the strain that yielded the highest L-ASNase activity (540 U.L-1) and volumetric productivity (5.6 U.L-1.h-1). Carbon and Nitrogen sources were evaluated by a method of one-factor at a time (OFAT). From the gather results, sucrose, yeast extract and proline resulted in a maximal growth and highest enzyme production.The absence or low production of L-ASNase in medium with urea, ammonium chloride and ammonium sulfate suggests the presence of nitrogen catabolic repression (NCR). Carbon and nitrogen concentration were evaluated by full factorial design and yielded about ten times higher enzyme and volumetric productivity (4582.5 U.L-1 and 63.6 U.L-1.h-1, respectively). Initial inoculum concentration (X0), initial pH, temperature and concentration of seawater in the culture were evaluated by OFAT analysis and the best condition for L-ASNase production was: pH = 5.5 or 6.5, at 15 °C with addition of seawater (25-50 wt%). X0 was not considered a significant variable. Bioreactor assays (in batch regime) were performed in four different dissolved oxygen (DO) levels: (1) without DO control (DO remained under 20%), (2) without DO control (DO remained above 20%), (3) DO controlled at 80%, and (4) DO controlled at 20%.The results showed that DO is a key factor for growth of L. muscorum CRM 1648 and production of L-ASNase by this yeast and should be maintained above 35% for higher production of this enzyme.At this work, the medium and culture conditions were established to support the production of a novel glutaminase-free L-ASNase by a cold adapted yeast, opening a new path for further studies regarding its antileukemic potential and possible use as an alternative for ALL treatment

Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain

An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg²⁺, Mn²⁺, K⁺; Ca²⁺, Na⁺, Ba²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Sr²⁺ and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

Partial characterization of cold active amylases and proteases of Streptomyces sp. from Antarctica

The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100 percent identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20ºC, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20ºC. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures.

Antarctic marine bacterium Pseudoalteromonas sp. KNOUC808 as a source of cold-adapted lactose hydrolyzing enzyme

Psychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5¨¬C, and showed 0.867 unit of o-nitrophenyl ¥â-D-galactopyranoside(ONPG) hydrolyzing activity at 4¨¬C. The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20¨¬C, pH 6.8-7.2. The composition of major fatty acids in cell of KNOUC801 was C12:0 (5.48 percent), C12:0 3OH (9.21 percent), C16:0 (41.83 percent), C17:0 ¥ø8 (7.24 percent) and C18:1 ¥ø7 (7.04 percent). All suthese results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20¨¬C and 7.8, respectively. The enzyme was stable at 4¨¬C for 7 days, but its activity decreased to about 50 percent of initial activity at 37¨¬C in 7 days.

Analysis of the Structure-stability Relationship of Cold-adapted Lipase PsLip1 from Homology Modeling

Two initial models of cold-adapted lipase PsLip1 have been constructed, based on homology with the bacterial lipases Chromobacterium viscosum(CvLip) and Pseudomonas cepacia(PcLip), whose X-ray structures have been solved and refined to high resolution. The mature polypeptide chains of these lipases have 84% similarity. The models of Mod1 and Mod2 have been compared with the tertiary structures of CvLip and PcLip, respectively, and analyzed in terms of stabilizing interactions. Several structural aspects that are believed to contribute to protein stability have been compared: the number of conserved salt bridges, aromatic interactions, hydrogen bonds, helix capping, and disulfide bridges. The 3-dimensional structural model of PsLip1 has been constructed in order to elucidate the structural reasons for the decreased thermostability of the enzyme in comparison with its mesophilic counterparts.

Control of Influenza:Development of Live Vaccine / 감염과화학요법

Although trivalent subunit vaccine has been available, the influenza vaccine has been under-utilized because of cumbersome route of vaccination and low level of protection. Therefore, there has always been a great need to develop live attenuated influenza vaccine which can be administered through nasal route and elicit better immunogenicity. Through conventional repeated passage at low temperature, a live influenza vaccine carrier could be established. By reassortant formation between the 'cold- adapted' vaccine carrier and virulent strains, a prototype of trivalent live influenza vaccine is developed. Influenza A virus was adapted to replicate at low temperature. Serial passage at progressively lower temperature (30degrees C, 27degrees C and 24degrees C)resulted in the generation of cold-adapted (ca), temperature-sensitive (ts) mutant and attenuation (att) phenotype. This strain was evaluated for their ability to protect mice from challenge with same subtype and different subtype of influenza A virus. The study showed that vaccination of mice with live attenuated influenza virus provided complete protection against homologous and heterologous virus challenge. We also evaluated therapeutic potential of ca influenza virus. The mice infected with ca virus before challenge with wild type viruses or infected with simultaneously showed reduced clinical symptoms suggesting potential therapeutic effects.

Control of Influenza:Development of Live Vaccine / 감염과화학요법

Although trivalent subunit vaccine has been available, the influenza vaccine has been under-utilized because of cumbersome route of vaccination and low level of protection. Therefore, there has always been a great need to develop live attenuated influenza vaccine which can be administered through nasal route and elicit better immunogenicity. Through conventional repeated passage at low temperature, a live influenza vaccine carrier could be established. By reassortant formation between the 'cold- adapted' vaccine carrier and virulent strains, a prototype of trivalent live influenza vaccine is developed. Influenza A virus was adapted to replicate at low temperature. Serial passage at progressively lower temperature (30degrees C, 27degrees C and 24degrees C)resulted in the generation of cold-adapted (ca), temperature-sensitive (ts) mutant and attenuation (att) phenotype. This strain was evaluated for their ability to protect mice from challenge with same subtype and different subtype of influenza A virus. The study showed that vaccination of mice with live attenuated influenza virus provided complete protection against homologous and heterologous virus challenge. We also evaluated therapeutic potential of ca influenza virus. The mice infected with ca virus before challenge with wild type viruses or infected with simultaneously showed reduced clinical symptoms suggesting potential therapeutic effects.

Progress in the study of peculiar enzymes of antarctic microorganisms / 中国海洋药物

Increased research and exploitation of the low-temperature enzymes of antarctic microorganisms have occurred over recent years. There are a lot of study on protease, lipase and amylase, and so on. In antarctic microorganisms while some peculiar enzymes of antarctic microorganisms are still less researched. The recent development of the peculiar enzymes of the antarctic microorganisms was reviewed in this article. The properties of chitinase, dehydrogenas,?-galactosidase,RNA polymerase,DEAD-box RNA Helicase and metalloprotease etc. were introduced in detail, and the prospects of their application were also discussed.