Solvent tolerant enzymes in extremophiles: Adaptations and applications.

Non-aqueous enzymology has always drawn attention due to the wide range of unique possibilities in biocatalysis. In general, the enzymes do not or insignificantly catalyze substrate in the presence of solvents. This is due to the interfering interactions of the solvents between enzyme and water molecules at the interface. Therefore, information about solvent-stable enzymes is scarce. Yet, solvent-stable enzymes prove quite valuable in the present day biotechnology. The enzymatic hydrolysis of the substrates in solvents synthesizes commercially valuable products, such as peptides, esters, and other transesterification products. Extremophiles, the most valuable yet not extensively explored candidates, can be an excellent source to investigate this avenue. Due to inherent structural attributes, many extremozymes can catalyze and maintain stability in organic solvents. In the present review, we aim to consolidate information about the solvent-stable enzymes from various extremophilic microorganisms. Further, it would be interesting to learn about the mechanism adapted by these microorganisms to sustain solvent stress. Various approaches to protein engineering are used to enhance catalytic flexibility and stability and broaden biocatalysis's prospects under non-aqueous conditions. It also describes strategies to achieve optimal immobilization with minimum inhibition of the catalysis. The proposed review would significantly aid our understanding of non-aqueous enzymology.

Organic solvent tolerance of an alkaline protease from salt-tolerant alkaliphilic Streptomyces clavuligerus strain Mit-1.

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60-80 degrees C and displayed optimum activity at 70 degrees C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37 degrees C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.

Secretion of an alkaline protease from a salt- tolerant and alkaliphilic, Streptomyces clavuligerus strain Mit-1

An alkaliphilic and salt- tolerant actinomycete, Streptomyces clavuligerus strain Mit-1, was isolated from Mithapur, the western coast of India. The organism was Gram-positive, having filamentous, long thread like structure. The sporulation started after two days of growth and the optimum level of alkaline protease (130 U/ml) was produced during the early stationary phase. The strain could grow and produce protease with 0-10 percent NaCl (w/v), the optimum being 5 percent NaCl (w/v). Growth and protease production was optimum at pH 9 with substantial decline at neutral pH. Sucrose and gelatin were the best carbon and nitrogen sources respectively, whereas gelatin broth was the preferred medium for protease production. Mit-1 produced substantial protease with various amino acids, when employed as the sole nitrogen sources. Crude substrates, such as molasses, whey and wheat flour had significant effect on enzyme production. The results are quite valuable, as only few actinomycetes, particularly salt-tolerant alkaliphilic ones, have so far been explored for their enzymatic potential and process optimization.

Uma cepa halotolerante e alcalifílica de Streptomyces clavuligerus, Mit-1, foi isolada em Mithapur, na costa oeste da Índia. Esse microrganismo é Gram positivo e apresenta estrutura filamentosa na forma de longas cordas. A esporulação iniciou após dois dias de cultivo e o nível ótimo de produção de protease alcalina (130 U/ml) foi atingido no início da fase estacionária de crescimento. A cepa foi capaz de multiplicar com 0-10 por cento NaCl (w/v), com um ótimo de 5 por cento NaCl (w/v). O ótimo de crescimento e produção de protease foi atingido em pH 9, apresentando declínio substancial em pH neutro. Sacarose e gelatina foram as melhores fones de carbono e nitrogênio, respectivamente, enquanto o caldo gelatina foi o melhor meio para produção de protease. A cepa Mit-1 produziu bastante protease quando vários aminoácidos foram empregados como única fonte de nitrogênio. Substratos crus, como melaço, soro de leite e farinha de trigo, tiveram um efeito significativo na produção da enzima. Os resultados são bastante interessantes, considerando que somente poucos actinomicetos, especialmente os halotolerantes, já foram explorados por seu potencial de produção de enzimas e otimização de processos.

Two-step purification of a highly thermostable alkaline protease from salt-tolerant alkaliphilic Streptomyces clavuligerus strain Mit-1.

An alkaline protease from a salt-tolerant alkaliphilic Streptomyces clavuligerus was purified to homogeneity by 141-fold with a yield of 12% using two-step method of salt precipitation and ion exchange chromatography on DEAE cellulose. The apparent molecular mass was 49+/-2 kDa and the enzyme appeared as monomer based on SDS and Native-PAGE. The temperature optimum was 70 degrees C with significant stability at 60-80 degrees C for more than 60 min. The enzyme was active over the pH range of 8.5-11, with an optimum at 10-11. The serine nature of the protease was confirmed by PMSF inhibition. The enzyme was highly resistant against chemical denaturation and displayed varied effects towards metal ions. The results are significant as extremozymes are difficult to purify and therefore, a two-step purification of alkaline protease from relatively less explored group of actinomycetes is quite appealing.