Abstract Employing Illumina Hiseq whole genome metagenome sequencing approach, we studied the impact of Trichoderma harzianum on altering the microbial community and its functional dynamics in the rhizhosphere soil of black pepper (Piper nigrum L.). The metagenomic datasets from the rhizosphere with (treatment) and without (control) T. harzianum inoculation were annotated using dual approach, i.e., stand alone and MG-RAST. The probiotic application of T. harzianum in the rhizhosphere soil of black pepper impacted the population dynamics of rhizosphere bacteria, archae, eukaryote as reflected through the selective recruitment of bacteria [Acidobacteriaceae bacterium (p = 1.24e-12), Candidatus koribacter versatilis (p = 2.66e-10)] and fungi [(Fusarium oxysporum (p = 0.013), Talaromyces stipitatus (p = 0.219) and Pestalotiopsis fici (p = 0.443)] in terms of abundance in population and bacterial chemotaxis (p = 0.012), iron metabolism (p = 2.97e-5) with the reduction in abundance for pathogenicity islands (p = 7.30e-3), phages and prophages (p = 7.30e-3) with regard to functional abundance. Interestingly, it was found that the enriched functional metagenomic signatures on phytoremediation such as benzoate transport and degradation (p = 2.34e-4), and degradation of heterocyclic aromatic compounds (p = 3.59e-13) in the treatment influenced the rhizosphere micro ecosystem favoring growth and health of pepper plant. The population dynamics and functional richness of rhizosphere ecosystem in black pepper influenced by the treatment with T. harzianum provides the ecological importance of T. harzianum in the cultivation of black pepper.

Três formas distintas (Lip A, Lip B e Lip C) de lipases extracelulares (EC - 3.1.1.3), produzidas por Candida rugosa em fermentação no estado sólido (SSF) foram purificadas e caracterizadas. SSF foi conduzida em colunas de vidro, utilizando torta de óleo de coco e farelo de trigo. A enzima foi purificada do extrato aquoso do material fermentado por precipitação com sulfato de amônio, diálise, ultra - filtração e filtração em gel com Sephadex - 200 para uma purificação de 43 vezes e 64.35 - mg / ml de atividade específica. SDS - PAGE da enzima purificada revelou três faixas distintas, enquanto indicando a existência de três formas isômeras, Lip A, Lip B e Lip C com peso molecular aparente de aproximadamente 64,000, 62,000 e 60,000 Da, respectivamente. Todos as três formas isômeras eram ativas opticamente a 35-40ºC e pH 7-8. Elas mostraram diferenças marcantes nos valores de Km com diferentes triacilgliceróis saturados e insaturados. Ag + + e Hg + + inibiram fortemente a atividade enzimática de todas as três formas isômeras, Mn + + não produziu efeito e Ca + + e Mg + + aumentaram a atividade. EDTA inibiu fortemente as atividades enzimáticas dos isômeros. Porém, atividades de todas as três lipases eram completamente inibidas por inibidores de protease serina como 3,4 - dicloroisocomarina, pefabloc e parcialmente através de fluoreto de fenilmetanesulfonil. Este é o primeiro relatório que descreve a purificação e caracterização de lipases isoméricas produzidas por C. rugosa em culturas sólidas. Estes isômeros de lipase com características diversas produzidas em culturas sólidas podem obter aplicação potencial em campo biomédico.

Three distinct forms (Lip A, Lip B and Lip C) of extra-cellular lipases (EC- 3.1.1.3), produced by Candida rugosa in solid state fermentation (SSF) were purified and characterised. SSF was carried out in glass columns using coconut oil cake and wheat bran. The enzyme was purified from the aqueous extract of fermented matter by ammonium sulphate precipitation, dialysis, ultra-filtration and gel filtration using Sephadex-200 to a 43-fold purification and 64.35-mg/ml specific activity. SDS-PAGE of purified enzyme revealed three distinct bands, indicating the existence of three iso-forms, Lip A, Lip B and Lip C with apparent molecular weight about 64,000, 62,000 and 60,000 Da, respectively. All the three iso-forms were optimally active at 35-40°C and pH 7-8. They showed marked differences in their Km values with different saturated and unsaturated triacyl glycerols. Ag++ and Hg++ strongly inhibited enzyme activity of all the iso-forms, Mn++ has no effect and Ca++ and Mg++ enhanced the activity. EDTA also strongly inhibited the enzyme activities of iso-forms. However, activities of all the three lipases were completely inhibited by serine protease inhibitors such as 3,4-dichloroisocoumarin, pefabloc and partially by phenylmethanesulphonyl fluoride. To the best of our knowledge, this is the first report describing the purification and characterisation of C. rugosa lipase iso-forms from solid cultures. These lipase iso-forms with diverse characteristics produced in solid cultures may find potential application in biomedical field.

Três formas distintas (Lip A, Lip B e Lip C) de lipases extracelulares (EC- 3,1,1,3), produzidas por Candida rugosa em fermentação no estado sólido (SSF) foram purificadas e caracterizadas. A fermentação foi realizada em colunas de vidro usando como substrato bolo de óleo de coco e o farelo de trigo. O enzima obtida no extrato aquoso do material fermentado foi precipitada com do sulfato de do amônio, dialisada , ultra-filtrada e filtrada em gel usando Sephadex-200 obtendo assim um nível de purificação de 43-fold e uma atividade específica de 64,35 mg/ml. SDS-page da enzima purificada revelou três bandas distintas, indicando a existência de três iso-formas distintas, a banda A, a banda B e a banda C com pesos moleculares aparentes de 64.000, 62.000 e 60.000 Da, respectivamente. Todas as três iso-formas foram oticamente ativas entre 35-40ºC e a pH 7-8. Mostraram diferenças marcantes em seus valores do Km com triacyl glycerols saturado e não saturado. A atividade das enzimas de todas as três iso-formas foi fortemente inibida pelo Ag++ e Hg++. O Mn++ não apresentou nenhum efeito e o Ca++ e Mg++ realçaram a atividade das enzimas. O EDTA também inibiu fortemente a atividades das iso-formas da enzima . Entretanto, as atividades de todos as três lipases foram inibidas completamente por inibidores da serina protease como 3,4-dichloroisocoumarin, por pefabloc e parcialmente pelo fluoreto de phenylmethanesulphonyl. Segundo nossos conhecimentos, este é o primeiro estudo que descreve a purificação e a caracterização de iso-formas de lipases da C. rugosa em cultura sólida. Essas iso-formas de lipases com as características diversas produzidas em culturas sólida podem encontrar a aplicações em potencial no campo da biomedicina.

Three distinct forms (Lip A, Lip B and Lip C) of extra-cellular lipases (EC- 3.1.1.3), produced by Candida rugosa in solid state fermentation (SSF) were purified and characterised. SSF was carried out in glass columns using coconut oil cake and wheat bran. The enzyme was purified from the aqueous extract of fermented matter by ammonium sulphate precipitation, dialysis, ultra-filtration and gel filtration using Sephadex-200 to a 43-fold purification and 64.35-mg/ml specific activity. SDS-PAGE of purified enzyme revealed three distinct bands, indicating the existence of three iso-forms, Lip A, Lip B and Lip C with apparent molecular weight about 64,000, 62,000 and 60,000 Da, respectively. All the three iso-forms were optimally active at 35-40ºC and pH 7-8. They showed marked differences in their Km values with different saturated and unsaturated triacyl glycerols. Ag++ and Hg++ strongly inhibited enzyme activity of all the iso-forms, Mn++ has no effect and Ca++ and Mg++ enhanced the activity. EDTA also strongly inhibited the enzyme activities of iso-forms. However, activities of all the three lipases were completely inhibited by serine protease inhibitors such as 3,4-dichloroisocoumarin, pefabloc and partially by phenylmethanesulphonyl fluoride. To the best of our knowledge, this is the first report describing the purification and characterisation of C. rugosa lipase iso-forms from solid cultures. These lipase iso-forms with diverse characteristics produced in solid cultures may find potential application in biomedical field.