Activity Of Crude Enzyme Research Articles

Statistical Optimisation of Fermentation Media for Novel Clostridium novyi -NT Using Response Surface Methodology.

ABSTRACT Clostridial collagenase is recognized as one of the important proteolytic enzymes used in treatment of varieties of fibro-proliferative disorders. The present work aimed to optimise the biomass concentration and collagenase enzyme activity of novel bacilli Clostridium novyi-NT. The response surface methodology tool was used to identify the optimal fermentation parameters. Central composite design (CCD) was applied with respect to three influencing factors - pH, proteose peptone and trypticase soya broth. These factors showed significant effect on the overall biomass concentration and collagenase enzyme activity (p<0.05). The maximum biomass concentration in terms of absorbance and collagenase activity (of crude enzyme) was achieved and recorded as 0.8309±0.0012 and 298.88±1.36 units/mg respectively after 22 hours of fermentation period while optimisation of media factors with help of response surface quadratic model. This is the first study to report maximum biomass concentration and collagenase activity by Clostridium novyi-NT till date by combining statistical designs.

Metagenomics Investigation of Agarlytic Genes and Genomes in Mangrove Sediments in China: A Potential Repertory for Carbohydrate-Active Enzymes

Monosaccharides and oligosaccharides produced by agarose degradation exhibit potential in the fields of bioenergy, medicine, and cosmetics. Mangrove sediments (MGSs) provide a special environment to enrich enzymes for agarose degradation. However, representative investigations of the agarlytic genes in MGSs have been rarely reported. In this study, agarlytic genes in MGSs were researched in detail from the aspects of diversity, abundance, activity, and location through deep metagenomics sequencing. Functional genes in MGSs were usually incomplete but were shown as results, which could cause virtually high number of results in previous studies because multiple fragmented sequences could originate from the same genes. In our work, only complete and nonredundant (CNR) genes were analyzed to avoid virtually high amount of the results. The number of CNR agarlytic genes in our datasets was significantly higher than that in the datasets of previous studies. Twenty-one recombinant agarases with agarose-degrading activity were detected using heterologous expression based on numerous complete open-reading frames, which are rarely obtained in metagenomics sequencing of samples with complex microbial communities, such as MGSs. Aga2, which had the highest crude enzyme activity among the 21 recombinant agarases, was further purified and subjected to enzymatic characterization. With its high agarose-degrading activity, resistance to temperature changes and chemical agents, Aga2 could be a suitable option for industrial production. The agarase ratio with signal peptides to that without signal peptides in our MGS datasets was lower than that of other reported agarases. Six draft genomes, namely, Clusters 1–6, were recovered from the datasets. The taxonomic annotation of these genomes revealed that Clusters 1, 3, 5, and 6 were annotated as Desulfuromonas sp., Treponema sp., Ignavibacteriales spp., and Polyangiaceae spp., respectively. Meanwhile, Clusters 2 and 4 were potential new species. All these genomes were first reported and found to have abilities of degrading various important polysaccharides. The metabolic pathway of agarose in Cluster 4 was also speculated. Our results showed the capacity and activity of agarases in the MGS microbiome, and MGSs exert potential as a repertory for mining not only agarlytic genes but also almost all genes of the carbohydrate-active enzyme family.

Evaluation of maifanite and silage as amendments for green waste composting

Composting is a popular method for recycling organic solid wastes including agricultural and forestry residues. However, traditional composting method is time consuming, generates foul smells, and produces an immature product. The effects of maifanite (MF; at 0%, 8.5%, and 13.5%) and/or silage (SG; at 0%, 25%, and 45%) as amendments on an innovative, two-stage method for composting green waste (GW) were investigated. The combined addition of MF and SG greatly improved composting conditions, reduced composting time, and enhanced compost quality in terms of composting temperature, bulk density, water-holding capacity, void ratio, pH, cation exchange capacity, ammonia nitrogen content, dissolved organic carbon content, crude fibre degradation, microbial numbers, enzyme activities, nutrient contents, and phytotoxicity. The two-stage composting of GW with 8.5% MF and 45% SG generated the highest quality and the most mature compost product and did so in only 21 days. With the optimized composting, the degradation rate of cellulose and hemicellulose reached 46.3 and 82.3%, respectively, and the germination index of Chinese cabbage and lucerne was 153 and 172%, respectively, which were all far higher than values obtained with the control. The combined effects of MF and SG on GW composting have not been previously explored, and this study therefore provided new and practical information. The comprehensive analyses of compost properties during and at the end of the process provided insight into underlying mechanisms. The optimized two-stage composting method may be a viable and sustainable alternative for GW management in that it converts the waste into a useful product.

Prospecting the gut fluid of giant African land snail, Achatina fulica for cellulose degrading bacteria

The rapidly increasing human population poses a serious risk of fast depletion of natural resources like fossil fuels. Therefore, production of ecofriendly renewable alternatives like biofuels from waste materials has sparked more curiosity from the industrialists as well as academicians. Similarly, production of bioenergy from agricultural wastes is gaining interest worldwide. In view of the above objective, cellulose degrading bacterium G9-KDP isolated from the crop fluid of giant African land snail, Achatina fulica was identified as Bacillus tequilensis based on 16S rRNA gene sequencing. The B. tequilensis G9 produced cellulase with the highest activity of 956.9 IU/ml extract on 8th day of incubation when grown in BMS medium induced by sugarcane bagasse. The optimal temperature and pH of the organism were 60 °C and 6.6, respectively. It was observed that the activity of crude enzyme was affected to various extents by different metal ions. The activity was inhibited by Co2+ and Ca2+ at 5 mM concentrations but enhanced by K+ and Mn2+ ions. However, EDTA at 1% concentration increased the activity by 94% while SDS reduced it by 57% at the same concentration. Scanning electron microscopy of the filter paper used as substrate revealed significant structural modifications after the treatment with bacteria. The degradation of the various cellulosic compounds into simpler sugars by this isolate brands its potential applications in industrial biotechnology.

Cloning of Partial β-Mannanase Gene from Indonesia Marine Bacteria Bacillus Subtilis LBF-005

The strain LBF-005 from marine bacteria have already isolated and screened for mannanase degrading enzyme in submerged fermentation process. This strain was further identified by using 16S rRNA showed that bacterium is belong to Bacillus subtilis that could produce mannanase with activity around 9.5 U/mL. The optimum pH and temperature for the activity of crude enzyme for mannanase was 6.0 and 50 oC. Cloning of mannanase gene from B. subtilis was conducted using six primers set designed based on the homology analysis conserve region several mannanase from bacteria (Bacillus sp.) glycosyl hydrolase (GH) family 26. Optimization of PCR conditions was performed by gradient PCR to obtained PCR product of b-mannanase gene. The PCR product was obtained by third primer combination and was estimated to be around 972-bp. Analysis of the nucleotide sequence showed that sequences has similarity with mananase gene from other Bacillus sp., such as the B. subtilis strain WLY-12, B. subtilis strain WL-8, B. subtilis strain CICC 10260, B.subtilis strain CD-25, B.subtilis strain G1, and Bacillus sp. SWU60 were about 98%, 98%, 98%, 98%, 97% and 95%, respectively. The next research will to obtain a whole gene encoding β-mannanase by using in vitro cloning method, characterization of recombinant mannanase and application this enzyme for mannooligosaccharides production.

Effect of copper ion and soil humic acid on biodegradation of decabromodiphenyl ether (BDE-209) by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a good environmental microorganism capable of degrading decabromodiphenyl ether (BDE‐209). This paper studied the effect of Cu2+ and humic acid (HA) extracted from e‐waste contaminated soils on biodegradation of BDE‐209 by P. aeruginosa. The adsorption isotherms of Cu2+ on HA, the crude enzyme activity, cell surface morphology, and biodegradation pathway were also investigated. The results showed that BDE‐209 biodegradation by P. aeruginosa was inhibited at Cu2+ concentrations above 5 mg L−1, but exhibited the best effect at the condition of 40 mg L−1 Cu2+ + 3 g L−1 HA. At the condition of 40 mg L−1 Cu2+ + 3 g L−1 HA, 97.35 ± 2.33% of the initial BDE‐209 was degraded after 5 days, debromination efficiency was 72.14 ± 1.89%, crude enzyme activity reached the maximum of 0.519 ± 0.022U g−1 protein, cell surface of P. aeruginosa was smooth with normal short‐rod shapes, and biodegradation pathway mainly include debromination, hydroxylation, and cleavage of the diphenyl ether bond. It was suggested that soil HA could eliminate the toxic effect of high Cu2+ concentrations and biodegradation of BDE‐209 was improved by synergistic effect of HA and Cu2+.

Isolation and identification of Novel Lipase-producing Bacterium Aneurinibacillus aneurinilyticus LP-II

The aim of this research work was to isolate novel strain of lipase producer from local oil mill soil at Gangakhed. The lipase can be catalysing the transesterification of algae oil in to biodiesel. A sample of soil was collected from oil mill and enrichment the soil sample with basal lipase production media containing 1% Algae oil as a carbon source. Lipase producing microbes were screened on tributyrin agar plates, the activity of crude enzyme were determined. The isolate produced lipase and showed maximum activity 1.5 U/mL at 40°C and at 7.0 PH, after 48 hours of incubation period at 150 rpm. Isolate identified by morphological, biochemical and 16s r-RNA sequencing gene. The 16s r-RNA sequencing revealed it is a new strain of Aneurinibacillus aneurinilyticus LP-II.

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance.

Backgroundβ-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used.ResultsA newly identified β-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75 °C and pH 4.5), good thermostability (maintaining stable at 60 °C), and high catalytic performance (with a specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively). However, the narrow stability of Bgl3A at pH 4.0–5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0–10.0). Besides, with better stability under pH 5.0 and 50 °C compared with wild type Bgl3A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent saccharification performance to commercial β-glucosidase Novozyme 188 with identical β-glucosidase activity, suggesting its great prospect in biofuels production.ConclusionsIn this study, we overexpressed a novel β-glucosidase Bgl3A with high specific activity and high catalytic efficiency in P. pastoris. We further proved the negative effect of excessive O-glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing the O-glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0560-8) contains supplementary material, which is available to authorized users.

Optimization of the Medium for the Production of Extracellular Amylase by the Pseudomonas stutzeri ISL B5 Isolated from Municipal Solid Waste.

The management of municipal solid waste is one of the major problems of the present world. The use of microbial enzymes for sustainable management of the solid waste is the need of the time. In the present study, we have isolated a potent amylase producing strain (ISL B5) from municipal solid waste. The strain was identified as Pseudomonas stutzeri (P. stutzeri) both biochemically and by 16S rDNA sequencing. The optimization studies revealed that the strain ISL B5 exhibited maximum activity in the liquid media containing 2% starch (2.77 U/ml), 0.8% peptone (2.77 U/ml), and 0.001% Ca2+ ion (2.49 U/ml) under the pH 7.5 (2.59 U/ml), temperature 40°C (2.63 U/ml), and 25 h of incubation period (2.49 U/ml). The highest activity of crude enzyme has also been optimized at the pH 8 (2.49 U/ml).

The Study of Food-Grade Induced Expression and Enzymatic Properties of L-Arabinose Isomerase from &amp;lt;i&amp;gt;Lactobacillus plantarum WU&amp;lt;/i&amp;gt;14 with High D-Tagatose Yield

L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60&degC, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60&degC. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50&degC, pH 7.17 and D-galactose concentration was 0.6 mol/L.

Utilization of Rice Straw Waste in The Production of Crude Cellulase by Aspergillus Niger for Biodeingking Processes

Rice straw is agricultural waste that contains cellulose and has not been used optimally. The agricultural wastes contain cellulose which can be utilized for the production of cellulase enzymes. Cellulase produced from cellulose hydrolysis process in such materials through enzymatic hydrolysis. This study aimed to determine the effect of substrate type and temperature delignification on cellulase enzyme production and determine the effect of the number of additional enzymes of each substrate at biodeinking process. Variable used is the temperature used in the delignification process (100oC and 120oC), the addition of substrate (2,3,4,5,6%), additional doses of the application biodeinking (1,2,3,4,5%). Research conducted by softening rice straw to 30 mesh size media then delignification using 100 ° C and 120 ° C for 150 minutes, and fermented with Aspergillus niger in liquid inoculum, growth curves, and the crude cellulase production. The resulting crude cellulase was applied to the process biodeinking. Fieldwork results showed that at a temperature of 120oC delignification can reduce lignin content in straw was 48.7%. Highest crude cellulase activity of delignification results 120oC 4% resulting from the addition of media on the production of the enzyme activity of crude enzyme with 118.055 units / ml. Biodeinking application results showed that the addition of 3% crude cellulase with medium straw delignification results of 120oC can raise the value of paper brightness of 35.39% to 42.86% or 17:42%.

Optimization Studies on Cellulase Production from Bacillus Anthracis and Ochrobactrum Anthropic (YZ1) Isolated from Soil

The present study was carried out to demonstrate the optimization of growth conditions of bacteria with high cellulase activity. Cellulose degrading bacteria were isolated from soil samples collected from different areas of Guntur district, A.P. The bacteria were isolated using serial dilution and pour plate methods. The isolated bacteria were identified by morphological, biochemical and molecular procedures. The isolated bacterial species were screened for cellulase production in sub-merged fermentation process. The two tested bacterial species showed maximum yield for cellulase production. These two bacteria were identified as Bacillus anthracis and Ochrobactrum anthropi (YZ1). Supplementation of glucose, peptone, tyrosine and EDTA to the fermentation medium is favoured enzyme secretion. The optimum pH and temperature for the activity of crude enzyme was 8 and 45°C, respectively for Ochrobactrum anthropi (YZ1) while for Bacillus anthracis, it was 8 and 4°C, respectively.14% of inoculum level and 96 h of incubation period showed the maximum yield by both the species bacteria for cellulase production. The results of present study indicated that favorable fermentation conditions and the selection of a suitable growth medium played a key role in the production of cellulase from newly isolated Bacillus anthracis and Ochrobactrum anthropi (YZ1).Int J Appl Sci Biotechnol, Vol 3(2): 272-284 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12616

Digestive alkaline proteinases from Serranus scriba viscera: Characteristics, application in the extraction of carotenoproteins from shrimp waste, and evaluation in laundry commercial detergents

Abstract In this study, alkaline proteases from the viscera of the Serranus scriba were extracted and characterized. Interestingly, the crude alkaline protease showed optimum activity at pH 11.0. The relative activities at pH 10.0 and 12.0 were about 79% and 80.5% of that at pH 11.0, respectively. In addition, the crude protease was stable over a broad pH range (7.0–12.0) retaining more than 80% of its initial activity after incubation for 1 h at 25 °C. The optimum temperature for the crude enzyme activity was 50 °C. The crude alkaline protease extract showed high stability towards various surfactants and bleach agents. In addition, the crude enzyme showed high stability and compatibility with various solid and liquid commercial detergents. Further, proteases from S. scriba viscera were found to be effective in the extraction of carotenoproteins from shrimp waste. Extracted carotenoproteins were found to exhibit antioxidant activity evaluated through DPPH radical scavenging activity and inhibition of β-carotene bleaching assay. These results suggest that proteases from S. scriba viscera could be used as additive in laundry detergents and in the deproteinisation of shrimp wastes to extract carotenoproteins.

Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose.

Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)8 TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na(+) or K(+). The enzyme showed strong ability to hydrolyze cellobiose with a K m and V max of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K i of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Using electric field to enhance the activity of anammox bacteria.

Electric field was applied to enhance the activity of anaerobic ammonium oxidation (anammox) bacteria for nitrogen removal in this study. The effect of different distributive modes with the total electric field time of 6 h/24 h (R2) and 18 h/24 h (R3) at 2 V/cm was determined through long-term tests. With the continuous application time increasing from 3 to 9 h in R3 (1 to 3 h in R2), the total nitrogen removal rate (NRR) showed an acceleration trend. When the electric field was applied continuously for 9 h (R3, mode 3, application-rest time: 9 h-3 h), the NRR dramatically increased 38.7 % over that of control on day 160. Besides, it was demonstrated that the increase of crude enzyme activities and the cell quantities were the main reasons for the enhancement of nitrogen removal performance of the anammox process. Additionally, transmission electron microscope observation proved the morphological change of anammox biomass under electric field application.

Extraction, Purification and Characterization of Protease from Aspergillus Niger Isolated from Yam Peels

Protease was obtained from Aspergillus niger isolated from yam peels; a food waste, purified and characterized. Purification was achieved using ion exchange DEAE column and gel filtration (Sephadex G-200) chromatography. Effects of temperature; pH and production time on protease production were investigated. Also, physicochemical characteristics of the purified enzyme were investigated. The optimum production of protease was at temperature, pH and time of 37oC, 7.0 and 42 hrs respectively. The results showed that purified protease had more specific enzymatic activity than crude samples from Aspergillus Niger. whereas the specific activity of crude enzyme was 0.51 (U/mg), while the purified enzyme had an improved specific activity of 8.51 (U/mg).Optimum temperature and pH values of the purified protease were found to be 50°C and 10.0, respectively. pH stability of the enzyme ranged from 3.0- 12.0. At 3.0 and 10.0 it retained 70% and 60% of its activity after 5 hrs of incubation. Temperature stability ranged between 30oC and 90oC but most stable at 50oC retaining 94% of its activity after 1 h of incubation. The enzyme exhibited maximum activity on casein, among other protein substrates. EDTA, Cu2+, Fe2+, Mg 2+, and Ca2+ inhibited its activity while Na+ enhanced it. The enzyme was purified 16.60-fold, had a yield of 10.96 and the apparent molecular weight was 46.90 kDa. The study revealed that protease from A. niger can be exploited for protein conversion biotechnologies.

Enzymes extracted from apple peels have activity in reducing higher alcohols in Chinese liquors.

As the unavoidable byproducts of alcoholic fermentation, higher alcohols are unhealthy compounds widespread in alcoholic drinks. To investigate the activity of apple crude enzymes toward higher alcohols in liquors, five kinds of apple peels, namely, Fuji, Gala, Golden Delicious, Red Star, and Jonagold, were chosen to prepare enzymes, and three kinds of Chinese liquors, namely, Xifeng (containing 45% ethanol), Taibai (containing 50% ethanol), and Erguotou (containing 56% ethanol), were tested. Enzymes were prepared in the forms of liquid solution, powder, and immobilized enzymes using sodium alginate (SA) and chitosan. The treatment was carried out at 37 °C for 1 h. The relative amounts of different alcohols (including ethanol, 1-propanol, isobutanol, 1-butanol, isoamylol, and 1-hexanol) were measured using gas chromatography (GC). Conditions for preparing SA-immobilized Fuji enzymes (SA-IEP) were optimized, and the obtained SA-IEP (containing 0.3 g of enzyme) was continuously used to treat Xifeng liquor eight times, 20 mL per time. Significant degradation rates (DRs) of higher alcohols were observed at different degrees, and it also showed enzyme specificity according to the apple varieties and enzyme preparations. After five repeated treatments, the DRs of the optimized Fuji SA-IEP remained 70% for 1-hexanol and >15% for other higher alcohols.

Draft genomic DNA sequence of strain Halomonas sp. FS-N4 exhibiting high catalase activity

Halomonas sp. FS-N4 is a bacterium, which can grow in the medium Marine Broth 2216 with 5M initial hydrogen peroxide concentration, shows a strong oxidation resistance, and the crude enzyme activity can reach as high as 13.33katal/mg. We reported the draft genome sequence of H. sp. FS-N4, showing that it contains 3434 protein-coding genes, including the genes putatively involved in the response to the oxidative stress, among which a phytochrome-like gene might be a key point to survive in the environment with high concentration of hydrogen peroxide and exhibit high catalase activity.

Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.

Quantitative approach to track lipase producing Pseudomonas sp. S1 in nonsterilized solid state fermentation

Proliferation of the inoculated Pseudomonas sp. S1 is quantitatively evaluated using ERIC-PCR during the production of lipase in nonsterile solid state fermentation an approach to reduce the cost of enzyme production. Under nonsterile solid state fermentation with olive oil cake, Pseudomonas sp. S1 produced 57·9 IU g(-1) of lipase. DNA fingerprints of unknown bacterial isolates obtained on Bushnell Haas agar (BHA) + tributyrin exactly matched with that of Pseudomonas sp. S1. Using PCR-based enumeration, population of Pseudomonas sp. S1 was proliferated from 7·6 × 10(4) CFU g(-1) after 24 h to 4·6 × 10(8) CFU g(-1) after 96 h, which tallied with the maximum lipase activity as compared to control. Under submerged fermentation (SmF), Pseudomonas sp. S1 produced maximum lipase (49 IU ml(-1) ) using olive oil as substrate, while lipase production was 9·754 IU ml(-1) when Pseudomonas sp. S1 was grown on tributyrin. Optimum pH and temperature of the crude lipase was 7·0 and 50°C. Crude enzyme activity was 71·2% stable at 50°C for 360 min. Pseudomonas sp. S1 lipase was also stable in methanol showing 91·6% activity in the presence of 15% methanol, whereas 75·5 and 51·1% of activity were retained in the presence of 20 and 30% methanol, respectively. Thus, lipase produced by Pseudomonas sp. S1 is suitable for the production of biodiesel as well as treatment of oily waste water. This study presents the first report on the production of thermophilic organic solvent tolerant lipase using agro-industry waste in nonsterile solid state fermentation. Positive correlation between survival of Pseudomonas sp. S1 and lipase production under nonsterile solid state fermentation was established, which may emphasize the need to combine molecular tools and solid state fermentation in future studies. Our study brings new insights into the lipase production in cost-effective manner, which is an industrially relevant approach.