Extracellular alpha‐amylase from halophilic bacteria Marinobacter sp. LES TG5: Isolation, optimization, and characterization

Thermostable amylases are the most important enzymes in present with potential industrial applications. The main objective of this study was to isolate and characterize thermophilic amylases from Bacilli found in starch rich soil. Amylase producing bacilli were isolated and their enzymes were also characterized. Effect of temperature, pH, substrate and salt concentration on amylases activity were determined. All amylases produced by different isolates were hydrolyzed greater than 91% of starch after 60 h of fermentation. There was no significant (P ≥ 0.05) variation in enzyme productivity along with fermentation time. Amylase producing isolates were designated as Isolate-1, Isolate-2, Isolate-3, Isolate-4, Isolate-5 and Isolate-6. Amylases activities of all isolates were reached their optimum at 60°C. Amylases from all bacilli isolates were shown hydrolysis capacity of starch ranging from 91.4 to 95.7%. The optimum enzyme activity of amylase from Isolate-2 was extended from pH 7 to 8 with starch hydrolysis efficiency of 98% but other isolates enzyme activity reaches 99.5 to 100% at pH 8. The crude amylase extract has an activity with inversely proportional with substrate concentration. The bacterial dry weight increases as the course of incubation time increases and NaCl concentration greater than 5 molar significantly decreases the activity of the crude amylase extract. Amylases of this finding with thermophilic, alkalophilic and halophilic characteristics have wide range of huge potential for industrial applications. Besides, further purification of the crude extract could be conducted to meet thermophilic amylase enzyme requirements of pharmaceuticals and clinical sectors. Key words: Alkalophilic, amylase, bacillus, industrial application, thermostable.

Abstract. Chlorella sp. has the ability of mixotrophs, and the optimization of its culture medium has significant effect on growth. This paper investigated the effects of organic carbon source and nitrogen source on the accumulated biomass of Chlorella sp. 1067. In order to select the optimal organic carbon source, nitrogen source, and concentration combination; then concluded a regression equation of the effect on dry weight. It aimed at analyzing the mixotrophic growth of Chlorella 1067, and laying the foundation for further study of the relationship between autotrophic and heterotrophic. The results showed that glucose was the best organic carbon source to promote the accumulated biomass efficiently while carbamide was the optimal nitrogen source. In addition, the interaction of organic carbon source and nitrogen source has been studied. The experiment investigated the combined effect of glucose and carbamide on dry weight, and concluded the regression equation. The optimal solution was 50 mmol/L glucose and 35.4 mmol/L carbamide. In this case, the maximum theoretical value of dry weight could be up to 0.726 g/L.

Peptones are accepted as one of the most expensive medium components of microorganisms. The present study was undertaken to investigate the effect of chicken feather peptone (CFP) on enzyme (lipase and amylase) production by Bacillus licheniformis 016. In order to assess its effectiveness on enzyme production, CFP was compared with commercial fish peptone (FP) and protease peptone (PP). The optimum concentration of CFP for lipase and amylase production was determined as 5 and 6 g/L, respectively. The optimum concentration of both FP and PP was found as 4 g/L for lipase production and 5 g/L for amylase production. In all the peptone media, the optimal incubation times for amylase and lipase production were determined as 24 and 48 h, respectively. CFP was found to be more favorable for lipase and amylase production. In CFP, PP and FP media, the maximum lipase activities were 1870, 1582 and 1831 U/L, and the maximum amylase activities were 1680, 1505 and 632 U/L, respectively. On the other hand, better cell growth performance was achieved in CFP media compared to PP and FP media. The least pH change was detected in CFP-containing media. CFP was also found to prevent starch aggregation in the medium in contrast to FP and PP. This study exhibited that CFP was a better nitrogen source or an inducer for lipase and amylase production as well as cell growth in comparison to the tested commercial peptones.

A polyhydroxyalkanoate (PHA)-producing strain was isolated from propylene oxide (PO) saponification wastewater activated sludge and was identified as Brevundimonas vesicularis UJN1 through 16S rDNA sequencing and Biolog microbiological identification. Single-factor and response surface methodology experiments were used to optimize the culture medium and conditions. The optimal C/N ratio was 100/1.04, and the optimal carbon and nitrogen sources were sucrose (10g/L) and NH4Cl (0.104g/L) respectively. The optimal culture conditions consisted of initial pH of 6.7 and an incubation temperature of 33.4°C for 48h, with 15% inoculum and 100mL medium at an agitation rate of 180rpm. The PHA concentration reached 34.1% of the cell dry weight and increased three times compared with that before optimization. The only report of PHA-producing bacteria by Brevundimonas vesicularis showed that the conversion rate of PHAs using glucose as the optimal carbon source was 1.67%. In our research, the conversion rate of PHAs with sucrose as the optimal carbon source was 3.05%, and PHA production using sucrose as the carbon source was much cheaper than that using glucose as the carbon source.

Atrazine is a persistent organic pollutant in the environment which affects not only terrestrial and aquatic biota but also human health. Since its removal from the environment is needed, atrazine biodegradation is achieved in the present study using the bacterium Rhodococcus sp. BCH2 isolated from soil, long-term treated with atrazine. The bacterium was capable of degrading about 75 % atrazine in liquid medium having pH 7 under aerobic and dark condition within 7 days. The degradation ability of the bacterium at various temperatures (20-60 °C), pH (range 3-11), carbon (glucose, fructose, sucrose, starch, lactose, and maltose), and nitrogen (ammonium molybdate, sodium nitrate, potassium nitrate, and urea) sources were studied for triumph optimum atrazine degradation. The results indicate that atrazine degradation at higher concentrations (100 ppm) was pH and temperature dependent. However, glucose and potassium nitrate were optimum carbon and nitrogen source, respectively. Atrazine biodegradation analysis was carried out by using high-performance thin-layer chromatography (HPTLC), Fourier transform infrared spectroscopy (FTIR), and liquid chromatography quadrupole time-of-flight (LC/Q-TOF-MS) techniques. LC/Q-TOF-MS analysis revealed formation of various intermediate metabolites including hydroxyatrazine, N-isopropylammelide, deisopropylhydroxyatrazine, deethylatrazine, deisopropylatrazine, and deisopropyldeethylatrazine which was helpful to propose biochemical degradation pathway of atrazine. Furthermore, the toxicological studies of atrazine and its biodegraded metabolites were executed on earthworm Eisenia foetida as a model organism with respect to enzymatic (SOD and Catalase) antioxidant defense mechanism and lipid peroxidation studies. These results suggest innocuous degradation of atrazine by Rhodococcus sp. BCH2 in nontoxic form. Therefore the Rhodococcus sp.BCH2 could prove a valuable source for the eco-friendly biodegradation of atrazine pesticide.

This study was carried out to investigate the effects of various carbon and nitrogen sources on mycelial biomass production of Macrolepiota procera and Polyporus squamosus, important edible mushrooms in Turkey, in submerged cultures. Seven carbon (dextrose, glucose, lactose, maltose, mannitol, sucrose and xylose) and six nitrogen sources ((NH4)2HPO4, NH4NO3, Ca(NO3)2, malt extract, peptone and yeast extract) were used in the study. The Sabouroud (SB) and Dextrose Yeast Extract (DYE) liquid media without nitrogen and carbon were considered as the control. All carbon and nitrogen sources promoted significantly (p<0.01) mycelial biomass production in M. procera and P. squamosus. The optimum carbon and nitrogen sources for mycelial biomass production showed changes depending on mushroom species. As a result, the most suitable carbon source for mycelial biomass production in M. procera was dextrose and followed by glucose, mannitol, sucrose and lactose, while the medium with xylose was in P. squamosus. Peptone and malt extract as a nitrogen source in M. procera gave the best result for the biomass production, whereas yeast extract was the most favorable nitrogen source in P. squamosus. The lowest mycelial biomass production for the both mushroom species was determined in the control medium without carbon and nitrogen. In conclusion, the basic informations obtained from this study could be useful in the optimization of submerged culture conditions and nutritional requirements for mycelial biomass production in M. procera and P. squamosus.

Cellulose demonstrates unique properties and suitable for many different applications. In the present study an acetic acid bacterial strain has been isolated from rotten apple and investigated as cellulose producer. The strain was identified using morphological, biochemical characterization and 16s rRNA gene sequencing and named as Komagataeibacter hansenii (K. hansenii AS.5) under identity percentage 99%. Culture conditions for BC production by AS.5 were screened and compared with reference one Gluconacetobacter hansenii ATCC 23769 under static condition. Optimization of key production parameters has been carried out using OVAT (One Variable At Time) approach. Effect of media composition, inoculum size, pH, temperature, incubation time, different carbon and nitrogen sources were evaluated. The highest production of cellulose (3.75 g/l) was obtained after 10 days, 8% inoculum size, incubation 25◦C by K. hansenii AS.5 using Yamanaka medium with glucose and yeast extract as a sole carbon and nitrogen source, respectively. On contrast, G. hansenii ATCC 23769 exhibits the maximal BC production (2.18 g/l) under the modified GEM medium composed of mannitol and yeast extract as the optimum carbon and nitrogen source after 7 days at 25 ◦C and inoculum size 6%. It is clearly noticed the Cellulose production by the local isolate is higher than the reference one by 1.7- fold.

By investigating the effects of carbon source, nitrogen source, carbon-nitrogen ratio and inorganic salts on oil production of strains, the optimum carbon source, nitrogen source, carbon-nitrogen ratio, types and concentration of inorganic salts were determined. By investigating the effects of liquid loading, culture temperature and initial value of medium on oil production of strains, the optimum culture conditions were determined.

Bacillus subtilis is potential to play an important role on biological control of aflatoxin-producing fungi. The liquid fermentation medium of Bacillus subtilis LN for production of bioactive metabolites inhibitory to mycelial growth and aflatoxin biosynthesis was optimized by single factor experiments. The results showed that optimum carbon source was glucose, the optimum nitrogen source was soy peptone, and the optimum inorganic salt was MgSO4.These screened optimal carbon, nitrogen and inorganic salt sources could be used for high yield fermentation of bioactive metabolites for developing biopesticide against aflatoxin contamination in foods.

Aims: The effect of agitation speed and incubation time on Bacillus species growth and amylase production isolated from malted and fermented maize were investigated.&#x0D; Study Design: An experimental study.&#x0D; Methodology: Bacillus species were screened for amylase production using starch hydrolysis method on starch agar. Bacillus sp.IBM21 was observed to exhibit highest hydrolytic activity with zone of clearance of diameter 41.6mm. Process parameters optimizations were evaluated using submerge fermentation techniques.&#x0D; Results: The effect of agitation speed on growth and amylase production was varied from 100-250 rpm. It was observed that, the amylolytic Bacillus species were able to grow optimally at agitation speed of 100 rpm while their highest amylase activities were recorded at speed of 150 rpm for Bacillus sp.IBM22, 10.16Um-1 and 200 rpm for Bacillus sp.IBM21, 5.59Um-1. The effect of incubation time was varied from 12-72h, amylase activities as well as bacterial growth increases with increase in incubation time until peaks were attained at 36h (10.16Uml-1, OD540nm, 2.98) for Bacillus sp.IBM22 and 48h ( 5.5Uml-1, OD540nm 1.55) for Bacillus sp.IBM21.&#x0D; Conclusion: The results of this study indicate that Bacillus species isolated from malted/fermented maize produced amylase maximally at optimal agitation speed and incubation time ranges of 150-200rpm and 36-48h respectively.

Background: Various chemicals and dyes used in paper production are xenobiotic in nature and pose significant environmental concerns. Objective: In this study, a fungal strain was isolated from contaminated paper mill effluent and identified as Aspergillus niger through standard microbiological and morphological analyses. Methodology: The strain was screened for ?-amylase production using banana peel—a low-cost agro-residue—as the sole carbon source. Optimization of physicochemical parameters, including incubation temperature (30 °C), initial pH (6.0), and incubation time (72 h), yielded maximal enzyme activity. Crude ?-amylase activity was quantified spectrophotometrically by measuring reducing sugars released from soluble starch. Partial purification was achieved via ammonium sulfate precipitation, with 50% saturation providing the highest specific activity and yield. Application: The applicability of the partially purified ?-amylase was demonstrated by de-inking waste office paper; treatment with the enzyme significantly improved paper brightness and reduced ink load, as confirmed by standard gravimetric and spectrophotometric assays. Conclusion: These findings indicate that banana peel is a feasible and cost-efficient substrate for ?-amylase production by A. niger. The ?-amylase has promising potential for sustainable paper waste management and bioremediation in the pulp and paper industry.

The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing.

At present, there are production processes to produce protein by Escherichia coli (E. coli) fermentation. Research on the design and optimization of the plasmid fermentation medium, however, is less advanced. The fermentation medium that is optimized for plasmid DNA production is different from the medium that is optimized for protein production. So, establishing a scientific and rational method to optimize the fermentation medium used for plasmid production is very important. Previously, our laboratory developed a novel therapeutic DNA vaccine (named pSVK-HBVA) for hepatitis B based on the alphavirus replicon, and found that E. coli XL10-Gold was the optimal host strain for the production of plasmid pSVK-HBVA. The aim of this study was to establish a scientific and rational method to optimize the fermentation medium used for plasmid production, and investigate the effect of growth medium composition on the production of plasmid pSVK-HBVA harboured in E. coli XL10-Gold, as well as to optimize the medium composition.The one-factor-at-a-time experiments demonstrated that Luria-Bertani (LB) was the optimal basic medium. The optimal carbon source and nitrogen source were glycerol and home-made proteose peptone, respectively. Based on the Plackett–Burman (PB) design, proteose peptone, glycerol and NH4Cl were identified as the significant variables, which were further optimized by the steepest ascent (descent) method and central composite design. Growth medium optimization in 500-mL shake flasks by response surface methodology resulted in a maximum volumetric yield of 13.61 mg/L, which was approximately 2.5 times higher than that obtained from the basic medium (LB).

The application of amylase is relatively large, used in various industries: textiles, pulp and paper, feed, detergents, and food. Therefore, the isolation and optimization of amylase production from various microorganisms has been ongoing. Optimization of amylase production from Aspergillus sp. LBKURCC304 was carried out in liquid media with variable sources of nitrogen (soy flour, tempeh flour or catfish flour) and minerals (FeSO47H2O; CaCl2 2H2O; MgSO4 7H2O; MnSO4 H2O, BaCl2 2H2O) incubated at 50⁰C for 11 days with an agitation speed of 150 rpm. The amylase activity produced was determined by the Nelson-Semogyi method, the protein content by the Lowry method and the specific activity was calculated from the ratio of the amylase activity to the protein content. The research data were statistically tested using ANOVA and the multiple-distance Duncan method at a significance level of 5%. The results of the study showed that the highest amylase activity of 0.0084±0.0014 U/mL was produced in a medium with a nitrogen source of tempeh, the mineral was MgSO4.7H2O, a concentration of 0.05%. The protein content was 0.5111±0.0073 mg/ml and the specific activity was 0.0164±0.00 U/mg.

Glucose oxidase (GOx) has several industrial applications. It is believed that there are several species of fungi that have the ability to produce this enzyme, most of which are unexplored. This work aimed to investigate the production of glucose oxidase (EC 1.1.3.4) by fungi isolated from soil samples of the Amazonian forest. Filamentous fungi were isolated from soil samples from the Adolpho Ducke Forest Reserve, located in Manaus, Amazonas. Strains were subjected to submerged bioprocessing to select for the best GOx producers. Those selected for the production of isolated enzymes were subjected to kinetic tests that evaluated production of the enzyme and consumption of the biomass substrate by the isolates. In addition, experiments to evaluate the optimal carbon, nitrogen and phosphorus sources as well as the influence of the bioprocess factors were carried out. Finally, GOx production was investigated in a semi-continuous system for 7 days. The most frequent isolates isolated from soil samples belonged to the genera Aspergillus, Penicillium and Trichoderma. Aspergillus niger LMM01 was the best GOx producer. Glucose, peptone and KH2PO4 were demonstrated to be the optimal carbon, nitrogen and phosphorus sources, respectively. Multivariate experiments demonstrated that the parameters with the greatest effect on GOx production were pH and agitation. Stable expression results for GOx (7.74 U/ml) were obtained over 7 days in a semi-continuous process. In this context, the new Amazonian source of this enzyme (A. niger LMM01), and enzyme production in a semi-continuous process, demonstrates the importance of the present work. Key words: Amazon, fungi, production, glucose oxidase.