Immobilization of alkaliphilic Bacillus sp. cells for xylanase production using batch and continuous culture

Agar-immobilized alkaliphilic Bacillus sp. AR-009 cells were used for xylanase production using batch and continuous culture. In a batch culture, maximum enzyme production was observed after 48 h and remained high up to 72 h. In repeated batch cultivation, immobilized cells produced an appreciable level of xylanase activity in seven consecutive batches without any significant decline in productivity. For continuous xylanase production, immobilized cells were packed in a jacketed glass column and sterile medium was continuously pumped. A stable continuous production of xylanase was observed over a period of 1 mo. The volumetric productivity of the continuous culture was 17-fold higher than the batch culture using free cells.     

Continuous production of L-aspartic acid

For the continuous production ofl-aspartic acid from fumaric acid and ammonia by the action of aspartase, the enzyme extracted fromEscherichia coli orE. coli cells having high aspartase activity were immobilized by various methods. In 1973 we succeeded in the industrial production ofl-aspartic acid usingE. coli cells immobilized with polyacrylamide gel. For the improvement of this process, we developed a novel technique using κ-carrageenan as the immobilizing matrix forE. coli cells. Further, EAPc-7 strain, having higher aspartase activity, was contracted from the parentE. coli by continuous cultivation with a definite medium. The aspartase activity was about seven times higher than that of the parent cells. In 1982 we changed from the conventional method to the improved method, using EAPc-7 strain immobilized with κ-carrageenan.     

Immobilization of α-amylase from Bacillus circulans GRS 313 on coconut fiber

A simple and inexpensive method for immobilizing alpha-amylase from Bacillus circulans GRS 313 on coconut fiber was developed. The immobilization conditions for highest efficiency were optimized with respect to immobilization pH of 5.5, 30 degrees C, contact time of 4 h, and enzyme to support a ratio of 1:1 containing 0.12 mg/mL of protein. The catalytic properties of the immobilized enzyme were compared with that of the free enzyme. The activity of amylase adsorbed on coconut fiber was 38.7 U/g of fiber at its optimum pH of 5.7 and 48 degrees C, compared with the maximum activity of 40.2 U/mL of free enzyme at the optimum pH of 4.9 and 48 degrees C. The reutilization capacity of the immobilized enzyme was up to three cycles.     

Screening of a growing cell immobilization procedure for the biosynthesis of thermostable α-amylases

Studies were carried out on α-amylase production with immobilized cells of twoBacillus strains. High yields of thermostable αamylases were obtained byBacillus licheniformis 44MB82-G, resistant to glucose catabolite repression and a thermophileBacillus brevis 174, after repeated batch cultivation (270–600 h) of the immobilized biocatalysts. Various cell immobilization techniques were compared, including entrapment in gel matrices (Ca-alginate,x-carrageenan, agar, and their combinations with polyethylene oxide), adsorption on cut disks of polymerized polyethylene oxide, and fixation on formaldehyde activated acrylonitrile-acrylamide membranes. The optimal immobilization parameters (gel and biocatalyst concentration, initial cell quantity) were determined. Among the gels and supports tested, agar,x-carrageenan, agar/polyethylene oxide gels, and the membranes were found to be suitable for immobilization and biocatalysts with high operational stabilities were obtained. An enzyme yield of 2750 U/mL culture medium was reached in the fifth repeated batch run with membrane-immobilizedBacillus licheniformis cells. This activity represented 176% of the corresponding yield obtained in batch fermentation with free cells. Higher amylase yields than the activity of the control were reached in all experiments and repeated batch runs with immobilizedBacillus brevis cells.     

Improved Enzyme Stability in Lipase-Catalyzed Synthesis of Fatty Acid Ethyl Ester from Soybean Oil

In this work, we describe the optimization of the ethanolysis of soybean oil by the enzyme Lipozyme™ TL-IM in the lipase-catalyzed biodiesel synthesis and the improvement of the enzyme stability over repeated batches. The studied process variables were: reaction temperature, substrate molar ratio, enzyme content, and volume of added water. Fractional factorial design was used to analyze the variables so as to select those with higher influence on the reaction and then perform a central composite design to find the optimal reaction conditions. The optimal conditions found were: temperature, 26 °C; substrate molar ratio, 7.5:1 (ethanol/oil); enzyme content, 25% in relation to oil weight; and added water, 4% in relation to oil weight. Under these conditions, the yield conversion obtained was 69% in 12 h. The enzyme stability assessment in repeated batches was carried out by washing the immobilized enzyme with different solvents (n-hexane, water, ethanol, and propanol) after each batch. In the treatment with n-hexane, around 80% of the enzyme activity still remains after seven cycles of synthesis, suggesting its economical application on biodiesel production.     

Statistical Optimization of Alpha-Amylase Production with Immobilized Cells of <Emphasis Type="Italic">Streptomyces erumpens</Emphasis> MTCC 7317 in <Emphasis Type="Italic">Luffa cylindrica</Emphasis> L. Sponge Discs

The purpose of this investigation was to study the effect of Streptomyces erumpens cells immobilized in various matrices, i.e., agar–agar, polyacrylamide, and luffa (Luffa cylindrica L.) sponge for production of α-amylase. Luffa sponge was found to be 21% and 51% more effective in enzyme yield than agar–agar and polyacrylamide, respectively. Response surface methodology was used to evaluate the effect of three main variables, i.e., incubation period, pH, and temperature on enzyme production with immobilized luffa cells. The experimental results showed that the optimum incubation period, pH, and temperature were 36h, 6.0, and 50 °C, respectively. The repeated batch fermentation of immobilized cells in shake flasks showed that S. erumpens cells were more or less equally physiologically active on the support even after three cycles of fermentation (3,830–3,575 units). The application of S. erumpens crude enzyme in liquefying cassava starch was studied. The maximum hydrolysis of cassava starch (85%) was obtained with the application of 4ml (15,200 units) of crude enzyme after 5 h of incubation.     

Continuous production of butanol with immobilized cells ofClostridium acetobutylicum

Spores ofClostridium acetobutylicum were immobilized in calcium alginate. An active gel preparation was obtained after outgrowth of the spores to vegetative cells within the gel matrix. A 100 mL column containing the immobilized cells was used for continuous production. At steady-state conditions the productivity of butanol was 67 g/L reactor volume/day.     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.     

Mutagenesis and analysis of mold Aspergillus niger for extracellular glucose oxidase production using sugarcane molasses

Aspergillus niger ORS-4.410, a mutant of A. niger ORS-4, was generated by repeated ultraviolet (UV) irradiation. Analysis of the UV treatment dose on wild-type (WT) A. niger ORS-4, conidial survival, and frequency of mutation showed that the maximum frequency of positive mutants (25.5%) was obtained with a 57% conidial survival rate after the second stage of UV irradiation. The level of glucose oxidase (GOX) production from mutant A. niger ORS-4.410 thus obtained was 149% higher than that for WT strain A. niger ORS-4 under liquid culture conditions using hexacyanoferrate (HCF)-treated sugarcane molasses (TM) as a cheaper carbohydrate source. When subcultured monthly for 24 mo, the mutant strain had consistent levels of GOX production (2.62±0.51 U/mL). Mutant A. niger ORS-4.410 was markedly different from the parent strain morphologically and was found to grow abundantly on sugarcane molasses. The mutant strain showed 3.43-fold increases in GOX levels (2.62±0.51 U/mL) using HCF-TM compared with the crude form of cane molasses (0.762±0.158 U/mL). The results reported herein were obtained while the author was working at the Department of Biotechnology, Indian Institute of Technology, Roorkee-247667, India.     

Thermostable phytase production by Thermoascus aurantiacus in submerged fermentation

Phytases act on phytic acid, an antinutrient factor present in animal feeds, and release inorganic phosphate. We optimized the production parameters for phytase production using Thermoascus aurantiacus (TUB F 43), a thermophilic fungal culture, by submerged fermentation. A semisynthetic medium containing glucose, starch, peptone, and minerals supplemented with 3.75% (w/v) wheat bran particles was found to be the best production medium among the various combinations tried. Further supplementation of this medium with surfactants such as Tween-20 and Tween-80 considerably enhanced the enzyme yield. A maximum phytase activity (468.22 U/mL) was obtained using this production medium containing 2% (v/v) Tween-20 after 72 h of fermentation at 45°C in shake-flask cultures with a rotation of 150 rpm. Herein we present details of a few of the process parameter optimizations. The phytase enzyme was found to be thermostable, and the optimal temperature for phytase activity was found to be 55°C. However, 80% of the activity still remained when the temperature was shifted to 70°C.     

Statistical optimization of conditions for protease production fromBacillus sp. and its scale-up in a bioreactor

A statistical approach, response surface methodology (RSM), was used to study the production of extracellular protease fromBacillus sp., which has properties of immense industrial importance. The most influential parameters for protease production obtained through the method of testing the parameters one at a time were starch, soybean meal, CaCl₂, agitation rate, and inoculum density. This method resulted in the production of 2543 U/mL of protease in 48 h fromBacillus sp. Based on these results, face-centered central composite design falling under RSM was employed to further enhance protease activity. The interactive effect of the most influential parameters resulted in a 1.50-fold increase in protease production, yielding 3746 U/mL in 48 h. Analysis of variance showed the adequacy of the model and verification experiments confirmed its validity. On subsequent scale-up in a 30-L bioreactor using conditions optimized through RSM, 3978 U/mL of protease was produced in 18 h. This clearly indicated that the model remained valid even on a large scale. RSM is a quick process for optimization of a large number of variables and provides profound insight into the interactive effect of various parameters involved in protease production.     

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.     

High production of β-glucosidase from a marine Aspergillus niger immobilized on towel gourd vegetable sponges

To produce β-glucosidase by consecutive batch fermentation, a marine Aspergillus niger was immobilized on a natural carrier, towel gourd vegetable sponges. The immobilized mycelia were 0.15 g/g carrier with the immobilized biomass percentage of over 95%. The immobilized mycelia possessed the long durability(22.5 days). The maximum production occurred 1.5 day earlier by the immobilized mycelia than by the free mycelia. β-Glucosidase production of five consecutive batches was over 110 U/m L. At high salinity,the activity and stability of β-glucosidase from the marine A. niger increased remarkable. Immobilizing the marine A. niger on the novel natural carrier achieved the efficient production of β-glucosidase.     

Cloning, expression, and sequence analysis of diaminopropionate ammonia lyase gene from a nonvirulentsalmonella typhimurium PU011

Background: Seeds ofLathyrus sativus, a legume plant, contain 3-oxalyl and 2,3-dioxalyl DAP (O-DAP), neurotoxins which when consumed causes Neurolathyrism or Osteolathyrism, in humans, affecting nervous system and bone formation respectively. Some microorganisms viz virulent and non-virulentSalmonella typhimurium, Salmonella typhi and Pseudomonad have been shown to detoxifyL-α,β-diaminopropionate (DAP), the immediate precursor of O-DAP. Result: The gene coding for diaminopropionate ammonia lyase (DAPAL) which detoxifies DAP was cloned from nonvirulentS. typhimurium PU011 intoEscherichia coli DH5α and the nucleotides sequenced (1212 bp). Whereas the specific enzyme activity of DAPAL obtained from recombinantE. coli PU018 was 0.346 U/mg, the specific activity of the enzyme from nonvirulentS. typhimurium PU011 was 0.351 U/mg. The DAPAL corresponding to 43 kDa protein was found both in nonvirulentS. typhimurium PU011 andE. coli PU018. The Km value was found to be 0.740 mM and 0.680 mM forS. typhimurium PU011 and 0.741 mM and 0.683 mM forE. coli PU018 when grown in minimal medium (MM+DAP) andL. sativus seed extracts respectively, indicating that both of them were capable of utilizing the neurotoxins present inL. sativus seeds. The biomass, enzyme production and the effect of pH and temperature on DAPAL enzyme activity from both non-virulentS. typhimurium PU011 andE. coli PU018 were found to be similar. Conclusion: The recombinantE. coli PU018 as well as non-virulentS. typhimurium PU011 are as good as pathogenicS. typhimurium in detoxifying DAP, the immediate precursor of O-DAP present inL. sativus seeds.     

Immobilization of glucose isomerase and its application in continuous production of high fructose syrup

Bilayer glucose isomerase was immobilized in porousp-trimethylamine-polystyrene (TMPS) beads, through a molecular deposition technique. Some of the factors that influence the activity of immobilized glucose isomerase were optimized, with the enzyme concentration of 308 IU/mL, enzyme:matrix ratio of 924 IU/g wet carrier, and hexamethylenebis(trimethylammonium iodine) concentration of 15 mg/mL, giving the maximum catalytic activity (2238 IU/g dry gel) of the immobilized bilayer glucose isomerase, retaining 68.5% of the initially added activity. The half-life of the immobilized bilayer glucose isomerase was approx 45 d at pH 8.5, 60°C, with 50% (w/v) glucose as substrate. The specific productivity of the immobilized bilayer glucose isomerase was 223 g dry D-glucose/g dry immobilized enzyme per day.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.     

Immobilization of glucose isomerase and its application in continuous production of high fructose syrup

Bilayer glucose isomerase was immobilized in porousp-trimethylaminepolystyrene (TMPS) beads through a molecular deposition technique. Some of the factors that influence the activity of immobilized glucose isomerase were optimized, with the enzyme concentration of 308 IU/mL, enzyme-to-matrix ratio of 924 IU/g wet carrier, and hexamethylene bis(trimethylammonium iodine) concentration of 15 mg/mL giving the maximum catalytic activity (2238 IU/g dry gel) of the immobilized bilayer glucose isomerase, retaining 68.5% of the initially added activity. The half-life of the immobilized bilayer glucose isomerase was approx 45 d at pH 8.5, 60°C, with 50% (w/v) glucose as substrate. The specific productivity of the immobilized bilayer glucose isomerase was 223 g dry D-glucose/g dry immobilized enzyme per d.     

Cellulose filter paper immobilized α-glucosidase and its application to screening inhibitors from traditional Chinese medicine

In this study, α-glucosidase was successfully immobilized on cellulose filter paper and further applied to screening inhibitors from traditional Chinese medicines combined with capillary electrophoresis analysis. For α-glucosidase immobilization, a cellulose filter paper was used as the carrier and grafted with amino groups by coating chitosan, then α-glucosidase was covalently bonded on the amino-modified carrier via epoxy ring-opening reaction using polyethylene glycol diglycidyl ether as the crosslinker. Several parameters influencing the enzyme immobilization were optimized and the optimal values were enzyme concentration of 4 U/mL, polyethylene glycol diglycidyl ether concentration of 1.25%, chitosan concentration of 7.5 mg/mL, immobilization pH 7.0, crosslinking time of 4 h and immobilization time of 2 h. The immobilized α-glucosidase exhibited good batch-to-batch reproducibility (RSD = 2.1%, n = 5), excellent storage stability (73.5% of its initial activity after being stored at 4°C for 15 days), and reusability (75% of its initial activity after 10 repeated cycles). The Michaelis constant of immobilized α-glucosidase and half-maximal inhibitory concentration of acarbose were calculated to be 1.12 mM and 0.38 μM, respectively. Finally, the immobilized α-glucosidase was used for screening inhibitors from 14 kinds of Traditional Chinese Medicine extracts, and Sanguisorbae Radix showed the strongest inhibitory effect on α-glucosidase.     

Synthesis of Pure <Emphasis Type="Italic">meso</Emphasis>-2,3-Butanediol from Crude Glycerol Using an Engineered Metabolic Pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

meso-2,3-Butanediol (meso-2,3-BDO) is essential for the synthesis of various economically valuable biosynthetic products; however, the production of meso-2,3-BDO from expensive carbon sources is an obstacle for industrial applications. In this study, genes involved in the synthesis of 2,3-BDO in Klebsiella pneumoniae were identified and used to genetically modify Escherichia coli for meso-2,3-BDO production. Two 2,3-BDO biosynthesis genes—budA, encoding acetolactate, and meso-budC, encoding meso-SADH—from K. pneumoniae were cloned into the pUC18 plasmid and introduced into E. coli. In 2 l batch culture, the SGSB03 E. coli strain yielded meso-2,3-BDO at 0.31 g/g_glucose (with a maximum of 15.7 g/l_culture after 48 h) and 0.21 g/g_{crude glycerol} (with a maximum of 6.9 g/l_culture after 48 h). Batch cultures were grown under optimized conditions (aerobic, 6% carbon source, 37 °C, and initial pH 7). To find the optimal culture conditions for meso-2,3-BDO production, we evaluated the enzyme activity of meso-SADH and the whole cell conversion yield (meso-2,3-BDO/acetoin) of the E. coli SGSB02, which contains pSB02. meso-SADH showed high enzyme activity at 30–37 °C and pH 7 (30.5–41.5 U/mg of protein), and the conversion yield of SGSB02 E. coli was highest at 37–42 °C and a pH of 7 (0.25–0.28 g _meso-2,3-BDO/g_acetoin).