DEAE-cellulose Ion-exchange Chromatography Research Articles

Production optimization, purification and characterization of a heat-tolerant acidic pectinase from Bacillus sp. ZJ1407

Medium compositions for a heat-tolerant acidic pectinase production from Bacillus sp. ZJ1407 were optimized via response surface methodology (RSM) and its enzymatic properties were investigated. A 2-level factorial design was used to estimate the main effect of factors, and to screen the significant factors. A central composite design was used to find out the optimal concentrations of screened key factors. Lactose, tryptone and (NH4)2SO4 were found to have a significant influence on the pectinase activity (p <0.05). The optimal medium compositions were as follows: lactose 44.8g/l, tyrptone 30.9g/l, (NH4)2SO4 1.35g/l, MnSO4·H2O 0.2g/l, MgSO4 0.4g/l and NaCl 3.5g/l. Pectinase was purified to homogeneity by ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephadex G-100 size-exclusion chromatography. The molecular weight of the purified pectinase determined by SDS-PAGE was about 23kDa, and its final specific activity was 110.47U/mg. Its optimal temperature and pH were 37°C and 5.0, respectively. Pectinase was very stable within a pH range of 3.0–5.0, and showed a high thermo-stability at 80 and 90°C. Ba2+ could significantly promote the activity of pectinase, and Mn2+ heavily inhibited its activity. This study provides new insight into the future development and use of pectinase from Bacillus sp. ZJ1407.

Purification, physicochemical and thermodynamic studies of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated Aspergillus griseoaurantiacus KX010988

In our search for chitinase and chitosanase producer from unconventional sources, the marine-derived fungus Aspergillus griseoaurantiacus KX010988 was obviously the best producer of the highest chitinase and chitosanase activities by solid state fermentation of potato shells. Chitinase was purified in three steps involving ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephacryl S-300 gel chromatography. 12.55 fold increase in purity with a recovery of 17.6 was obtained. The molecular mass of the purified chitinase was found to be 130kDa. It was optimally active at pH 4.5 and 40°C. Km and Vmax values were 0.22mgmL−1 and 19.6μmolemin−1mg−1 respectively. Mn2+ and Zn2+ ions lead to increased chitinase activity. While Fe2+and Cu2+ions strongly inhibited the chitinase activity. The thermodynamics of pure chitinase including activation energy for thermal denaturation (Ea,d), change of free energy (ΔGd), enthalpy(ΔHd), entropy(ΔSd) and half life values (T1/2) at 40, 50 and 60°C were determined. Chitinase showed antifungal activity against pathogenic fungus Fusarium solani. Chitosanase was partially purified by acetone precipitation (50–75%) v/v concentration. The hydrolytic products of moderate molecular weight of chitosan by chitosanase were analyzed by thin layer chromatography (TLC) after 12 and 24h respectively. Chitosan-oligosaccharides showed good antibacterial and antioxidant activities.

Biodiesel production from lipid of carbon dioxide sequestrating bacterium and lipase of psychrotolerant Pseudomonas sp. ISTPL3 immobilized on biochar

An extracellular lipase was purified and characterized from psychrotolerant bacterium Pseudomonas sp. ISTPL3 isolated from Pangong lake. Lipase was purified by sequential methods of ammonium sulphate precipitation, dialysis, DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration chromatography, resulting in a purification fold of 6.53 and yield of 5.45%. The molecular weight was approximately 31kDa. The purified lipase was used for transesterification of lipids produced by oleaginous chemolithotrophic bacterium Serratia sp. ISTD04 for production of biodiesel. Upon biochemical characterization, lipase was found to be alkalophilc, thermostable, active in organic polar solvents and sensitive to detergents. Further, lipase was immobilized on activated biochar to assess its transesterification efficiency during biodiesel production. Immobilized lipase gave the highest yield of fatty acid methyl esters (FAMEs) (92.23%)>unimmobilized lipase>NaOH. The immobilized lipase was assessed for its reusability and retained 75.11% of its activity after 3 cycles of biodiesel production.

ISOLATION, PURIFICATION, AND OPTIMIZATION OF THERMOPHILIC AND ALKALIPHILC PROTEASE ORIGINATING FROM HOT WATER SPRING BACTERIA

Objective: The main objective of this study is to investigate the industrial applications of a thermophillic alkaline protease from a hot water spring bacterial isolate “A” and to study its production, optimization, and purification.Methods: The alkaline protease was produced using shake flask studies maintaining a pH of 9.0 and a temperature of 50°C. Optimization studies of the enzyme were carried out using variable pH, temperature, organic carbon, and nitrogen sources followed by purification of the enzyme using DEAE-cellulose ion exchange chromatography technique. Stability of the enzyme was analyzed in the presence of organic solvents and surfactants. The efficiency of the enzyme in the removal of proteinaceous stains in the presence of strong detergents under extreme conditions was assessed. The fibrinolytic activity of the enzyme in dissolving the blood clot was confirmed.Results: The isolated alkaline protease was purified to homogeneity with a 16-fold increase. Media optimization studies revealed that 1% glucose and 1 % casein-induced the production of alkaline protease. The purified enzyme retained stability in the presence of ethanol, methanol, and acetone and surfactants such as 0.5% (w/v) sodium dodecyl sulfate (SDS) and 0.5% (v/v) Triton-X-100. The isolated alkaline protease successfully removed the proteinaceous stains and showed significant results in the dissolution of blood clot.Conclusion: The above experimental results confirm that the isolated enzyme has both thermophilic and alkaliphilic protease properties. Thereby the enzyme finds promising industrial applications even in extreme conditions.

PROTEASE INHIBITOR FROM WHITE CRANBERRY BEANS (PHASEOLUS VULGARIS): ISOLATION, PURIFICATION AND CHARACTERIZATION

Objective: The main objective of the study was isolation, purification and characterization of protein protease inhibitor from the seeds of Phaseolus vulgaris and analysis of its antimicrobial potential.Methods: The protease inhibitor was extracted by homogenizing seeds of Phaseolus vulgaris in 0.1 M phosphate buffer (pH-7.0). The crude extract of the inhibitor was purified by using ammonium sulphate precipitation followed by DEAE Cellulose ion exchange chromatography. The protease inhibitor was characterized to determine its optimum pH and pH stability, optimum temperature and temperature stability, stability in the presence of chemical modifiers, thermal stabilizers, metal ions, detergents, oxidising and reducing agents. The antimicrobial potential of the inhibitor against various bacterial species was confirmed using agar well diffusion method.Results: The extracted protease inhibitor was purified to homogeneity with a 1.4 fold increase in the specific activity and 56 % purification yield. Inhibitor was optimally active at pH 7.0 and a temperature of 50 °C as well as showed considerable stability over pH ranging from 4.0-11.0 and up to a temperature of 70 °C for 4 h duration. The inhibitor was substantially active and stable in the presence of surfactants 1 % (v/v) Tween 20, 4 % (v/v) of oxidizing agents such as dimethyl sulphoxide (DMSO) and hydrogen peroxide (H2O2), 0.8 % (v/v) of reducing agents β-mercaptoethanol and Sodium thioglycolate. Metal ions Mg++, Ca++and Zn++enhanced the activity of inhibitor while CaCl2, glycine and glycerol promoted thermal stability of the inhibitor. Chemical modification of amino acids at the active site by diethyl pyrocarbonate (DEPC) and phenyl methyl sulphonyl fluoride (PMSF) led to decrease in the inhibitory activity. The stoichiometry of trysin-protease inhibitor interaction was 1:2 while 216 μg of the inhibitor effected 50 % inhibition. The inhibitor displayed antimicrobial activity against Aeromonas hydrophilla, Citrobacter freundii and Acinetobacter baumanii.Conclusion: The experimental results confirmed the anti proteolytic action of protease inhibitor extracted from P. vulgaris as well as its promising antimicrobial properties. Thus isolated protease inhibitor has significant industrial and therapeutic potential.

Bioefficiency of Indigenous Microbial Rhodanese in Clean-up of Cyanide Contaminated Stream in Modakeke, Ile-Ife, Osun State, Nigeria

Cyanide pollution of aquatic environment has become a great concern in Nigeria because of the increase in cassava cultivation. In Nigeria, cassava processing milling plants are usually situated around streams or rivers such that the waste from each stages of processing easily find their way into these water bodies as effluents and waste waters. Extracellular rhodanese of Klebsiella edwardsii isolated from Atutulala stream, Modakeke, where cassava is being processed, was assessed for its bioremediation potential. Cyanide concentration of the stream was analysed for six months. Four bacterial isolates were screened for their ability to degrade free cyanide and the best strain was further screened for rhodanese producing ability. The enzyme was purified by 85% ammonium sulphate precipitation and diethyl aminoethyl-cellulose ion-exchange chromatography. The pure enzyme had a specific activity of 0.0473 Rhodanese Unit mg-1 with a purification fold of 4.56 and a percentage yield of 30.30%. The enzyme demonstrated a broad pH range but the optimum pH was at 6.0 while the optimum temperature was 60°C. The bioremediation potential of the enzyme was assessed under various conditions such as the field pH and temperature as well as optimum pH and temperature using the cyanide contaminated water as substrate source in a typical assay protocol. The enzyme was able to convert 1.6481 μmol of cyanide to thiocyanate in the water sample at optimum pH and temperature of the enzyme. It could be concluded from the study that at optimum pH and temperature, rhodanese exhibited remediation activity in cyanide contaminated aquatic ecosystems and thus, can be used for its restoration.

Purification and Characterization of New Alkaline L-methioninase from Aspergillus ustus AUMC 1051 Grown under Solid-State Fermentation Conditions.

bran) under solid state fermentation (SSF) of wheat bran. The enzyme was purified 15.83-fold with 62.63% yield after three steps of purification involved ammonium sulfate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. The purified enzyme had a molecular mass of 46 kDa under denaturating conditions and an isoelectric point of 6. Maximal activity was recorded at pH 8.5 and 35

Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839.

Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24 h of incubation with 2.5 % oil seed waste concentration. O. xanthineolytica was found to produce two proteases—P1 and P2. The proteases were purified using 60 % cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular weight of P1 and P2 was 36 and 24 kDa, respectively. P1 and P2 were optimally active at pH 7.0 and pH 7.5 at temperature 35 and 40 °C, respectively. Analysis of hydrolyzed product of P1 and P2 by HPLC reveals that the P1 has endoprotease and P2 has exoprotease activity. The treated soy milk with immobilized proteases showed increased shelf life and removal of off flavor.

Production and Characterization of Chitinases from Thermophilic Bacteria Isolated from Prataan Hot Spring, East Java

Thermophilic bacteria producing chitinase were collected from Prataan hot spring, East Java, Indonesia and screened. The isolated bacterium was analyzed using 16S rRNA gene sequencing analysis and identified as Paenibacillus sp. The molecular identification was confirmed through morphological and physiological analyses. The production of chitinase was conducted at various incubation times, temperatures, pH and concentrations of colloidal chitin. The optimum condition of the isolate to produce the highest chitinase was 0.9% (w/v) of colloidal chitin (pH 7.0) at 48 °C for 24 hours. The obtained chitinases were optimally active at 55 °C and pH 6.0-7.0. The chitinases were gradually purified by ammonium sulfate precipitation, Sephadex G-100 gel filtration, followed by DEAE"“cellulose ion exchange chromatography (IEC). The purification method gave a purification factor of 9.43 and a yield of 2.68%. Two protein fractions were obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 68 and 82 kDa.

A thermostable-endo-β-(1,4)-mannanase from Pediococcus acidilactici (M17): purification, characterization and its application in fruit juice clarification

A Turkish fermented sausage was bought from a local market in Erzurum and was used for isolation and characterization of lactic acid bacteria. As the result of 16S rRNA analysis, this bacterium was determined as Pediococcus acidilactici (Genbank number KF895384) at a rate of 99 %. The mannanase from P. acidilactici (M17) was purified using the (NH4)2SO4 precipitation, DEAE-cellulose ion exchange and Sephacryl S200 gel filtration chromatography techniques. The yield and purification fold of mannanase were 10.6 % and 106.1, respectively. The molecular mass of the enzyme (38 kDa) was determined by SDS-PAGE and gel filtration chromatography. In order to identify, the resistance of the purified mannanase to Mn2+, Co2+, Cu2+, Zn2+ and Ca2+ was examined, and all the metal ions activated the enzyme at a significant level. The V max and K m values for locust bean gum were calculated as 78.74 mmol/min mg and 0.592 mM, by Lineweaver–Burk. Purified mannanase was also used to clarify some fruit juices. The studied variables were, the incubation time (from 0 to 60 min), the enzyme concentration (17 EU/L), and the temperature (60 °C). It was observed that the purified mannanase had maximum yield on orange juice with 161.1 %. Also maximal clarification was obtained as 84.65 %, and the clarification for grape juice and sugar reduction was obtained as 19.26 mg/mL for apple juice. Consequently, it was concluded that the P. acidilactici (M17) mannanase could be successfully used in the fruit juice industry.

Human Salivary Aldehyde Dehydrogenase: Purification, Kinetic Characterization and Effect of Ethanol, Hydrogen Peroxide and Sodium Dodecyl Sulfate on the Activity of the Enzyme.

Human salivary aldehyde dehydrogenase (hsALDH) enzyme appears to be the first line of defense in the body against exogenous toxic aldehydes. However till date much work has not been done on this important member of the ALDH family. In this study, we have purified hsALDH to homogeneity by diethylaminoethyl-cellulose (DEAE-cellulose) ion-exchange chromatography in a single step. The molecular mass of the homodimeric enzyme was determined to be approximately 108kDa. Four aromatic substrates; benzaldehyde, cinnamaldehyde, 2-naphthaldehyde and 6-methoxy-2-naphthaldehyde were used for determining the activity of pure hsALDH. K m values for these substrates were calculated to be 147.7, 5.31, 0.71 and 3.31μM, respectively. The best substrates were found to be cinnamaldehyde and 2-naphthaldehyde since they exhibited high V max /K m values. 6-methoxy-2-naphthaldehyde substrate was used for further kinetic characterization of pure hsALDH. The pH and temperature optima of hsALDH were measured to be pH 8 and 45°C, respectively. The pure enzyme is highly unstable at high temperatures. Ethanol, hydrogen peroxide and SDS activate hsALDH, therefore it is safe and beneficial to include them in mouthwashes and toothpastes in low concentrations.

Mitigation of acrylamide by l-asparaginase from Bacillus subtilis KDPS1 and analysis of degradation products by HPLC and HPTLC.

The use of bacterial l-asparaginase (LA) is one of the alternative approaches for acrylamide reduction in food stuffs as it catalyzes the conversion of l-asparagine to l-aspartic acid and ammonia. In present investigation, purification of extracellular LA from isolate of Bacillus subtilis sp. strain KDPS-1 was carried out by solid state fermentation process. The effects of solid substrates, initial moisture content, moistening agents, temperature, and incubation time on LA production was studied, and the highest asparaginase activity (47 IU/ml) was achieved in the medium having orange peel as substrate. The enzyme was purified to homogeneity by diethylaminoethyl (DEAE) cellulose ion exchange chromatography; with 84.89 % yield and 12.11 fold purity. LA showed stimulant activity against β-mercaptoethanol and was greatly inhibited by Zn2+ and Hg2+ metal ions. Reduction of acrylamide in fried potatoes was detected by high performance liquid chromatography, which showed clear degradation of acrylamide by height and area (%) in the chromatograms of standard sample to that of the test sample. Hydrolysates analysis by high performance thin layer chromatography confirmed the test sample to be LA.

Extraction and Purification of L-Asparaginase Produced by Acinetobacter Baumannii and their Antibiofilm Activity Against Some Pathogenic Bacteria

L-asparaginase is an enzyme catalyzing the hydrolysis of L-asparagine and formation of L-aspartate and ammonia and widely used as anticancer drug in pharmaceutical and food industry. This amino acid (L-asparagine) has an important role for development of cancerous; in contrast the normal cells don’t need this amino acid. Eight Acinetobacter baumannii isolates of were isolated from different blood and sputum samples and it was found that high isolation rate of Acinetobacter baumannii isolates from sputum was consisted with their association with lower respiratory tract infections. All these eight isolates were screened for higher L-asparaginase production and found that among all these isolates, Acinetobacter baumannii Sp3 gave higher asparaginase activity of 7.32 U/ml. L-asparaginase was purified to homogeneity by sequential chromatographic steps involved ammonium sulfate at 45% saturation followed by DEAE-cellulose ion exchange chromatography and sephadex G-100 gel filtration chromatography with a recovery yield of 68% and 22.65 fold of purification. L-asparaginase had antibiofilm activity against all tested biofilm forming pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Acinetobacter baumannii) after using Congo red agar and Microtitration plates methods for detecting biofilm formation ability. Highly antibiofilm of L-asparaginase recorded against Klebsiella pneumoniae followed by Pseudomonas aeruginosa with reduction of biofilm formation ratio to 32 and 41% ,respectively compared with (100)% of control. Thus we can conclude that L-asparaginase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria that have multidrug resistance.

Purifi cation and Characterization of Protease From Bacillus sp. TBRSN- 1

Potato Cyst Nematode (PCN), Globodera rostochiensis, is one of the important potato’s pests and causedeconomic looses up to 70% in the several centrals of potato plantations in Indonesia. PCN’s shell componentof egg shell containing chitin (inner layer) and viteline/ protein (outer layer). The purpose of this researchwas to purify of protease Bacillus sp. TBRSN-1, isolate from tomato’s rhizosfer in Yogyakarta province. Thepurifi ed protease could be used for cutting the life cycle of PCN. Results showed that Bacillus sp. TBRSN-1could produce extracellular protease and purifi cation using DEAE-cellulose ion-exchange chromatographyand Sephacryl S-300 gel fi ltration chromatography resulted in specifi c activity 4.31 fold and 1.68% recovery.Analysing using SDS-PAGE 12.5% and molecular weight 48.1 kDa. Km and Vmax values of the protease forcasein substrate were 7.83 mg/ml and 4.03 μg/h, respectively. The optimum activity at the temperature30oC and pH 7.0. Keywords : protease, purifi cation, indigenous Bacillus sp. TBRSN-1

Significance of Penicillium ochrochloron chitinase as a biocontrol agent against pest Helicoverpa armigera

Penicillium ochrochloron chitinase purified by DEAE-cellulose ion exchange chromatography was evaluated for its antifeedant and growth inhibitory activities against Helicoverpa armigera at different concentrations of 2000, 1000, 500, 250 and 100UmL−1.It reduced the successful pupation and increased larval and pupal mortality, adult emergence in a dosage-dependent manner when applied topically. The highest mortalities were recorded for groups treated with 2000UmL−1 chitinase activity. The studies showed P.ochrochloron chitinase can affect the growth of H.armigera larvae. Since this insect pest species has developed resistance and resurgence to chemical insecticides, only alternate is the usage of enzyme-based pesticide formulations as an environmentally friendly pest management tool.

Isolation and purification of trypsin inhibitors from the seeds of Abelmoschus moschatus L.

Four trypsin inhibitors, AMTI-I, AMTI-II, AMTI-III, and AMTI-IV, have been isolated and purified to homogeneity from the seeds of Abelmoschus moschatus following ammonium sulphate fractionation, DEAE-cellulose ion exchange chromatography and gel permeation on Sephadex G-100, and their molecular weights were determined to be 22.4, 21.2, 20.8 and 20.2 kDa respectively by SDS-PAGE. While all the four inhibitors were very active against bovine trypsin, two of them (AMTI-III and AMTI-IV) showed moderate activity towards bovine chymotrypsin. AMTI-I and AMTI-II were found to be glycoproteins with neutral sugar content of 2.8 and 4 %, respectively, and all the four inhibitors were devoid of free sulphhydryl groups. The inhibitors were quite stable up to 80 °C for 10 min and were not affected at alkaline as well as acidic conditions tested. Treating them with 8 M urea and 1 % SDS for 24 h at room temperature did not result in any loss of their antitryptic activities. However, they lost considerable antitryptic activity when treated with 6 M guanidine hydrochloride. Activities of the inhibitors were unaffected even after their reduction with DTT suggesting that disulphide bonds are not needed for their inhibitory activities.

Purification and characterization of polygalacturonase from ripened fruits of Musa acuminata cultivar from Kerala (Musa acuminata cv. Palayankodan)

Bananas are the fruit crop with strong economic and nutritional relevance. This fruits have a very short shelf life due to the softening of the pulp during ripening. Fruit softening is an important aspect of ripening process in fleshy fruits and is caused by the cumulative action of a group of cell wall modifying enzymes. The present study reports characterization of polygalacturonase (PG) isoforms from ripened fruits of Musa acuminata cv. Palayankodan. Three isoforms of PG was purified by ammonium sulphate fractionation followed by DEAE cellulose ion exchange chromatography and gel filtration using Sephadex G 100. Three isoforms showed two subunits on SDS-PAGE and a single band on native PAGE. Enzyme showed maximum activity at pH 4 (PG1, PG2) and 3.5 (PG3). Fe3+ activated PG1 and PG2, while Ca2+ reduced the activity of all isoforms. Km value for substrate polygalacturonic acid was 0.013, 0.015 and 0.011 % for PG1, PG2 and PG3 respectively.

Preparation of polyclonal antibody against porcine beta defensin 2 and identification of its distribution in tissues of pig.

Porcine β-defensin 2 (pBD2) is an antimicrobial peptide in pigs that plays an important role in the immune system by preventing bacterial invasion. To produce an anti-pBD2 antibody, which is not commercially available, we expressed and purified a soluble, his-tagged version of pBD2 (his-pBD2). Purified pBD2 was injected into New Zealand white rabbits to generate polyclonal antiserum. Anti-pBD2 antibodies were purified by ammonium sulfate precipitation, followed by diethylaminoethyl cellulose ion-exchange chromatography. The purified polyclonal antibody showed high sensitivity, with a titer as high as 204,800 by enzyme-linked immunosorbent assay, and it also showed high specificity for both his-pBD2 and native pBD2, as assessed by western blotting. Furthermore, immunohistochemistry analysis using the purified antibody revealed that pBD2 protein is distributed in the tongue, liver, kidney, small intestine, and large intestine of pigs. These results indicate that the prepared polyclonal antibody will be a useful tool for further studies of the function and mechanism of pBD2.

Screening and characterization of alkaline protease produced by a pink pigmented facultative methylotrophic (PPFM) strain, MSF 46

Abstract Among the various bacterial isolates, the strain MSF 46 isolated from thorn forest soil samples, Tamil Nadu, India, was screened and characterized for its proteolytic activity. While the 16S rRNA sequencing and biochemical characterization revealed that the strain closely resembles Methylobacterium sp., methylotrophy of the strain was confirmed by the sequence homology of mxaF gene with other relative Methylobacterium sp. The alkaline protease was purified to homogeneity using DEAE cellulose ion exchange chromatography, with a 5.2-fold increase in specific activity and 34% recovery. The apparent molecular weight of the enzyme was determined as 40 kDa by SDS–PAGE study. The pH and temperature optima were 9.0 and 50 °C respectively with maximum protease activity of 1164 U/ml. Protease of MSF 46 was active in a broad pH range 7.0–11.0 with a maximum at pH 8.5 and exhibited thermostability at 50 °C. The enzyme activity was inhibited by PMSF but showed stability with Tween 20, Triton X-100 and hydrogen peroxide. Nearly 30% reduction in enzyme activity was observed in the presence of EDTA and DTT. The enzyme was effective in hydrolyzing gelatin, skimmed milk and blood clots and exhibited the potency for dehairing of goat skin and removing blood stain from cotton fabric. Significant morphological changes were observed under scanning electron microscope between cells grown in normal and casein amended medium. This first detailed report on the production of alkaline protease by a PPFM strain appears promising toward development of protocols for mass production, study of the molecular mechanism and other applications.

Detection of tilapia metallothionein using antibody-immobilized quartz crystal microbalance sensor

Metallothionein (MT) was induced in tilapia by three subcutaneous injections of CdCl2 and the recovered MT protein was purified to homogeneity through DEAE-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. For use as biological component, a polyclonal anti-tilapia MT antibody was prepared using the purified tilapia MT. During the preparation of the tilapia MT sensor, topological changes pertinent to immobilization of the thiolated antibody, subsequent blocking of the surface with BSA and final immune reaction were observed by AFM imaging. Specificity of the sensor was confirmed through comparison of the responses of defective and intact sensor chips, and the responses of the sensor chips that were immobilized with the antibody and BSA. Concentration-dependent responses of the sensor showed a linear relationship of Y (log10 sensor response)=0.3438X (log10 tilapia MT)+0.7368 (r=0.9606) in double-logarithmic plot with a limit of detection of 20ng/mL. When the serum from a tilapia induced with CdCl2 was compared with the control serum, induction of MT was detectable even at 20,000-fold dilution of tilapia serum.