A Novel Method of Magnetic Nanoparticles Functionalized with Anti-Folate Receptor Antibody and Methotrexate for Antibody Mediated Targeted Drug Delivery.

Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections to analyze the binding efficacy and intracellular tracking of synthesized nanomedicines. MNP-MTX-FR Ab demonstrated good cytotoxicity along all the nanocomposites, which confirms that the antibody-coated medicine possesses the potential affinity to destroy cancer cells in the targeted drug delivery process. Immunohistochemical approaches and fluorescent study further confirmed their uptake by FRs on the tumor cells' surface in antibody-mediated endocytosis. The current approach is a useful addition to targeted drug delivery for better management of cancer therapy along with immunotherapy in the future.

A novel method of affinity purification and characterization of polygalacturonase of Aspergillus flavus by galacturonic acid engineered magnetic nanoparticle.

Pectinases hydrolyze pectin and make up 25% of global food processing enzyme sales. In this study, we aimed to purify exo-polygalacturonase (Exo-PG) by using galacturonic acid conjugated magnetic nanoparticles (MNPs) and examined its application in juice purification. The submerged fermentation was carried out in the presence of apple pectin (1%) to promote production of exo-PG from Aspergillus flavus. Maximum exo-PG activity was observed after 4 days (30 °C and pH 5.0). A single protein band (66 kDa) of purified exo-PG was observed in SDS-PAGE. Purification of exo-PG enzyme was âˆ¼ 10 fold with a yield of 29%. The enzyme retained 98% activity in the presence of 15 % glycerol at 4 °C. The purified exo-PG using MNPs yielded a 10-12% increase in juice production as compare to without treated fruit juice. To the best of our knowledge, this is the first report of affinity purification of exo-PG enzyme, using engineered magnetic nanoparticles.

Probing Transferrin Receptor Overexpression in Gastric Cancer Mice Models.

Exposure to carcinogenic chemicals, Helicobacter pylori infection, and high dietary salt are the risk factors associated with gastric cancer. Mice models of gastric cancer are key to understanding the cancer mechanism, to discerning the role played by different factors, and to determining therapeutic effects of different treatments. The goal has been to find targets which are only expressed with cancer so that they can be targeted specifically without harming normal cells. One such target could be the transferrin receptor, a glycoprotein receptor that is expressed many-folds on rapidly growing cells due to the greater demand of iron. In this study, gastric cancer was developed in mice (BALB/c) with human cancer-associated risk factors by feeding them with tumor-inducing concentration of methyl nitrosourea, dietary salt, and H. pylori along with normal feed and water. Three strategies were adopted to induce gastric cancer; (1) use of N-methyl-N-nitrosourea (MNU) with high dietary salt (NaCl), (2) infection with H. pylori (isolated from human gastric tissue), and (3) use of MNU along with high concentration of NaCl after H. pylori infection. Mice were dissected after induction, and histological study of gastric tissue was done with Hematoxylin and Eosin staining. A diagnostic probe comprising transferrin conjugated with cadmium sulfide quantum dots was prepared and characterized. It was used to study the transferrin receptor overexpression in gastric tissue of cancer-induced mice relative to the normal mice. Mice of group 3 showed the highest rate of the cancer incidence ratio (96%) along with a high expression of transferrin receptors among the three groups. Histochemical studies showed that different types of gastric cancer depend upon the cancer-induction conditions. The mouse model of group 3 has the potential to be used in the future to study the therapeutic effects of cancer medicines, and overexpression of transferrin receptors could be identified through the designed probe to be used as diagnostics.

Purification of transferrin by magnetic nanoparticles and conjugation with cysteine capped gold nanoparticles for targeting diagnostic probes.

Transferrin is an iron binding glycoprotein actively involved in the growth and maintenance of cell cycle. The transferrin receptors expression is increased on growing cancer/tumor cells for absorption of iron through transferrin and participation in biological activity. In this study, a novel method for the purification of transferrin by using magnetic nanoparticles (MNP) is developed and compared with reported method. Magnetic nanoparticles were synthesized by co-precipitation method under hydrothermal conditions in the presence of ammonium hydroxide. MNP were characterized by FTIR, VSM, DLS, TEM, and SEM. Purified transferrin was characterized by SDS-PAGE, MALDI-TOF, ELISA, Western blot, and its activity was further confirmed by iron binding assay and receptor binding assays. Purified transferrin was also conjugated with cysteine capped gold nanoparticles (GNP) and characterized by UV-Vis spectra, TEM, DLS, and fluorescent spectrophotometry. Transferrin conjugated cysteine capped GNP used as a targeted fluorescent probe on gastric cancer, tumor tissue and MDA-MB 231 cancer cells to confirm transferrin receptor binding activity and application as diagnostic probe. The purified transferrin showed stability and activity up to 36 months. The results indicated that the synthesized superamagnetic MNP are good for the purification of transferrin. A good yield of transferrin was purified by this method, good quality and showed active biological activity. GNP conjugated transferrin has a potential to be used in cancer diagnosis as targeted diagnosis probe in vivo and in vitro. Experiments are underway for utilizing transferrin as carrier for targeting drug delivery.

Stable Silver Nanoparticles Synthesis by Citrus Sinensis (Orange) and Assessing Activity Against Food Poisoning Microbes / 生物医学与环境科学（英文）

Silver nanoparticles are considered as good antimicrobial agent. AgNPs were synthesized by mixing silver nitrate solution with citrus sinesis extract for 2 h at 37 °C and analyzed by UV-visible spectra, SEM, XRD, and FTIR. AgNPs were tested against B. subtilis, Shigella, S. aureus, and E. coli. Minimum inhibitory concentration of AgNPs was 20 µg/mL for B. subtilis and Shigella and 30 µg/mL for S. aureus and E. coli. Antibiofilm activity (80% to 90%) was observed at 25 µg/mL. AgNPs were stable for five months with sustained antimicrobial activity. Biosynthesized AgNPs can be used to inhibit food poisoning microbial growth.

Anticancer medicines (Doxorubicin and methotrexate) conjugated with magnetic nanoparticles for targeting drug delivery through iron.

The uptake of iron is increased by cancer cells. Iron magnetic nanoparticles (MNP) can be used as a nanovehicle for immobilization of anticancer medicines and to integrate them at a target site. The anticancer medicines doxorubicin (DOX) and methotrexate (MTX) were immobilized separately and in combination onto MNP by a glutaraldehyde activation method and confirmed by magnetic nanoparticles linked immunosorbent assay (MagLISA) and Fourier-transform infrared (FTIR) spectroscopy. The phenol peaks of DOX and MTX at 2896.6 cm⁻¹ to 2912.5 cm⁻¹ in FTIR spectra of immobilized medicines indicated the conjugation. Affinity-purified anti-DOX and anti-MTX antibodies were used to evaluate the coupling of DOX and MTX onto MNP, and the binding was found 34.6% to 37.2% and 51.8% to 54.3% separately, respectively. The immobilization of DOX and MTX in combination onto MNP was 18% and 27%, respectively. HeLa and B cells were cultured with DOX-MNP, MTX-MNP, and DOX-MNP-MTX separately, and MagLISA indicated that the binding of DOX-MNP/MTX-MNP was 41.5% to 45% with HeLa cells and 20% to 26% with B cells. No significant difference was observed in binding of DOX-MNP-MTX with HeLa and B cells. Results also indicated that the release of medicines at pH 5.0 is more (39% to 44%) than at pH 7.4 (3.7% to 10.2%). Sixteen to 22% more killing effect was observed on HeLa cells than on B cells. In immunohistochemical staining, more deposition of brown color on HeLa cells than on B cells may be due to more expression of iron-binding sites on cancer cells. The dual property of MNP can be used for binding of medicines and for targeting drug delivery.

Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b.

Interferon alpha 2b (IFNα-2b) is an important cytokine and used for antiviral and anticancer treatment. The low cost production of IFNα-2b with high biological activity is necessary to provide the interferon therapy to the hepatitis patients in Pakistan. In the present study, human interferon alpha 2b (hIFNα-2b) gene from a healthy person was cloned and overexpressed in E. coli BL21(DE3). The molecular weight of the expressed hIFNα-2b is 19 kDa. The over expressed recombinant hIFNα-2b was checked by ELISA using antibodies raised against commercially available hIFNα-2b. The biocomputational analysis of recombinant hIFNα-2b gene showed the 99.9% nucleotide sequence and 100% deduced amino acid sequence homology with reported sequences of IFNα-2b. The predicted 3D-structure showed mainly five α-helices, one 310 helix and two disulfide bonds at Cys1-Cys98 and Cys129-Cys138. The amino acid sequence alignment indicated that the disulfide linkage position is conserved in all IFNα family members. On the basis of sequence homology among interferon alpha family, new potent variants of hIFNα-2b with enhance efficacy can be produced. Indigenous production of IFNα-2b from gene of local population will reduce the cost and increase tolerability of interferon therapy.

Molecular epidemiology of hepatitis C virus genotypes in different geographical regions of Punjab Province in Pakistan and a phylogenetic analysis.

BACKGROUND: Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. In Pakistan, more than 10 million people are living with HCV. Very little is known about the genotype distribution in Punjab Province, the largest province of Pakistan. Pretreatment genotype identification is very important, as different HCV genotypes respond differently to interferon therapy. METHODS: In this study we performed HCV genotyping for 1537 HCV-infected patients from different districts of Punjab Province, Pakistan. Sequencing of partial HCV NS5B sequences from 14 samples belonging to genotypes 3 and 1 was also done. A sequence comparison was made of our reported sequences with those reported in the National Center for Biotechnology Information (NCBI), and a phylogenetic tree was constructed. RESULTS: Our study showed that the most frequent HCV genotype was 3a (in 88.1% of infected individuals), followed by 1a (3.5%), 3b (3.0%), 1b (0.8%), and 2a (1.0%). A mixed genotype infection was found in 3.6% of infected individuals, with 0.3% living with 1a + 1b co-infection, 3.1% with 3a+3b, and 0.2% suffering from 3a+1b co-infection. The sequence comparison showed that HCV NS5B motif B residues G283, T287, and N291 were highly conserved in both genotype 1 and genotype 3 sequences, while the motif B residue T286 was mutated to proline in all the genotype 3 sequences. The GDD motif, which forms the catalytic pocket and binding site for the divalent cations, was highly conserved in all the reported sequences. The phylogenetic tree suggests clustering of genotype 1 sequences with sequences from the USA, UK, Germany, and France, while genotype 3 sequences are clustered with sequences from Japan and the UK. CONCLUSIONS: The major prevalent genotype in Punjab Province of Pakistan was genotype 3a, followed by genotype 1a, and only 3.6% of infected individuals had a mixed genotype infection. Sequencing of the HCV NS5B gene suggested that the active site residues were highly conserved in all the reported sequences. Our sequences, which are clustered with sequences from the USA, UK, France, and Japan, show the diversity in origin of the different genotypes prevalent in Pakistan.

Identification and purification of antigenic 34 kDa outer membrane protein of Salmonella typhi.

BACKGROUND: Salmonella Typhi is a pathogenic bacterium that causes a number of infectious diseases such as gastroenteritis and typhoid fever. In this study, an antigenic (34 kDa) protein was identified, purified, and characterized from outer membrane of Salmonella typhi. METHODS: Immunoblot analysis was used to screen antigenic proteins from outer membrane of Salmonella Typhi. Proteins from outer membrane were isolated and resolved on SDS-PAGE. In immunoblot analysis, four proteins with the following molecular weights of 60 kDa, 54 kDa, 34 kDa, and 26 kDa were identified as highly antigenic against the serum of patients suffering from typhoid fever. One of these outer membrane proteins, with a molecular mass of 34 kDa, was selected for this study. The 34 kDa protein was purified and characterized by a combination of anion exchange chromatography and gel permeation chromatography. The molecular weight of 34 kDa was determined using SDS-PAGE electrophoresis. The antigenic nature of the purified 34 kDa protein was determined by ELISA against serum proteins of patients suffering from typhoid fever and finally confirmed by immunoblot analysis. Antisera against the purified 34 kDa outer membrane protein of Salmonella typhi was produced and was used to recognize the epitope on the surface of an intact Salmonella typhi bacterium. RESULTS: The antigenic 34 kDa protein from the outer membrane of Salmonella typhi was identified, purified and characterized. The antigenecity of purified protein was confirmed by using antibodies present in serum of patient suffering from typhoid fever. It was also observed that antibody against 34 kDa outer membrane protein recognizes intact Salmonella typhi cells. CONCLUSIONS: It is established from the study that 34 kDa protein is antigenic in nature and antibody against this protein can also recognize epitopes on intact Salmonella typhi cells. Furthermore, this protein can be a good source material to produce vaccine against typhoid fever.

Inhibitory effect of Allium sativum and Zingiber officinale extracts on clinically important drug resistant pathogenic bacteria.

BACKGROUND: Herbs and spices are very important and useful as therapeutic agent against many pathological infections. Increasing multidrug resistance of pathogens forces to find alternative compounds for treatment of infectious diseases. METHODS: In the present study the antimicrobial potency of garlic and ginger has been investigated against eight local clinical bacterial isolates. Three types of extracts of each garlic and ginger including aqueous extract, methanol extract and ethanol extract had been assayed separately against drug resistant Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Shigella sonnei, Staphylococcusepidermidis and Salmonella typhi. The antibacterial activity was determined by disc diffusion method. RESULTS: All tested bacterial strains were most susceptible to the garlic aqueous extract and showed poor susceptibility to the ginger aqueous extract. The (minimum inhibitory concentration) MIC of different bacterial species varied from 0.05 mg/ml to 1.0 mg/ml. CONCLUSION: In the light of several socioeconomic factors of Pakistan mainly poverty and poor hygienic condition, present study encourages the use of spices as alternative or supplementary medicine to reduce the burden of high cost, side effects and progressively increasing drug resistance of pathogens.

Biocatalytic activity of recombinant human ß-mannosidase immobilized onto magnetic nanoparticles for bioprocess.

Recombinant human ß-mannosidase (rhMANB) is an important glycosidase enzyme that degrades mannose-linked glycoproteins and mannan polysaccharides. rhMANB was purified and covalently immobilized onto magnetic nanoparticles. The immobilization of the enzyme was confirmed by Fourier-transform infrared spectroscopy (FTIR) and magnetic nanoparticles linked immunosorbent assay (MagLISA). Antibodies against rhMANB were raised, purified and characterized for MagLISA. The binding of rhMANB onto magnetic nanoparticles was found to be 65%. The V( max ) and K( m ) of immobilized rhMANB was observed 3.0-fold higher and 2.024-fold lower, respectively, as compared to unbound rhMANB. The stability and activity of immobilized enzyme was observed at different pH, temperature, and after storage at 4°C. Metal chelators (oxalic acid, citric acid, and ascorbic acid) did not affect the enzyme activity of immobilized enzyme, whereas ethylenediamine tetraacetic acid reduced the activity. The results obtained from thin-layer chromatography indicate that immobilized rhMANB is more efficient than the unbound form to hydrolyze mannobiose, mannotriose, mannotetraose, mannopentose, galactoglucomannan, and locust bean gum. Magnetic nanoparticles suspended gel-permeation chromatography showed that 29% locust bean gum hydrolyzed efficiently during flow in the column. The immobilization of rhMANB will be a good process for gelling and saccharification of mannan polymers at industrial scale.

Determination of oxygen derived free radicals producer (xanthine oxidase) and scavenger (paraoxonase1) enzymes and lipid parameters in different cancer patients.

BACKGROUND: Cancer is a major cause of death throughout the world. Xanthine oxidase (XO) is considered an oxygen derived free radicals producer and paraoxonasel (PON1) is known as a free radicals scavenger. This study was undertaken to determine the activity of PON1 and XO and their concentration along with the lipid profile, lipid peroxide, and thiol level in cancer patients and healthy persons. METHODS: Antigen competitive ELISA was used to determine the level of paraoxonasel and xanthine oxidase in plasma. Paraoxonase and arylesterase activity of the PON1 enzyme and the activity of the XO enzyme were also determined. PON1 and XO activity, cholesterol, LDL-C, HDL-C, lipid peroxides, and thiol level in patients with breast cancer (n = 50), prostate cancer (n = 20), lung cancer (n = 12,), cervix cancer (n = 15), Non-Hodgkin lymphoma (n = 10), and acute lymphoblastic lymphoma (n = 8) and total age matched healthy persons (n = 115) were studied. RESULTS: Low plasma paraoxonase/arylesterase activities (p < 0.05) and the concentration of the PON1 enzyme were observed in all cancer patients. An elevated level of XO activity was observed in cancer patients. Among different cancers, comparatively high level of XO activity was noted in acute lymphoblastic lymphoma patients (0.18 +/- 0.03, p = 0.001) except cervix cancer patients (0.053 +/- 0.03, p = 0.0029) where a low level was observed. Low HDL-C (p < 0.01), cholesterol (p < 0.01), triglycerides (p < 0.01), thiol levels (p < 0.01) and high lipid peroxide levels (p > 0.05) were observed in cancer patients. CONCLUSIONS: Lower PON1 and high XO enzyme activities cause an imbalance of the free radical system which enhances the lipid peroxidation and other pathological conditions. Lower HDL-C level is also indicative of the low level of PON1 enzyme activity.

PCR assay targeting virulence genes of Helicobacter pylori isolated from drinking water and clinical samples in Lahore metropolitan, Pakistan.

Helicobacter pylorus is considered for chronic gastritis, gastric ulcers and adenocarcinoma and its high infection rate is observed in overcrowded and lower socioeconomic groups in developing countries. This study was designed to identify the role of drinking water in the transmission and prevalence of H. pylori (HP). Selective HP medium was developed for enrichment and presumptive identification of H. pylori by urease, catalase and species specific 16S rRNA tests. The virulence genes (vacA 's' and 'm' regions and cagA) of H. pylori in 90 out of 225 H. pylori positive drinking water samples were present (40%). Ten out of 18 biopsies (55.55%) and 15 out of 50 vomiting fluids of gastric disease patients (30%) were also positive for virulence genes. Anti-H. pylori antibodies were also detected in 31 out of 50 patients' sera. The presence of virulence genes was also directly confirmed by hybridization studies using non-radioactive DNA probes of 16S rRNA, vacA and cagA genes. The presence of H. pylori in water is due to poor sanitary conditions, improper waste disposal and lack of public health education. PCR-based analysis and colony hybridization can be used for detection of H. pylori in clinical and environmental samples.

Immunobiochemical analysis of Paraoxonase1 (anti-oxidant), xanthine oxidase (oxidant) enzymes and lipid profile of cardiac disease patients in Lahore Metropolitan, Pakistan.

Cardiac diseases are the major cause of death. Paraoxonase1 (PON1) is known as free radicals scavenger/anti-atherosclerosis, whereas xanthine oxidase (XO) is a free radicals generator. This study was undertaken to determine and compare the Paraoxonase and arylesterase activities of PON1 enzyme and activity of XO enzyme. The concentration of XO and PON1 enzymes along with lipid profile, lipid peroxides, and thiol level in plasma of cardiac patients (n=200) and healthy persons (n=200) of Lahore metropolitan, Pakistan was also determined. Anti-PON1 and anti-XO antibodies were developed, purified, and used to measure the concentration of PON1 and XO by competitive ELISA. It is observed that low paraoxonase (P=0.0073)/arylesterase activity (P=0.0038) of PON1 enzyme and its low concentration (P=0.0049) were observed in cardiac patients, whereas elevated level of XO activity (P=0.0129) and its concentration (P=0.0097) was observed in cardiac patients as compared with healthy persons. Low levels of HDL (P=0.0013), thiol (P=0.0014) and high level of cholesterol (P=0.0025), triglycerides (P=0.0018), LPO (P=0.0014), and LDL level (P=0.05) were observed in cardiac patients admitted in intensive care unit as compared with hypertensive patients and control subjects. It is concluded that overall low PON1 and high XO activities do cause imbalance of free radical system which ultimately leads to or enhance the cardiac pathological conditions.

Synthesis of cholesterol-conjugated magnetic nanoparticles for purification of human paraoxonase 1.

Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe(3)O(4) nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm(-1), and at 2,334.9 cm(-1) confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg(-1) protein, and the purification fold was 515 with 73% yield. The K (m) values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg(+2), Mn(+2), glycerol, (NH(4))(2)SO(4), PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb(+2), Co(+2), Zn(2+), ethanol, beta-mercaptoethanol, and acetone reduced the activity while Ni(2+), Cd(2+), Cu(2+), iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml(-1), respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.

PCR targeting of antibiotic resistant bacteria in public drinking water of Lahore metropolitan, Pakistan.

OBJECTIVE: To investigate the prevalence of kanamycin (kan) and ampicillin (amp) resistant bacteria in public drinking water. METHODS: Bacteria containing kan and amp resistant genes were amplified by PCR and further characterized by colony hybridization and transformation studies. The genus of kan and amp resistant bacteria was determined with standard methods. RESULTS: Among the 625 drinking water samples, 400 contained kan and amp resistant bacteria and the percentage was 42.5% and 57.5%, respectively, which was further confirmed by the amplification of a 810 bp kan resistant gene and a 850 bp amp resistant gene. Of the 170 kan resistant bacteria, 90 were Gram negative and 80 were Gram positive. Of the 230 amp resistant bacteria, 160 were Gram negative while 70 were Gram positive. Salmonella, Shigella, Staphylococcus, Streptococcus, and E.coli were detected as 13%, 11%, 17%, 30%, and 29%, respectively. Bacterial strain DH5alpha transformed with plasmids isolated from kan and amp resistant bacteria confirmed that the antibiotic resistant genes were mediated by plasmids. CONCLUSION: Drinking water is contaminated with kan and amp resistant bacteria due to poor sanitary conditions.

Cloning, sequence, expression and characterization of human beta-mannosidase.

Beta-mannosidase (EC 3.2.1.25, MANB) dissects the non-reducing end of N-linked mannose moieties of glycoproteins in eukaryotic cells. The human beta-mannosidase gene was amplified by RT-PCR, cloned and sequenced. The DNA sequence was compared with reported human beta-mannosidase DNA sequence and sixteen nucleotide differences were found. The deduced amino-acid sequence showed that seven codons coded the same amino acids and nine codons coded different amino acids with reference to nucleotide substitution positions but did not affect recombinant MANB enzyme activity. No splice mutation was observed after comparison with reported MANB DNA sequences. A 75% homology of deduced amino-acid sequence was observed with mouse, goat and bovine beta-mannosidase amino-acid sequences. The cloned beta-mannosidase gene was subcloned into pET22b+ and pET28a+ expression vectors to transform the BL21-codon plus cells for expression of recombinant MAN22 and MAN28 enzymes, respectively. The optimized conditions for overexpression of recombinant beta-mannosidase enzyme were induction with 1 mM IPTG for 12 h at 37 degrees C. The expressed beta-mannosidase enzyme was purified to homogeneity by a combination of DEAE-ion exchange and size exclusion chromatography. The molecular mass of MAN22 and MAN28 enzymes is 97 kDa by SDS/PAGE and is confirmed by western blot analysis. The recombinant enzymes are active at 37 degrees C and at pH 5.0 and showed activity with p-nitrophenyl-beta-d-mannopyranoside and not with p-nitrophenyl-alpha-d-mannopyranoside. The K(m) value of enzymes was 2.53 mM. The enzyme activity was inhibited by Zn(2+), Co(2+), Cu(2+), Pb(2+), Ag(1+), iodoacetate, SDS, DMF, DMSO and ethanol. Fe(3+), Ca(2+) Mg(2+), Mn(2+), Triton X-100 and PMSF did not inhibit the enzyme activity. Northern blot analysis showed a transcript of about 3.7 kb in all cells and tissues studied. This is the first report on the expression and characterization of recombinant human MANB enzyme.