Recombinant production and biochemical and in silico characterization of lactate dehydrogenase from Geobacillus thermodenitrificans DSM-465

Background: Lactate dehydrogenase (LDH) is an enzyme of glycolytic pathway, ubiquitously found in living organisms. Increased glycolysis and LDH activity are associated with many pathologic conditions including inflammation and cancer, thereby making the enzyme a suitable drug target. Studies on conserved structural and functional domains of LDH from various species reveal novel inhibitory molecules. Our study describes Escherichia coli production and characterization of a moderately thermostable LDH (LDH-GT) from Geobacillus thermodenitrificans DSM-465. An in silico 3D model of recombinant enzyme and molecular docking with a set of potential inhibitors are also described. Results: The recombinant enzyme was overexpressed in E. coli and purified to electrophoretic homogeneity. The molecular weight of the enzyme determined by MALDI-TOF was 34,798.96 Da. It exhibited maximum activity at 65°C and pH 7.5 with a KM value for pyruvate as 45 µM. LDH-GT and human LDH-A have only 35.6% identity in the amino acid sequence. On the contrary, comparison by in silico structural alignment reveals that LDH-GT monomer has approximately 80% identity to that of truncated LDH-A. The amino acids "GEHGD" as well as His179 and His193 in the active site are conserved. Docking studies have shown the binding free energy changes of potential inhibitors with LDH-A and LDH-GT ranging from −407.11 to −127.31 kJ mol−1 . Conclusions: By highlighting the conserved structural and functional domains of LDH from two entirely different species, this study has graded potential inhibitory molecules on the basis of their binding affinities so that they can be applied for in vivo anticancer studies

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro / 中国天然药物

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro / 中国天然药物

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Effect of Pichia pastoris host strain on the properties of recombinant Aspergillus niger endoglucanase, EglB

Aims@#The methylotrophic yeast Pichia pastoris is widely used to express foreign proteins fused to secretion signals. As the effect of the expression host on the final protein product is unclear, we compared the properties of an endoglucanase (eglB of Aspergillus niger) expressed in two different P. pastoris strains. @*Methodology and results@#Full-length cDNA encoding endoglucanase of A. niger strain ATCC10574 was isolated and expressed in P. pastoris X33 (the methanol utilisation plus phenotype, Mut+) and P. pastoris GS115 (slow methanol utilisation, MutS). EglB-GS115 showed the highest activity and stability at 60 °C while EglB-X33 was most active at 50 °C. EglB-X33 was active towards other substrates such as arabinogalactan, guar gum and locust bean gum besides its specific substrate, carboxymethyl cellulose (CMC). However, EglB-GS115 was only active on CMC. The affinity of EglB-X33 towards CMC (Km = 7.5 mg/mL and specific activity 658 U/mg) was higher than that of EglB-GS115 (Km = 11.57 mg/mL, specific activity 144 U/mg). @*Conclusion, significance and impact of study@#Although eglB was cloned in the same expression vector (pPICZαC), two different characteristics of enzymes were recovered from the supernatant of the different hosts. Thus, expression of recombinant enzyme in different P. pastoris strains greatly affects the physical structure and biochemical properties of the enzyme.

Expression and characterization of a keratinase encoding gene gm2886 in Streptomyces pactum ACT12 strain / 生物工程学报

By bioinformatics analysis, a putative keratinase gene gm2886 (Accession number: KY368946) was discovered in the genome of a feather-degrading strain, Streptomyces albidoflavus Fea-10. gm2886 was ligated into integrative Escherichia coli-Streptomyces shuttle vector pSET152 under the promoter PermE and added with C-terminal His-tag. The expression vector was transformed into Streptomyces pactum ACT12 by conjugal transfer and the recombinant protein GM2886-His6 was detected in fermentation broth. GM2886-His6 was purified and characterized. Its size was nearly 36 kDa. GM2886-His6 showed proteolytic activity towards a variety of substrates and could even degrade insoluble substrates, such as azure keratin and chicken feathers. The optimal pH and temperature of GM2886-His6 for proteolysis of casein was pH 10.0 and 50 ℃, respectively. The enzyme activity was inhibited by PMSF, but not EDTA, indicating that GM2886-His6 was a serine proteinase. Our results laid the foundation for the research of the molecular biological mechanism on feather-degrading and for the further utilization of Fea-10.

Gene cloning, expression, and characterization of the Bacillus amyloliquefaciens PS35 lipase

Abstract Lipases are enzymes of immense industrial relevance, and, therefore, are being intensely investigated. In an attempt to characterize lipases at molecular level from novel sources, a lipase gene from Bacillus amyloliquefaciens PS35 was cloned, heterologously expressed in Escherichia coli DH5α cells and sequenced. It showed up to 98% homology with other lipase sequences in the NCBI database. The recombinant enzyme was then purified from E. coli culture, resulting in a 19.41-fold purification with 9.7% yield. It displayed a preference for long-chain para-nitrophenyl esters, a characteristic that is typical of true lipases. Its optimum pH and temperature were determined to be 8.0 and 40 °C, respectively. The half-lives were 2.0, 1.0 and 0.5 h at 50 °C, 60 °C and 70 °C, respectively. The metal ions K+ and Fe3+ enhanced the enzyme activity. The enzyme displayed substantial residual activity in the presence of various tested chemical modifiers, and interestingly, the organic solvents, such as n-hexane and toluene, also favored the enzyme activity. Thus, this study involves characterization of B. amyloliquefaciens lipase at molecular level. The key outcomes are novelty of the bacterial source and purification of the enzyme with desirable properties for industrial applications.

Molecular cloning, characterization and expression of lipoxygenase 2 (lox-2) isozyme from Indian soybean [Glycine max (L.) Merrill] cv. Pusa 16.

The consumption of soybean is limited worldwide, despite being highly nutritious and having versatile uses, due to the presence of grassy, beany and rancid off-flavour. The lipoxygenase-2 (LOX-2) is the key enzyme responsible for the production of volatiles released from the beans, which cause off-flavour in soy products. In this study, a 2.6-kb full-length lox2 gene (NCBI accession No. JQ929619.1) was isolated and cloned from soybean (Glycine max L. Merril) cv. Pusa 16. The cloned cDNA sequence of lox2 gene showed the complete open reading frame (ORF) of a putative protein, having 866 amino acids with start codon present at the foremost position and stop codon at the end. The theoretical pI of predicted protein was 6.22. A hydropathy profile calculated from the amino acid sequence resembled those of dicot LOXs, suggesting conservation of the secondary structure of these enzymes. The LOX-2 showed conserved six Histidine residues within a span of 520 to 590 amino acid position, a signature element for the enzyme activity. The lox2 gene was expressed using pET vector in prokaryotic expression system. The recombinant LOX-2 protein was purified after induction with IPTG (isopentyl thiogalactoside). A prominent band of 97 kDa was observed, when affinity purified fractions were analyzed by SDS-PAGE. The purified protein was characterized for the enzyme activity, substrate preference and Km. Inhibitor studies with natural antioxidant molecules present in soybean revealed α-tocopherol to be the most effective inhibitor of LOX-2.

Prolonged hypotensive effect of human tissue kallikrein gene delivery and recombinant enzyme administration in spontaneous hypertension rats

To evaluate the feasibility of treating hypertension by human tissue kallikrein gene (KLK1) delivery and by enzyme (rK1) administration, two recombinant vectors expressing KLK1 cDNA were constructed for gene delivery (pcDNA-KLK1) and recombinant enzyme preparation (pOV-KLK1). Expression of the pcDNA-KLK1 vector in COS-1 cells was confirmed by immunofluorescence and in spontaneous hypertension rats (SHR) by enzymatic detection. Following intramuscular or intravenous injection with the pcDNA-KLK1 vector, systolic pressure of SHR was significantly decreased, which lasted for 20 d to two months depending on dose, route and/or time of injection. Egg white containing recombinant hK1 was prepared by injection of egg-laying hens with the oviduct-specific expression vector pOV-KLK1 and administered into SHR via oral gavage. Following administration, systolic pressure of the SHR was decreased to that of normal rats, which lasted for 3-5 d depending on the dosage used. These data suggest that both hKLK1 gene delivery and recombinant enzyme administration can be used as alternative strategies for treating human hypertension.