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Functional Nucleic Acid Sensors

Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non-aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross-linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).

http://largens.com/biochemlab/peroxidase2.pdf

Enzyme immobilization: The quest for optimum performance

Natural products have been a rich source of compounds for drug discovery. However, their use has diminished in the past two decades, in part because of technical barriers to screening natural products in high-throughput assays against molecular targets. Here, we review strategies for natural product screening that harness the recent technical advances that have reduced these barriers. We also assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products, and highlight recent examples of natural products in antimicrobial drug discovery and as inhibitors of protein-protein interactions. The growing appreciation of functional assays and phenotypic screens may further contribute to a revival of interest in natural products for drug discovery.

/pdf/the-re-emergence-of-natural-products-for-drug-discovery-in-1h0jebzmkp.pdf

The re-emergence of natural products for drug discovery in the genomics era

Utilization of polymers as biomaterials has greatly impacted the advancement of modern medicine. Specifically, polymeric biomaterials that are biodegradable provide the significant advantage of being able to be broken down and removed after they have served their function. Applications are wide ranging with degradable polymers being used clinically as surgical sutures and implants. In order to fit functional demand, materials with desired physical, chemical, biological, biomechanical and degradation properties must be selected. Fortunately, a wide range of natural and synthetic degradable polymers has been investigated for biomedical applications with novel materials constantly being developed to meet new challenges. This review summarizes the most recent advances in the field over the past 4 years, specifically highlighting new and interesting discoveries in tissue engineering and drug delivery applications.

/pdf/biomedical-applications-of-biodegradable-polymers-2db9gcyzxw.pdf

Biomedical Applications of Biodegradable Polymers

CYP51, a sterol 14α-demethylase, is one of the key enzymes involved in sterol biosynthesis and requires electrons transferred from its redox partners. A unique CYP51 from Nocardia farcinica IFM10152 forms a distinct cluster with iron-sulfur containing NADPH-P450 reductase (FprD) downstream of CYP51. Previously, sequence alignment of nine reductases from N. farcinica revealed that FprC, FprD, and FprH have an additional sequence at their N-termini that has very high identity with iron-sulfur clustered ferredoxin G (FdxG). To construct an artificial self-sufficient cytochrome P450 monooxygenase (CYP) with only FprD, CYP51, and iron-sulfur containing FprD were fused together with designed linker sequences. CYP51-FprD fusion enzymes showed distinct spectral properties of both flavoprotein and CYP. CYP51-FprD F1 and F2 in recombinant Escherichia coli BL21(DE3) catalyzed demethylation of lanosterol more efficiently, with k(cat) /K(m) values of 96.91 and 105.79 nmol/min/nmol, respectively, which are about 35-fold higher compared to those of CYP51 and FprD alone.

Novel iron-sulfur containing NADPH-reductase from Nocardia farcinica IFM10152 and fusion construction with CYP51 lanosterol demethylase.

Rational engineering of ornithine decarboxylase with greater selectivity for ornithine over lysine through protein network analysis.

In this paper, the possibility of using a mutation of ftsZ as a pseudo-spore mutant is investigated. ftsZ, which is essential for cell division and sporulation of Bacillus subtilis, was placed under the spac promoter, which is inducible with isopropyl thiogalactose (IPTG). Cell growth of the ftsZ mutant and its β-galactosidase activity under the aprE promoter were compared with the wild type. In the presence of 1 mM IPTG, cell growth of the ftsZ mutant was almost the same as that of the wild type and its sporulation frequency was slightly lower than that of the wild type. However, under uninduced conditions, cell growth of ftsZ mutant was severely impaired. When induced with 0.2 mM IPTG, the ftsZ mutant showed about 13 times higher β-galactosidase activity than the wild type. When the ftsZ mutant was used for secretory production of subtilisin, only three times higher extracellular subtilisin activity was measured, compared with the wild type. By real-time PCR investigation, it was revealed that the ftsZ mutant intracellular mRNA level for subtilisin was more than 16 times higher, compared with the wild type. However, it appears that the secretion pathway is somewhat damaged in the ftsZ mutant. These results suggest that the cell division mutant can also be used like a sporulation mutant to produce recombinant proteins, with a precise control of cell growth and induction.

The effects of ftsZ mutation on the production of recombinant protein in Bacillus subtilis.

Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobacter sp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 degrees C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (S)-2-hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2-oxo-4-phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M-1 min-1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)-phenyllactic acid (> 94% ee) and 95 mM of (S)-2-hydroxy-4-phenylbutanoic acid (> 94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2-oxo-4-phenylbutanoic acid.

Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K.

A spectrophotometric method that can monitor the trans-sialidase catalyzed reaction was devised by using a chromogenic substrate, o-nitrophenyl-β-d-galactopyranoside (ONPG), as a sialic acid acceptor. This method yielded results that were consistent with those from HPLC, and could be very conveniently used in the screening of sialic acid donor or inhibitor of trans-sialidase.

Byung-Gee Kim

Papers

Novel iron-sulfur containing NADPH-reductase from Nocardia farcinica IFM10152 and fusion construction with CYP51 lanosterol demethylase.

Rational engineering of ornithine decarboxylase with greater selectivity for ornithine over lysine through protein network analysis.

The effects of ftsZ mutation on the production of recombinant protein in Bacillus subtilis.

Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K.

Development of spectrophotometric method for monitoring trans-sialidase reaction and its application