Beaded reactive polymers. 3. Effect of triacrylates as crosslinkers on the physical properties of glycidyl methacrylate copolymers and immobilization of penicillin G acylase.

Various glycidyl methacrylate (GMA) copolymers were synthesized by suspension polymerization, using pentaerythritol triacrylate (PETA), trimethylolpropane triacrylate (TMPTA), and trimethylolpropane trimethacrylate (TRIM) as crosslinking comonomers. These copolymers were evaluated for the immobilization of penicillin G acylase. Broad pore-size distribution that was observed was in the range 5-300 nm. Both surface area and pore volume increased with increase in the mole fraction of crosslinking comonomer (increasing crosslink density). The pore volume of the copolymers was more than doubled by including lauryl alcohol as porogen. Binding of penicillin G acylase (PGA) was quantitative on highly crosslinked copolymers. The expression of bound PGA was better on the relatively more hydrophilic GMA-TMPTA and GMA-PETA copolymer supports compared to the GMA-TRIM copolymers. Among the different copolymers studied, GMA-TMPTA copolymer 7411 exhibited highest activity of immobilized penicillin G acylase (167.4 IU/g) with 35.1% expression.

Adsorption and expression of penicillin G acylase immobilized onto methacrylate polymers generated with varying pore generating solvent volume.

Adsorption and expression of penicillin G acylase was studied on macroporous methacrylate polymer beads of differing pore volume, generated with kerosene. The absorption and expression of the penicillin G acylase was dependent on pore volume. Maximum expression of 57% of adsorbed enzyme was obtained on beads synthesized with 40 mL of kerosene, indicating minimum pore-diffusion limitations.

Detection and correction of a migration anomaly on a 310 genetic analyzer.

During STR analysis on the 310 Genetic Analyzer, retarded migration of GS500ROX size standards and alleles in some samples was observed. The contribution of reagents, capillary and performance optimized polymer POP 4 to the observed anomaly was experimentally eliminated. Variation in electrophoresis temperature between 55 degrees C and 65 degrees C did not alter the rate of migration of GX500ROX size standard and sample alleles. An eroded connector for the cathode mounted on the heat plate assembly caused the abnormal migration. Hence, it is important to verify the mobility of all fragments in the size standard for each sample to avoid any erroneous allele calls by the automated data analysis software.

Immobilization of penicillin G acylase on methacrylate polymers.

Macroporous weak cation-exchange methacrylate polymers were synthesized for the immobilization of penicillin G (Pen G) acylase. The role of certain factors such as pore-generating solvent, cross-linking agent, cross-linking density, and comonomer, in enzyme adsorption and expression was studied. Kerosene was a superior pore-generating solvent to paraffin oil. Ethylene glycol dimethacrylate and acrylic acid served as the best cross-linking agent and comonomer, respectively, in the systems studied. 80.3% of the activity of the enzyme adsorbed onto polymer beads prepared with 0.05 mol of acrylic acid (polymer PM-39) was expressed. Properties of the Pen G acylase, immobilized on PM-39 by adsorption and cross-linking with glutaraldehyde (IME-PM-39) were studied. The optimum pH, optimum temperature and Km of Pen G acylase shifted from 8.0 to 7.5-7.8, 50 degrees C to 55 degrees C and 0.038 mol dm-3 to 2.4-3.0 mol dm-3, respectively, as a result of immobilization on PM-39. IME-PM-39 was used repeatedly for 15 cycles in the production of 6-amino penicillanic acid (6-APA).

Principles, Practice, and Evolution of Capillary Electrophoresis as a Tool for Forensic DNA Analysis.

Capillary electrophoresis (CE) is a versatile and widely used analysis platform with application in diverse areas such as analytical chemistry, chiral separations, clinical, forensics, molecular biology, natural products, organic chemistry, and the pharmaceutical industry. Forensic applications of CE include fragment analysis, DNA sequencing, SNP typing, and analysis of gunshot residues, explosive residues, and drugs. Fragment analysis is a widely used method for short tandem repeat (STR) profiling for human identification (HID) due to the single-base resolution capability of CE. This approach circumvents the tedious and expensive approach of DNA sequencing for STR typing. The high sizing precision, ability to detect fluorescence emitted from multiple dyes, automated electrophoretic runs, and data collection software are key factors in the worldwide adoption of CE as the preferred platform for forensic DNA analysis. The most common CE systems used in forensic DNA analysis include the ABI PRISM® 310, 3100, 3100 Avant, 3130, 3130xl, 3500, and 3500xL Genetic Analyzers (GAs). The 3500 series GAs are developed with features useful for forensic scientists, including a normalization feature for analysis of the data designed to reduce the variation in peak height from instrument to instrument and injection to injection. Other hardware and software features include improved temperature control, radio frequency identification (RFID) tags for monitoring instrument consumables, HID-focused software features, and security and maintenance.

Training of Forensic DNA Scientists - A Commentary.

For the past two decades, forensic DNA analysis has rapidly expanded in both utility and value to criminal investigations. As the number of crime scene and convict/arrestee samples has continued to grow, many forensic DNA laboratories find themselves struggling to test samples in a timely fashion. Agencies employ various methods for calculating their sample intake and processing capacity, yet database and casework sample backlogs continue to present a major challenge. One issue many forensic laboratories face is limited availability of resources for training new analysts. High-quality training enables analysts to effectively perform various aspects of DNA profiling, and as such, it is essential to ensuring consistent, high-quality results. This is well documented in the guidelines established in the FBI's Quality Assurance Standards for Forensic DNA Testing Laboratories in the United States as well as internationally by agencies like INTERPOL. A facility dedicated to training analysts on both theoretical and practical aspects of automated sample processing accelerates the establishment and expansion of high-throughput forensic DNA laboratories. The present article will discuss various aspects of training and agencies that provide such training programs.

Assessment of DNA Extracted from Forensic Samples Prior to Genotyping.

Quantification of human DNA has been an integral part of forensic DNA analysis. Hybridizationbased methods such as Quantiblot® kits were used extensively in the 1990s. These methods fulfilled the need at the time, since their sensitivity range was similar to the genotyping methods in use, such as restricted fragment length polymorphism. Later, the development of robust and more sensitive megaplex genotyping systems such as short tandem repeat profiling, mitochondrial DNA sequencing, and single nucleotide polymorphism typing, created the need not only for quantification of DNA at the picogram level but also for assessment of the quality of the DNA extract to make informed decisions to ensure the success of downstream analysis. Real-time PCR-based quantification methods fulfilled this need. The different real-time PCR methods developed range from singleplex reactions for quantification of human or mitochondrial DNA to multiplex systems that enable analysis of up to four targets for quantification of human DNA, human male DNA, mitochondrial DNA, detection of PCR inhibitors, or determination of the extent of DNA degradation. Incorporation of these assays into the workflow enables selection of appropriate genotyping systems and increases the first-pass success rate for obtaining a genotype using a minimal amount of evidence sample. The real-time PCR methods described here would also be useful as DNA assessment tools prior to other genotyping methods like copy number variation, insertion/deletion, and Alu dimorphism analysis as well as sequencing, etc., that are currently being investigated as additional informative tools for human identification purposes.

Extraction of DNA from Human Remains.

Improvements to analytical methods have made it possible for highly discriminative genotypic information to be gleaned from smaller and smaller amounts of sample material. This fact makes it practical to genotype samples or remains consisting of bone and tooth-samples that likely would not have yielded interpretable genotypic results a short time ago. In parallel, there have been improvements to protocols specifically designed to recover DNA from very old calcified tissues, i.e., ancient or compromised nature. This review discusses the current best practices for isolating and purifying DNA from bones and teeth with a focus on the processes of lysis and DNA purification linked together to yield DNA from these challenging samples. The mitochondrial and genomic DNA recovered from more recently developed techniques for isolation from skeletal remains and teeth, even very old samples, is surprisingly amenable to genotypic analysis.

Extraction of DNA from Forensic Biological Samples for Genotyping.

Biological forensic samples constitute evidence with probative organic matter. Evidence believed to contain DNA is typically processed for extraction and purification of its nucleic acid content. Forensic DNA samples are composed of two things, a tissue and the substrate it resides on. Compositionally, a sample may contain almost anything and for each, the type, integrity, and content of both tissue and substrate will vary, as will the contaminant levels. This fact makes the success of extraction one of the most unpredictable steps in genotypic analysis. The development of robust genotyping systems and analysis platforms for short tandem repeat (STR) and mitochondrial DNA sequencing and the acceptance of results generated by these methods in the court system, resulted in a high demand for DNA testing. The increasing variety of sample submissions created a need to isolate DNA from forensic samples that may be compromised or contain low levels of biological material. In the past decade, several robust chemistries and isolation methods have been developed to safely and reliably recover DNA from a wide array of sample types in high yield and free of PCR inhibitors. In addition, high-throughput automated workflows have been developed to meet the demand for processing increasing numbers of samples. This review summarizes a number of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples, including manual, semiautomated, and fully automated platforms.

Y-Short Tandem Repeat Multiplex Systems - Y-PLEX™ 6 and Y-PLEX™ 5.

Y-Chromosome short tandem repeats (Y-STRs) have become a very useful tool in forensic casework, paternity, and male lineage studies. In forensic casework, one can obtain the male profile from a mixture sample containing male and female DNA. Two Y-STR genotyping systems, Y-PLEX™ 6 and Y-PLEX™ 5, have been developed for use in human identification. Y-PLEX™ 6 enables simultaneous amplification of DYS393, DYS19, DYS389II, DYS390, DYS391, and DYS385; Y-PLEX™ 5 enables simultaneous amplification of DYS389I, DYS389II, DYS439, DYS438, and DYS392 loci. The Y-PLEX™ 6 and Y-PLEX™ 5 systems together provide analysis of all nine Y-STR loci generating minimal haplotype and two additional loci, DYS438 and DYS439. These systems also provide analysis for all 11 Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM) for forensic casework and population database studies. Both the systems were validated following the Federal Bureau of Investigation (FBI) Director's Quality Assurance Standards. Allelic ladders, which serve as a reference in genotyping, were generated. The nucleotide sequence of alleles in the allelic ladder was determined and the nomenclature is in accord with the recommendations of the International Society of Forensic Genetics (ISFG). The minimum sensitivity of the Y-PLEX™ 6 and Y-PLEX™ 5 systems was 0.2 and 0.1 ng of male DNA, respectively. The nonhuman study revealed that the primers in the Y-PLEX™ 6 and Y-PLEX™ 5 systems were specific for the DNA from humans and some higher primates. Mean stutter values ranged from 3.6 to 11.9%. The Y-PLEX™ 6 and Y-PLEX™ 5 systems were used in several forensic cases. The results from these multiplex systems have been admitted in various U.S. Courts. Thus, Y-PLEX™ 6 and Y-PLEX™ 5 genotyping systems are sensitive, reliable, and robust for use in human forensic and male lineage identification studies.

Biosynthesis of penicillin V acylase by Fusarium sp.: effect of culture conditions.

Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO4 was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55°C, respectively.

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase.

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K m for penicillin V remained at 10MM. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.

Purification and characterization of extracellular penicillin V acylase from Fusarium sp. SKF 235.

Penicillin V acylase from Fusarium sp. SKF 235 culture filtrate was purified to homogeneity. The enzyme was a glycoprotein and composed of single polypeptide chain with molecular weight of 83,200 Daltons. The pH and temperature optima were 6.5 and 55 degrees C, respectively. The KM for penicillin V was 10 mM but the enzyme was inhibited by penicillin V at concentrations above 50 mM. Products of reaction, 6-aminopenicillanic acid and phenoxyacetic acid inhibited the enzyme competitively and noncompetitively with Ki values of 18 mM and 45 mM, respectively. The enzyme specifically hydrolyzed penicillin V, cephalosporanic acid V and penicillin V sulphoxide. Other phenoxy acetyl amides studied were not hydrolysed. It is proposed that phenoxyacetyl moiety alone is not recognized by the penicillin V acylase and in addition, the beta-lactam structure contributes in formation of enzyme-substrate complex.

Aryl beta-galactosidase from Sclerotium rolfsii: physiological and biochemical studies.

Production of beta-galactosidase by Sclerotium rolfsii NCIM 1084 was studied under submerged fermentation conditions. The enzyme was produced extracellularly and constitutively on glucose. The enzyme production was enhanced when galactose, raffinose, cellobiose, sucrose, xylose, maltose, cellulose and pectin were used as carbon sources. Cellulose and diammonium hydrogen phosphate were best carbon and nitrogen sources, respectively. Surfactants such as Sag, Paraffin oil, Tween 20 and Tween 80 increased the enzyme production. Maximum yield of beta-galactosidase obtained was 3.8-4.2 nkat/ml. The optimum pH, optimum temperature and molecular weight of the beta-glactosidase were 2.7, 60 degrees C and 2,21,000 daltons, respectively. The enzyme is an aryl beta-glactosidase and did not hydrolyse lactose. The Km value for o-nitrophenyl beta-D-galactoside was 3.7 mM. Galactose and 2-mercaptoethanol inhibited the enzyme.

Enzymatic hydrolysis of cellulosic materials by Sclerotium rolfsii culture filtrate for sugar production.

The hydrolysis of purified celluloses (cotton, Avicel, Cellulose-123, Solka Floc SW40) and cellulosic wastes (rice straw, sugarcane bagasse, wood powders, paper factory effluents) by Sclerotium rolfsii CPC 142 culture filtrate was studied. Factors which effect saccharification such as pH, temperature, enzyme concentration, substrate concentration, produce inhibition, adsorption, and inactivation of enzyme and particle size were studied. Virtually no inhibition (less than 3%) of cellulose hydrolysis by the culture filtrate was observed by cellobiose and glucose up to 100 mg/mL. Filter paper degrading enzyme(s) (but neither carboxymethylcellulase nor beta-glucosidase) was adsorbed on cellulose. The n value in the S. rolfsii system was calculated to be 0.32 for Avicel P.H. 101 and 0.53 for alkali-treated (AT) rice straw indicating penetration of cellulase into AT rice straw. In batch experiments at 10% substrate level, solutions containing 6 to 7%, 3.8 to 4.7%, 4.0 to 5.1%, and 4.2 to 4.9% reducing sugars were produced in 24 to 48 from AT rice straw. AT bagasse, alkali - peracetic acid treated mesta wood and paper factory sedimented sludge effluent, respectively. The main constituent in the hydrolysate from cellulose was glucose with little or no cellobiose, probably due to the high cellobiase content in the culture filtrate.

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.

Amino acid sequence of porcine spleen cathepsin D.

The amino acid sequence of porcine spleen cathepsin D heavy chain has been determined and, hence, the complete structure of this enzyme is now known. The sequence of heavy chain was constructed by aligning the structures of peptides generated by cyanogen bromide, trypsin, and endo-proteinase Lys C cleavages. The structure of the light chain has been published previously. The cathepsin D molecule contains 339 amino acid residues in two polypeptide chains: a 97-residue light chain and a 242-residue heavy chain, with a combined Mr of 36,779 (without carbohydrate). There are two carbohydrate units linked to asparagine residues 70 and 192. The disulfide bond arrangement in cathepsin D is probably similar to that of pepsin, because the positions of six half-cystine residues are conserved. The active site aspartyl residues, corresponding to aspartic acid-32 and -215 of pepsin, are located at residues 33 and 224 in the cathepsin D molecule. The amino acid sequence around these aspartyl residues is strongly conserved. Cathepsin D shows a strong homology with other acid proteases. When the sequence of cathepsin D, renin, and pepsin are aligned, 32.7% of the residues are identical. The homology is observed throughout the length of the molecules, indicating that three-dimensional structures of all three molecules are similar.

High Cellobiase and Xylanase Production by Sclerotium rolfsii UV-8 Mutant in Submerged Culture.

Sclerotium rolfsii UV-8 mutant secretes high levels of cellobiase and xylanase in addition to having high cellulase production. The apparent K(m) and V(max) of cellobiase (grown in NM-2 + 2% corn steep liquor medium) with cellobiose as a substrate were 5.6 mM and 22.2 mumol of glucose liberated per min per ml of culture filtrate, respectively. The addition of 2% corn steep liquor to NM-2 medium increased endo-beta-glucanase, cellobiase, and xylanase yields by approximately 1.5-fold.

Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii.

A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-beta-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-beta-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.

Thermal stability of penicillin G acylase : effect of stabilizing agents.

The role of sugars, polyhydroxy compounds, phenylacetic acid and 6-aminopenicillanic acid in stabilization of immobilized penicillin G acylase (IMPGA) was studied. The loss in the activity of IMPGA at 50 degrees C, 2 h, after incorporation of sucrose and mannitol at 0.1 M concentration was 16 and 18% respectively; the loss in the activity of the enzyme under these conditions in the absence of stabilizing agents was 40%.