Kinetics of enzymatic solid-to-solid peptide synthesis: synthesis of Z-aspartame and control of acid-base conditions by using inorganic salts

Enzymatic peptide synthesis can be carried out efficiently in solid-to-solid reaction mixtures with 10% (w/w) water added to a mixture of substrates. The final reaction mass contains >/=80% (by weight) of product. This article deals with acid-base effects in such reaction mixtures and the consequences for the enzyme. In the Thermoase-catalyzed synthesis of Z-Asp-Phe-OMe, the reaction rate is strongly dependent on the amount of basic salts added to the system. The rate increases 20 times, as the KHCO(3) or K(2)CO(3) added is raised 2.25-fold from an amount equimolar to the Phe-OMe. HCL starting material. With further increases in KHCO(3) addition, the initial rate remains at the maximum, but with K(2)CO(3) it drops sharply. Addition of NaHCO(3) is less effective, but rates are faster if more water is used. With >1.5 equivalents of basic salt, the final yield of the reaction decreases. Similar effects are observed when thermolysin catalyzes the same reaction, or Z-Gln-Leu-NH(2) synthesis. These effects can be rationalized using a model estimating the pH of these systems, taking into account the possible formation of up to ten different solid phases.     

Development of small-size tubular-flow continuous reactors for the analysis of operational stability of enzymes in low-water systems

A very small-scale continuous flow reactor has been designed for use with enzymes in organic media, particularly for operational stability studies. It is constructed from fairly inexpensive components, and typically uses 5 mg of catalyst and flow rates of 1 to 5 mL/h, so only small quantities of feedstock need to be handled. The design allows control of the thermodynamic water activity of the feed, and works with temperatures up to at least 80 degrees C. The reactor has been operated with both nonpolar (octane) and polar (4-methyl-pentan-2-one) solvents, and with the more viscous solvent-free reactant mixture. It has been applied to studies of the operational stability of lipases from Chromobacterium viscosum (lyophilized powder or polypropylene-adsorbed) and Rhizomucor miehei (Lipozyme) in different experimental conditions. Transesterification of geraniol and ethylcaproate has been adopted as a model transformation.     

The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas

A comprehensive analysis of somatic and germline mutations related to DNA mismatch-repair (MMR) genes can clarify the prevalence and mechanism of inactivation in colorectal carcinoma (CRC). In the present study, 257 unselected patients referred for CRC resection were examined for evidence of defective DNA MMR. In particular, we sought to determine the frequency of hereditary defects in DNA MMR in this cohort of patients. MMR status was assessed by testing of tumors for the presence or absence of hMLH1, hMSH2, and hMSH6 protein expression and for microsatellite instability (MSI). Of the 257 patients, 51 (20%) had evidence of defective MMR, demonstrating high levels of MSI (MSI-H) and an absence of either hMLH1 (n=48) or hMSH2 (n=3). All three patients lacking hMSH2, as well as one patient lacking hMLH1, also demonstrated an absence of hMSH6. DNA sequence analysis of the 51 patients with defective MMR revealed seven germline mutations-four in hMLH1 (two truncating and two missense) and three in hMSH2 (all truncating). A detailed family history was available for 225 of the 257 patients. Of the seven patients with germline mutations, only three had family histories consistent with hereditary nonpolyposis colorectal cancer. Of the remaining patients who had tumors with defective MMR, eight had somatic mutations in hMLH1. In addition, hypermethylation of the hMLH1 gene promoter was present in 37 (88%) of the 42 hMLH1-negative cases available for study and in all MSI-H tumors that showed loss of hMLH1 expression but no detectable hMLH1 mutations. Our results suggest that, although defective DNA MMR occurs in approximately 20% of unselected patients presenting for CRC resection, hereditary CRC due to mutations in the MMR pathway account for only a small proportion of patients. Of the 257 patients, only 5 (1.9%) appear to have unequivocal evidence of hereditary defects in MMR. The epigenetic (nonhereditary) mechanism of hMLH1 promoter hypermethylation appears to be responsible for the majority of the remaining patients whose tumors are characterized by defective DNA MMR.     

Hereditary desmoid disease in a family with a germline Alu I repeat mutation of the APC gene

Two families with autosomal dominantly inherited desmoid tumors have recently been shown to have germline mutations at the 3' end of the APC gene. We subsequently identified an Amish family with autosomal dominantly inherited desmoid tumors. Genetic analysis performed on one family member, a 47-year-old man with multiple desmoid tumors and no colon polyps, revealed a protein truncating mutation in the middle of the APC gene. The truncating mutation is the result of a 337-bp insertion of an Alu I sequence into codon 1526 of the APC gene. The presence of a poly(A) tail at the 3' end of the insertion suggests that the Alu I sequence was inserted by a retrotranspositional event. Germline insertions of Alu I sequences have occasionally been reported to cause other genetic diseases including type I neurofibromatosis, hereditary site-specific breast cancer (BRCA2), and hemophilia B. However, this is the first report of a germline mutation of the APC gene resulting from an Alu I insertion.     

Supercritical fluids are superior media for catalysis by cross-linked enzyme microcrystals of subtilisin Carlsberg

We report on the performance of cross-linked enzyme microcrystals (CLECs) of subtilisin Carlsberg in supercritical fluids (SC-fluids). The catalytic activity of CLECs in SC-ethane was found to be 2- to 10-fold greater than in hexane under the same conditions, using CLECs dried by propanol washing. Air-dried CLECs and lyophilized powders showed much lower activities, reflecting the same hydration hysteresis effects as in organic solvents. Reaction rates were much lower in SC-CO(2), especially at higher water activity, probably as a result of acid-base effects of carbonic acid on the enzyme.     

Effect of pH on rate of interfacial inactivation of serine proteases in aqueous-organic systems

We studied the inactivation of trypsin and alpha- and beta-chymotrypsin by passage of droplets of tridecane though their aqueous solutions. The mechanism involves contact with the interface, because the loss of activity is proportional to the total area exposed. The rates of inactivation vary up to fivefold over the pH range 3 to 10. However, there is no clear maximum at the isoelectric point (pI) of each enzyme, where the amount of protein adsorbed is usually found to be highest. This is probably because, at the pI, there is also a minimum in structural alteration on adsorption. There may be a weak correlation with pH effects on foamability of the enzyme solutions, a parameter reported to reflect the "hardness" of different proteins, which controls their interfacial unfolding. The pH dependence of both inactivation and hardness cautions against attempts to correlate inactivation of different enzymes with a single value of a parameter such as adiabatic compressibility. There is no correlation between the effects of pH on interfacial inactivation and those reported in the literature on irreversible inactivation in concentrated urea or at high temperature.     

Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst

This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH(2) was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the "solid-to-solid" and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied.     

A new genus of Boletaceae from eastern North America

Bothia is described as a new genus in the Boletaceae based on Boletinus castanellus described by C.H. Peck from eastern North America. A widespread, occasionally encountered taxon, Bothia castanella possesses a combination of macro- and microscopic features that has prompted past placement in seven different genera. Yet, as a species it is readily recognizable with its chestnut brown, dry pileus, decurrent, pale brown hymenophore with radially elongated tubes, a short, sometimes eccentric, exannulate stipe, yellow brown spore deposit and constant association with Quercus. Phylogenetic analyses of large subunit rDNA and BLAST searches using the ITS region confirm the placement of B. castanella as a unique generic lineage in the Boletaceae.     

Tylopilus orsonianus sp. nov. and Tylopilus eximius from Guyana

Tylopilus orsonianus sp. nov. and Tylopilus eximius (Boletaceae, Basidiomycota) are described for the first time from the Pakaraima Mountains of Guyana. Both boletes occur in forests dominated by ectomycorrhizal trees in the genus Dicymbe (Caesalpiniaceae). A key to Tylopilus species distinguishes those known to occur in Guyana.     

Morphological and molecular evidence supporting an arbutoid mycorrhizal relationship in the Costa Ricanpáramo

This study examines evidence for a particular arbutoid mycorrhizal interaction in páramo, a high-altitude neotropical ecosystem important in hydrological regulation but poorly known in terms of its fungal communities. Comarostaphylis arbutoides Lindley (Ericaceae) often forms dense thickets in Central American páramo habitats. Based on phylogenetic classification, it has been suggested that C. arbutoides forms arbutoid mycorrhizae with diverse Basidiomycetes and Ascomycetes; however, this assumption has not previously been confirmed. Based on field data, we hypothesized an arbutoid mycorrhizal association between C. arbutoides and the recently described bolete Leccinum monticola Halling & G.M. Mueller; in this study, we applied a rigorous approach using anatomical and molecular data to examine evidence for such an association. We examined root samples collected beneath L. monticola basidiomes for mycorrhizal structures, and we also compared rDNA internal transcribed spacer (ITS) sequences between mycorrhizal root tips and leaf or basidiome material of the suspected symbionts. Root cross sections showed a thin hyphal sheath and intracellular hyphal coils typical of arbutoid mycorrhizae. DNA sequence comparisons confirmed the identity of C. arbutoides and L. monticola as the mycorrhizal symbionts. In addition, this paper provides additional evidence for the widespread presence of minisatellite-like inserts in the ITS1 spacer in Leccinum species (including a characterization of the insert in L. monticola) and reports the use of an angiosperm-specific ITS primer pair useful for amplifying plant DNA from mycorrhizal roots without co-amplifying fungal DNA.