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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.117, #2)

Suppressive activity of protease inhibitors from buckwheat seeds against human T-acute lymphoblastic leukemia cell lines by Sung-Soo Park; Hideki Ohba (pp. 65-74).

The buckwheat protease inhibitor designated BWI-1, a member of the potato inhibitor I family, inhibits trypsin, chymotrypsin, and subtilisin, whereas the buckwheat protease inhibitor designated BWI-2a, a novel protease inhibitor homologous to the vicilin family, inhibits only trypsin. We examined the suppressive activity of BWI-1 and BWI-2a against T-acute lymphoblastic leukemia (T-ALL) cells, such as JURKAT and CCRF-CEM, and human normal blood lymphocytes. Both inhibitors significantly suppressed the growth of T-ALL cells as judged by the soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (tetrazolium/formazan assay). JURKAT cells showed slightly higher susceptibility to buckwheat inhibitors than CCRF-CEM cells. Modification of Arg residue(s) in inhibitors by 1,2-cyclohexandione inactivated their trypsin inhibitory activity, considerably abolishing their suppressive activity. This suggests that the trypsin inhibitory activity is involved in the suppression of growth of human T-ALL cell lines. It was further found that both inhibitors triggered programmed cell death (apoptosis) of these cell strains with DNA fragmentation.

Keywords: Apoptosis; buckwheat; protease inhibitor; tumor hematopoietic stem cell; T-acute lymphoblastic leukemia

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus by M. I. Rajoka; Samia Khan; Farooq Latif; Riaz Shahid (pp. 75-92).

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y P/S) and volumetric productivity (Q P) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.

Keywords: β-Glucosidase; cellobiose; corn steep liquor; Kluyveromyces marxianus ; shake flask

Noncompetitive enzyme immunoassay for α-fetroprotein using flow injection chemiluminescence by Jiehua Lin; Feng Yan; Huangxian Ju (pp. 93-102).

A novel, direct noncompetitive flow injection enzyme immunoassay for α-fetoprotein (AFP) was developed by enhanced chemiluminescence detection. The method was based on off-line incubation of AFP and horseradish peroxidase (HRP)-labeled anti-AFP, and then trapping of the unbound enzyme conjugate by an immunoaffinity column filled with AFP-modified Sepharose. The immunocomplex formed in incubation passed through the column and then was directly detected by a postcolumn chemiluminescence technique. The optimal conditions for the immunoassay procedure and chemiluminescence detection were established. At a 1:10 dilution of enzyme conjugate solution, the linear range for chemiluminescence detection of AFP was from 2.0 to 75 ng/mL with a correlation coefficient of 0.993 and a coefficient of variation of 2.67% at 30 ng/mL. The detection limit was 0.5 ng/mL. This method was flexible, sensitive, and rapid. The immunoaffinity column of 200 µL could be repeatedly used 100 times without a single decrease. The whole assay time including the preincubation step was only 30 min for one sample.

Keywords: Flow injection analysis; enzyme immunoassay; noncompetitive immunoassay; immunoaffinity; α-fetoprotein; horseradish peroxidase; chemiluminescence

Dilute-acid pretreatment of corn stover using a high-solids percolation reactor by Yongming Zhu; Y. Y. Lee; Richard T. Elander (pp. 103-114).

Pretreatment of corn stover by dilute sulfuric acid was investigated using a laboratory percolation (flowthrough) reactor operated under high-solids conditions. The effects of reaction conditions and operating parameters on the performance of the percolation reactor were investigated seeking the optimal range in which acceptable levels of yield and sugar concentration could be attained. It was demonstrated that 70–75% recovery of xylose and 6 to 7% (w/w) xylose concentration were attainable. The high sugar concentration was obtained as a result of dense packing of dry corn stover and the low liquid throughput. Xylose was mostly unreacted, rather than decomposed. The cellulose and the unreacted xylan of treated corn stover were both effectively hydrolyzed by a “cellulase” enzyme preparation that also exhibits some activity on xylan. The xylose yield was affected significantly by the flow rate under the same reaction time and conditions. This behavior appears to be related to sugar decomposition, mass transfer resistance, and the fact that acid is neutralized by the buffering components of the biomass.

Keywords: Corn stover; dilute-acid hydrolysis; hemicellulose; percolation process; pretreatment; xylan

The “bringer” strategy by Andreas Bichet; Matthias Bureik; Natalie Lenz; Rita Bernhardt (pp. 115-122).

Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR). Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property. However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure. Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures. Primers binding to the β-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible. The functionality of this approach was demonstrated by mutating the α-peptide coding region of the lacZ gene.

Keywords: Error-prone polymerase chain reaction; α-peptide; ring-closed plasmids; Escherichia coli ; Bringer strategy

Interaction of an intermediate structure of Bacillus subtilis α-amylase with alkyl-substituted sepharose 4B by Hamid Reza Karbalaei-Heidari; Azadeh Ebrahim Habibi; Khosro Khajeh; Bijan Ranjbar; Mohsen Nemat-Gorgani (pp. 123-132).

An intermediate from of α-amylase from Bacillus subtilis was prepared following a previously reported procedure. Stabilization of this protein structure by various additives and its interaction with alkyl-substituted Sepharose 4B (Sepharose-lipid), used to mimic the role of the alkyl chains of the phospholipid bilayer, were investigated. Exposure of hydrophobic clusters in the protein structure on denaturation was indicated by a greater affinity of the intermediate form for interaction with the alkyl chains on the matrix, as compared with the original native structure. Near- and far-ultraviolet circular dichroism studies supported loss of tertiary structure with retention of secondary structure, as expected from molten globular intermediate forms. Based on the results presented, we suggest that interaction of a protein in its native and nonnative forms with an alkyl-substituted matrix may provide useful information regarding its capacity for insertion into and translocation across the biologic membrane.

Keywords: α-Amylase; intermediate structure; Sepharose-lipid; hydrophobic matrix; membrane insertion; membrane translocation

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.117, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.117, #2)