Applied Biochemistry and Biotechnology (v.90, #1)

A 13 amino acid sequence, CRVARGDWNDNYC, originated from disintegrin eristostatin, was introduced into an inactive human proinsulin molecule between the B29 and A2 sites to replace proinsulin C-peptide by molecular cloning techniques. The constructed Arg-Gly-Asp (RGD)-proinsulin gene was cloned into a temperature-inducible vector pBV220 and expressed in Escherichia coli. The expressed RGD-proinsulin was refolded and purified by Sephadex G50 and DEAE-Sephadex A25 separations. The chemical identity was confirmed by both amino acid composition and mass spectrometry analyses. This RGD-proinsulin showed an inhibitory activity of adenosine 5′-diphosphate-induced human platelet aggregation with an IC50 value of 200 nM. Its insulin receptor binding activity remained as low as 0.03% with native insulin as a control, and its insulin immune activity retained 27.6% compared with proinsulin.
Keywords: RGD motif; mutant proinsulin; RGD-proinsulin; recombinant; inhibition of platelet aggregation

Direct antigen capture by soluble scFv antibodies by Mrinalini Kala; Kiran Bajaj; Subrata Sinha (11-22).
For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag.
Keywords: Recombinant antibody; heat-stable alkaline phosphatase; reverse Western; enzyme-linked immunosorbent assay; affinity; phage display

d- or l-aspartic acid containing polyacrylamides were synthesized. Binary and ternary complex formation between these polymers with copper and bovine serum albumin was studied by spectroscopic and electrochemical measurement. Depending on the ratio of the polymer/copper and protein/polymer, the mixture exhibited water-soluble and insoluble character. A hypothetical structural scheme for the formation of ternary complexes is proposed. The effect of radiation on these complexes was also investigated.
Keywords: Ternary complexes; radiostability; biopolymer; electrochemistry of ternary complexes; polyacrylamide

An improved method for the extraction of β-carotene from Blakeslea trispora is described. The fermentation broth was steamed at 121°C for 15 min, and the liquid was centrifuged at 5000g for 20 min. β-Carotene was removed from the biomass by extraction with absolute ethanol at a ratio of 1:100 at 30°C for 2 h in a rotary shaker incubator at 300 rpm. The carotenoid pigment was completely removed from the cells after three repeated extractions. The removal of β-carotene from B. trispora was higher during the first stage (75%) whereas in the other stages it was very slow.
Keywords: β-Carotene; extraction; Blakeslea trispora

Comparative studies on a mesophilic and a thermophilic α-amylase by Khosro Khajeh; Mohsen Nemat-Gorgani (47-55).
A comparative study was performed on thermal stability of mesophilic and thermophilic α-amylases, and the effects of various denaturing agents, organic solvents, and stabilizers were investigated. As expected, the thermophilic enzyme showed higher resistance toward denaturation in water as its natural medium, but such a difference could not be detected in nonaqueous environments. Furthermore, stability of these molecules was improved by including various stabilizing agents. Of the compounds tested, sorbitol provided the highest degree of protection, which was found to be owing to its effect on increasing T m and its ability in totally preventing deamidation of amino acid residues in the protein molecules.
Keywords: α-Amylase; thermostability; deamidation; organic solvent; thermostabilizing additives

The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion of GOD excreted into the culture fluid reached similar high levels (≅ 90%) by using either glucose or xylose as substrate, whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD.
Keywords: Aspergillus niger ; glucose oxidase; gpdA-promoter

Nonproteolytic cleavage of aspartyl proline bonds in the cellulosomal scaffoldin subunit from Clostridium thermocellum by Raphael Lamed; Rina Kenig; Ely Morag; Sima Yaron; Yuval Shoham; Edward A. Bayer (67-73).
Previous work from our group [Morag (Morgenstern), E., Bayer, E. A., and Lamed, R. (1991), Appl. Biochem. Biotechnol. 30, 129–136] has demonstrated an anomalous electrophoretic mobility pattern for scaffoldin, the 210-kDa cellulosome-integrating subunit of Clostridium thermocellum. Subsequent evidence [Morag, E., Bayer, E. A., and Lamed, R. (1992), Appl. Biochem. Biotechnol. 33, 205–217] indicated that the effect could be attributed to a nonproteolytic fragmentation of the subunit into a defined series of lowermolecular-weight bands. In the present work, a recombinant segment of the scaffoldin subunit was employed to determine the site(s) of bond breakage. An Asp-Pro sequence within the cohesin domain was identified to be the sensitive peptide bond. This sequence appears quite frequently in the large cellulosomal proteins, and the labile bond may be related to an as yet undescribed physiological role in the hydrolysis of cellulose by cellulosomes.
Keywords: Cellulosome; multienzyme complex; cohesin domain; Asp-Pro bond; cellulases; Clostridium thermocellum

Quaternized wood as sorbent for hexavalent chromium by Kun-She Low; Chnoong-Kheng Lee; Chun-Yuan Lee (75-87).
The potential of quaternized wood (QW) chips in removing hexavalent chromium from synthetic solution and chrome waste under both batch and continuous-flow conditions was investigated. Sorption was found to be dependent on pH, metal concentration, and temperature. QW chips provide higher sorption capacity and wider pH range compared with untreated wood chips. The equilibrium data could be fitted into the Langmuir isotherm model, and maximum sorption capacities were calculated to be 27.03 and 25.77 mg/g in synthetic chromate solution and chrome waste, respectively. The presence of sulfate in high concentration appeared to suppress the uptake of chromium by QW chips. Column studies showed that bed depth influenced the breakthrough time greatly whereas flow rate of influent had little effect on its sorption on the column.
Keywords: Wood; chemical modification; biosorption; hexavalent chromium