Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.134, #1)

Optimization of an innovative hollow-fiber process to produce lactose-reduced skim milk by Winfried Neuhaus; Senad Novalin; Mario Klimacek; Barbara Splechtna; Inge Petzelbauer; Alexander Szivak; Klaus D. Kulbe (pp. 1-14).

The research field for applications of lactose hydrolysis has been investigated for several decades. Lactose intolerance, improvement for technical processing of solutions containing lactose, and utilization of lactose in whey are the main topics for development of biotechnological processes. We report here the optimization of a hollow-fiber membrane reactor process for enzymatic lactose hydrolysis. Lactase was circulated abluminally during luminal flow of skim milk. The main problem, the growth of microorganisms in the enzyme solution, was minimized by sterile filtration, ultraviolet irradiation, and temperature adjustment. Based on previous experiments at 23±2°C, further characterization was carried out at 8±2°C, 15±2°C (β-galactosidase), and 58±2°C (thermostable β-glycosidase) varying enzyme activity and flow rates. For a cost-effective process, the parameters 15±2°C, 240 U/mL of β-galactosidase, an enzyme solution flow rate of 25 L/h, and a skim milk flow rate of about 9 L/h should be used in order to achieve an aimed productivity of 360 g/(L·h) and to run at conditions for the highest process long-term stability.

Keywords: Lactose hydrolysis; hollow-fiber module; β-galactosidase; thermostable; CelB β-glycosidase; diffusional reactor

Enhancement of acid tolerance in Zymomonas mobilis by a proton-buffering peptide by David J. Baumler; Kai F. Hung; Jeffrey L. Bose; Boris M. Vykhodets; Chorng M. Cheng; Kwang-Cheol Jeong; Charles W. Kaspar (pp. 15-26).

A portion of the cbpA gene from Escherichia coli K-12 encoding a 24 amino acid proton-buffering peptide (Pbp) was cloned via the shuttle vector pJB99 into E. coli JM105 and subsequently into Zymomonas mobilis CP4. Expression of Pbp was confirmed in both JM105 and CP4 by HPLC. Z. mobilis CP4 carrying pJB99-2 (Pbp) exhibited increased acid tolerance (p<0.05) in acidified TSB (HCl [pH 3.0] or acetic acid [pH 3.5]), glycine-HCl buffer (pH 3.0), and sodium acetate-acetic acid buffer (pH 3.5) in comparison to the parent strain (CP4) and CP4 with pJB99 (control plasmid). Although the expression of Pbp influenced survival at a low pH, the minimum growth pH was unaffected. Growth of Z. mobilis in the presence of ampicillin also significantly increased acid tolerance by an unknown mechanism. Results from this study demonstrate that the production of a peptide with a high proportion of basic amino acids can contribute to protection from low pH and weak organic acids such as acetic acid.

Keywords: Z. mobilis ; acid tolerance; CbpA; ampicillin; pH homeostasis

Simulaaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration by Rui M. F. Bezerra; Albino A. Dias; Irene Fraga; António Nazaré Pereira (pp. 27-38).

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K m=21 mM, K ic=0.035 mM; K icl=1.5×10¹⁵mM; k _cat=12 h⁻¹) or assuming variable substrate (K m=16 mM, K ic=0.037 mM; K icl=5.8×10¹⁴ mM; k _cat=9 h⁻¹), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K icl. In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.

Keywords: Cellulase kinetics; ethanol inhibition; exoglucanase Cel7A; integrated Michaelis-Menten equations

Effect of lipase immobilization on resolution of (R, S)-2-octanol in nonaqueous media using modified ultrastable-Y molecular sieve as support by Dazhang Dai; Liming Xia (pp. 39-50).

The lipase from Penicillium expansum PED-03 (PEL) was immobilized onto modified ultrastable-Y (USY) molecular sieve and the resolution of (R, S)-2-octanol was carried out in a bioreactor in nonaqueous media by the immobilized lipase. It was found that the conversion rate, enantiomeric excess (ee) value, and enantioselectivity (E) value of the resolution catalyzed by PEL immobilized on modified USY molecular sieve were much higher than those of the reaction catalyzed by free PEL and PEL immobilized on other supports. Immobilized on modified USY molecular sieve, the PEL exhibited obvious activity within a wider pH range and at a much higher temperature and showed a markedly enhanced stability against thermal inactivation, by which the suitable pH of the buffer used for immobilization could be “memorized.” The conversion rate of the reaction catalyzed by PEL immobilized on modified USY molecular sieve reached 48.84%, with excellent enantio-selectivity (avarege E value of eight batches >460) in nonaqueous media at “memorial” pH 9.5, 50°C for 24 h, demonstrating a good application potential in the production of optically pure (R, S)-2-octanol.

Keywords: Modified ultrastable-Y molecular sieve; immobilized lipase; resolution; 2-octanol; catalytic property

A comparative study on biosorption characteristics of certain fungi for bromophenol blue dye by Youssef Zeroual; Beom Su Kim; Choel Sang Kim; Mohamed Blaghen; Kang Min Lee (pp. 51-60).

Laboratory investigations of the potential use of dried biomasses of Rhizopus stolonifer, Fusarium sp., Geotrichum sp., and Aspergillus fumigatus as biosorbents for the removal of bromophenol blue (BPB) dye from aqueous solutions were conducted. Kinetics studies indicated that the BPB dye uptake processes can be well described by the pseudo-second-order model. The fungal biomasses exhibited the highest dye biosorption at pH 2.0. The Langmuir adsorption model appears to fit the dye biosorption better than the Freundlich model, with maximum dye uptake capacities ranging from 526 to 1111 mg/g, depending on the biomass used.

Keywords: Biosorption; bromophenol blue; fungi; isotherms; kinetics

Proliferation, viability, and metabolism of human tumor and normal cells cultured in microcapsule by Xulang Zhang; Wei Wang; Yubing Xie; Ying Zhang; Xiuli Wang; Xin Guo; Xiaojun Ma (pp. 61-76).

In this study, we investigated the effect of the microenvironment provided by alginate-poly-l-lysine-alginate (APA) microcapsule with liquefied or gelled core on the proliferation, viability, and metabolism of human cells, including anchorage-dependent MCF-7 breast cancer cells and primary fibroblasts, and anchorage-independent K-562 leukemia cells; cells in conventional culture were used as control. The growth pattern of cells in microcapsule was examined by phase-contrast micrography. The cell viability, proliferation, organization, and gene expression were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, hematoxylin and eosin staining, live/dead staining, 5-bromo-20-deoxyuridine labeling, and immunohistochemistry, respectively. Cell metabolism was determined by measuring glucose and lactate concentrations in medium. The results demonstrate that APA microcapsule with liquefied core provides a microenvironment for both anchorage-dependent and anchorage-independent cells to grow into a large cell aggregate and maintain cell viability at a constant level for a period of time. In conclusion, cells in APA microcapsule are alive and have proliferation potential with lower metabolism rate. APA microcapsule may be a useful tool for in vitro tumor cell modeling and anticancer drug screening as well as for cancer gene therapy. In addition, it lays a solid foundation for the use of microencapsulation in cell culture in vitro and cell implantation in vivo.

Keywords: Alginate; microcapsule; cell encapsulation; poly-l-lysine; tumor cell

Thiol-dependent serine alkaline proteases from Bacillus sp. HR-08 and KR-8102 by Fatemeh Moradian; Khosro Khajeh; Hossein Naderi-Manesh; Rahim Ahmadvand; Reza H. Sajedi; Majid Sadeghizadeh (pp. 77-87).

Two Bacillus sp. strains, HR-08 and KR-8102, isolated from soil of the west and north parts of Iran were screened on gelatin agar medium for their ability to produce alkaline protease. The enzymes were active in a wide pH range (6.0–11.0) and stable in the alkaline range (7.0–12.0). The optimum temperatures for the protease from HR-08 and KR-8102 were 65 and 50°C, respectively. The irreversible thermoinactivation of HR-08 and KR-8102 proteases showed that the stability of HR-08 enzyme was higher than that of KR-8102 and the half-lives of these enzymes were 95 and 32 min at 50°C, respectively. In the presence of 10 mM Ca²⁺, HR-08 retained 100, 90, and 20% of its initial activity after heating for 30 min at 50, 60, and 70°C, respectively. Enzymes were inhibited by phenylmethylsulfonyl fluoride and iodoacetate. After inhibition by iodoacetate, both enzymes were reactivated by dithiothreitol. These data show that the enzymes seem to be thiol-dependent serine alkaline proteases. The enzymes especially from HR-08 were stable in the presence of H₂O₂, surfactants, and local detergents; their activities were enhanced in the presence of 5 mM Fe²⁺; and the presence of 5mM metal ions such as Mg²⁺, Cu²⁺, and Mn²⁺ produced almost no effect.

Keywords: Proteolytic enzyme; thermostability; thiol-dependent protease; commercial detergent; inhibition

Methyl jasmonate stimulates jaceosidin and hispidulin production in cell cultures of Saussurea medusa by Chun-Xiang Fu; Li-Qin Cheng; Xiao-Fen Lv; De-Xiu Zhao; Fengshan Ma (pp. 89-96).

Cell cultures of Saussurea medusa produce valuable secondary metabolites, and jaceosidin and hispidulin are the major bioactive compounds. In the present study, the cultures were challenged by methyl jasmonate (MJ). The highest jaceosidin and hispidulin concentrations (65.2±3.67 mg/L and 12.3±0.47 mg/L) were achieved with 5 μM MJ added to 9-d-old subcultures, being 2.2-fold and 4.2-fold, respectively, higher than those from controls. The elicitor had little influence on cell growth, indicating that the changed biological processes did not include alterations in cell division. Furthermore, we observed that the activities of phenylalanine ammonia lyase were transiently increased after treatment with MJ, which suggests that this elicitor modifies jaceosidin and hispidulin production by regulating the phenylpropanoid pathway.

Keywords: Jaceosidin; hispidulin; methyl jasmonate; phenylalanine ammonia lyase; Saussurea medusa

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.134, #1)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.134, #1)