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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.142, #1)

Effect of Flow Characteristics on Online Sterilization of Cheese Whey in UV Reactors by J. P. Singh; A. E. Ghaly (pp. 1-16).

An ultraviolet (UV) coil reactor was designed and used for the online sterilization of cheese whey. Its microbial destruction efficiency was compared to that of the conventionally used UV reactor. Both reactors have the same geometry (840 ml volume and 17 mm gap size) and were tested at 11 flow rates of 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, and 70 ml/min. The results obtained from this study showed that despite of its high turbidity, cheese whey could be sterilized using UV radiation if the proper reactor design and flow rate are used. The performances of the UV reactors were governed by the flow rate and the hydraulics of flow inside the reactor. The flow was laminar in both the reactors, as the Reynolds number was in the range of 1.39–20.10. The phenomenon of Dean Flow was observed in the coil reactor and the Dean number was in the range of 1.09–15.41. Dean vortices resulted in higher microbial destruction efficiency in the coil reactor in a shorter retention time. The rate of microbial destruction was found to be exponential in the conventional reactor and polynomial in the coil reactor. Increasing the flow rate from 5 ml/min to 70 ml/min decreased the microbial destruction efficiency of the conventional reactor from 99.40 to 31.58%, while the microbial destruction efficiency in the coil reactor increased from 60.77% at the flow rate of 5 ml/min to 99.98% at the flow rate of 30 ml/min and then decreased with further increases in flow rate reaching 46.2% at the flow rate of 70 ml/min. The maximum effluent temperatures in the conventional and coil reactors were 45.8 and 46.1°C, respectively. Fouling in the coil reactor was significantly less compared to the conventional reactor. The extent of fouling was influenced by flow rate and reactor’s hydraulics.

Keywords: Cheese whey; Sterilization; Ultraviolet radiation; Flow rate

Optimisation of Hydrocortisone Production by Curvularia lunata by Wenyu Lu; Lianxiang Du; Min Wang; Xiaoqiang Jia; Jianping Wen; Yuping Huang; Yawen Guo; Wei Gong; Huike Bao; Jing Yang; Bing Sun (pp. 17-28).

A new method for breeding the hydrocortisone overproducing strain Curvularia lunata by screening ketoconazole-resistance mutant was developed. A hydrocortisone overproducing mutant C. lunata KA-91 with ketoconazole-resistance marker was obtained from protoplasts treated with ultraviolet radiation. The hydrocortisone conversion rate of C. lunata KA-91 was increased by 42.1% compared to the original strain CL-114 at the substrate 17α-hydroxypregn-4-en-3, 20-dione-21-acetate addition concentration of 1.0 g/L. The by-products produced by KA-91 were fewer than those of the original strain. It was assumed that the higher cytochrome P450 content of ketoconazole-resistance mutant resulted in the increase of 11β-hydroxylation capacity. The culture conditions for biotransformation of 17α-hydroxypregn-4-en-3, 20-dione-21-acetate to hydrocortisone were optimized by response surface methodology. Plackett–Burman design was applied to elucidate the key factors affecting the hydrocortisone production, and the results indicated that glucose, initial pH, and glucose to total nitrogen sources ratio (ω) had significant effects on hydrocortisone production. Box–Behnken design was employed to search for the optimal parameters of those three key factors. According to the model, the trial checking at the optimal conditions showed a high hydrocortisone conversion rate of 82.67%.

Keywords: Curvularia lunata ; Cytochrome P450; Hydrocortisone; Ketoconazole-resistance screening; Optimization; Response surface methodology

Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism by Dimitris G. Hatzinikolaou; Diomi Mamma; Paul Christakopoulos; Dimitris Kekos (pp. 29-43).

Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOX_I) and the extracellular medium (GOX_II) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOX_I and GOX_II, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent K _m values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOX_I and GOX_II, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOX_II proved to be more stable than GOX_I at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOX_I at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOX_II were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOX_II was also more thermostable than GOX_I as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOX_I and GOX_II, at 45°C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX.

Keywords: Glucose oxidase isoform; Aspergillus niger BTL; GOX_I ; GOX_II

Removal of Nitrogen and Organic Matter in a Radial-Flow Aerobic-Anoxic Immobilized Biomass Reactor Used in the Posttreatment of Anaerobically Treated Effluent by Ajadir Fazolo; Eugenio Foresti; Marcelo Zaiat (pp. 44-51).

This work reports on the removal of organic matter and nitrogen in a radial-flow aerobic-anoxic immobilized biomass (RAIB) reactor fed with domestic sewage pretreated in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor. Polyurethane foam was used as support material for biomass attachment in both reactors. In batch experiments, a first-order kinetic model with residual concentration represented the organic matter removal rate, whereas nitrogen conversion followed a pseudo-first-order reaction in series model, with kinetic constants k ₁ (ammonium to nitrite) and k ₂ (nitrite to nitrate) of 0.25 and 6.62 h⁻¹, respectively. The RAIB reactor was operated in continuous-flow mode and changes in the airflow rate and hydraulic retention time were found to interfere in the apparent kinetic constants to the nitritation (k ₁) and nitratation (k ₂). Nitrification and denitrification were achieved in the partially aerated RAIB reactor operating with hydraulic retention times of 3.3 h and 2.7 h in the aerobic and anoxic zones, respectively. Ethanol was added in the anoxic zone of the reactor to promote denitrification. The effluent flow of the RAIB reactor presented a COD of 52 mg l⁻¹, and concentrations of 2 mg $$ { ext{N}} - { ext{NH}}^{ + }_{4} { ext{ l}}^{{ - 1}} $$ , 1.24 mg $$ { ext{N}} - { ext{NO}}^{ - }_{2} { ext{ l}}^{{ - 1}} $$ and 3.46 mg $$ { ext{N}} - { ext{NO}}^{ - }_{3} { ext{ l}}^{{ - 1}} $$ .

Keywords: Nitrification; Denitrification; Domestic sewage; Anaerobic treatment; Posttreatment

Combinational Biosynthesis of a Fluorescent Cyanobacterial Holo-α-Phycocyanin in Escherichia coli by Using One Expression Vector by Xiangyu Guan; Song Qin; Zhongliang Su; Fangqing Zhao; Baosheng Ge; Fuchao Li; Xuexi Tang (pp. 52-59).

The phycobiliproteins (PBSs) are large pigment proteins found in certain algae that play a central role in harvesting light energy for photosynthesis. Phycocyanin (PC) is one type of PBSs that gains increasing attention owing to its various biological and pharmacological properties. In this paper, an expression vector containing five essential genes in charge of biosynthesis of cyanobacterial C-phycocyanin (C-PC) holo-α subunit (holo-CpcA) was successfully constructed resulting in over-expression of a fluorescent holo-CpcA in E. coli BL21. The vector harbored two cassettes: one cassette carried genes hox1 and pcyA required for conversion of heme to phycocyanobilin (PCB), and the other cassette carried cpcA encoding CpcA along with cpcE and cpcF both of which were necessary and sufficient for the correct addition of PCB to CpcA. The vector system contained a His-tag for protein purification. The purified protein showed correct molecular weight on SDS-PAGE gel and emitted orange fluorescence by UV excitation. The maximum peak of absorbance spectrum was at 623 nm, and the maximum peak of fluorescence emission and excitation were at 648 and 633 nm, respectively, which were similar to those of native C-PC. This study provides an efficient method for large-scale production of the fluorescent holo-CpcA in biotechnological applications.

Keywords: Escherichia coli ; Phycocyanin; Fluorescence; Holo-HT-CpcA

Improved Cellulase Production by Trichoderma reesei RUT C30 under SSF Through Process Optimization by Reeta Rani Singhania; Rajeev K. Sukumaran; Ashok Pandey (pp. 60-70).

The major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which includes the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). The current study investigated the production of cellulase by Trichoderma reesei RUT C30 on wheat bran under SSF. Process parameters important in cellulase production were identified by a Plackett and Burman design and the parameters with significant effects on enzyme production were optimized for maximal yield using a central composite rotary design (CCD). Higher initial moisture content of the medium had a negative effect on production whereas incubation temperature influenced cellulase production positively in the tested range. Optimization of the levels of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold increase in production from 0.605 to 3.8 U/gds of cellulase. The optimal combination of moisture and temperature was found to be 37.56% and 30 °C, respectively, for maximal cellulase production by the fungus on wheat bran.

Keywords: Cellulase; Trichoderma reesei ; Solid state fermentation; Wheat bran; Plackett and Burman; Central composite design

Microencapsulated Engineered Lactococcus lactis Cells for Heterologous Protein Delivery: Preparation and In Vitro Analysis by Bisi Lawuyi; Hongmei Chen; Fatemeh Afkhami; Arun Kulamarva; Satya Prakash (pp. 71-80).

This article demonstrates the potential of encapsulated, engineered Lactococcus lactis as a vehicle for the oral delivery of therapeutic proteins. Using alginate-poly-l-lysine-alginate membrane-encapsulated L. lactis engineered to secrete the reporter protein Staphylococcal aureus nuclease, we show comparable viability and protein secretion between free and immobilized cells. After 12 h, microcapsules with a cell density of 4.8 × 10⁵ colony forming unit (CFU) ml⁻¹ grew to 2.2 × 10⁸ CFU ml⁻¹ and released 0.24 arbitrary unit (AU) ml⁻¹ of nuclease, producing similar results as free cells, which grew from 3.4 × 10⁵ to 1.9 × 10⁸ CFU ml⁻¹ and secreted 0.21 AU ml⁻¹ of nuclease. Moreover, encapsulated cells at a density of 4.4 × 10⁷ CFU ml⁻¹ grew to 2.2 × 10¹⁰ CFU ml⁻¹ in 12 h and secreted 15.3 AU ml⁻¹ of nuclease although 3.1 × 10⁷ CFU ml⁻¹ of free cells reached only 2.3 × 10⁹ CFU ml⁻¹ and released 5.6 AU ml⁻¹ of nuclease. We also show the sustained stability of the microcapsules during storage at 4°C over 8 weeks.

Keywords: Lactococcus lactis ; Live cell therapy; Microencapsulation; Protein secretion; Staphylococcal nuclease

Overexpression of an Endochitinase Gene (ThEn-42) in Trichoderma atroviride for Increased Production of Antifungal Enzymes and Enhanced Antagonist Action Against Pathogenic Fungi by Shiping Deng; Matteo Lorito; Merja Penttilä; Gary E. Harman (pp. 81-94).

Trichoderma is one of the most promising biocontrol agents against plant fungal diseases. In this study, a transgenic strain of Trichoderma atroviride was characterized. The transgenic strain contains an endochitinase gene (ThEn-42) driven by the cellulase promoter cbh1 of T. reesei for overexpression of ThEn-42. The culture filtrates of the transformant and the parental strain grown in eight different media were evaluated for chitinase and antifungal enzyme production based on activity gels, protein profiles, and antifungal activities. Results demonstrated that chitinases are important components and synergistic interactions play a key role in the antagonistic action of T. atroviride. Moreover, altering medium nutrient concentration and composition led to enhanced production of antifungal enzymes, a potential strategy for mass production. Two of the culture filtrates contained almost pure endochitinase, and could be excellent commercial sources for this enzyme. Several culture filtrates were highly antifungal. Two filtrates were so effective in biocontrol of a fungal pathogen, Penicillium digitatum, that they not only inhibited spore germination but destroyed the spores completely when 20 μl of culture filtrate (corresponding to approximately 104 μg of total protein) was applied in a total volume of 150 μl (approximately 0.7 mg protein ml⁻¹).

Keywords: Biocontrol; Chitinase; Gene expression; Synergistic interaction; Trichoderma spp

Evaluation of Enzymatic Reactors for Large-Scale Panose Production by Fabiano A. N. Fernandes; Sueli Rodrigues (pp. 95-104).

Panose is a trisaccharide constituted by a maltose molecule bonded to a glucose molecule by an α-1,6-glycosidic bond. This trisaccharide has potential to be used in the food industry as a noncariogenic sweetener, as the oral flora does not ferment it. Panose can also be considered prebiotic for stimulating the growth of benefic microorganisms, such as lactobacillus and bidifidobacteria, and for inhibiting the growth of undesired microorganisms such as E. coli and Samonella. In this paper, the production of panose by enzymatic synthesis in a batch and a fed-batch reactor was optimized using a mathematical model developed to simulate the process. Results show that optimum production is obtained in a fed-batch process with an optimum production of 11.23 g/l h of panose, which is 51.5% higher than production with batch reactor.

Keywords: Optimization; Panose; Enzymatic synthesis

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.142, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.142, #1)