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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #2)

Reactive Extraction of Citric Acid Using Tri-n-octylamine in Nontoxic Natural Diluents: Part 1—Equilibrium Studies from Aqueous Solutions by Amit Keshav; Prakriti Norge; Kailas L. Wasewar (pp. 197-213).

Use of cheap, nontoxic, and selective solvents could economically provide a solution to the recovery of carboxylic acids produced by the bioroute. In this regard in the present paper, reactive extraction of citric acid was studied. Problems encompassing the recovery of the acid ([H₃A] _aq ^o = 0.1–0.8) was solved by using tertiary amine (tri-n-octylamine, TOA) in natural diluents (rice bran oil, sunflower oil, soybean oil, and sesame oil). TOA was very effective in removal of acid providing distribution coefficient (D) as high as 18.51 (E% = 95 %), 12.82 (E% = 93 %), 15.09 (E% = 94 %), and 16.28 (E% = 94 %) when used with rice bran oil, sunflower oil, soybean oil, and sesame oil, respectively. Overall extraction constants and association numbers for TOA + rice bran oil, TOA + sunflower oil, TOA + soybean oil, and TOA + sesame oil were evaluated to be 35.48 (mol/l)^−1.46, 29.79 (mol/l)^−1.30, 33.79 (mol/l)^−1.51, and 37.64 (mol/l)^−1.65 and 1.46, 1.30, 1.51, and 1.65, respectively. Specific equilibrium complexation constants (K E(n/m₎) were also predicted using mathematical modeling.

Keywords: Reactive extraction; Citric acid; Tri-n-octylamine; Natural diluents; Toxicity

Novel Fungal Pelletization-Assisted Technology for Algae Harvesting and Wastewater Treatment by Wenguang Zhou; Yanling Cheng; Yun Li; Yiqin Wan; Yuhuan Liu; Xiangyang Lin; Roger Ruan (pp. 214-228).

A novel fungi pelletization-assisted bioflocculation technology was developed for efficient algae harvesting and wastewater treatment. Microalga Chlorella vulgaris UMN235 and two locally isolated fungal species Aspergillus sp. UMN F01 and UMN F02 were used to study the effect of various cultural conditions on pelletization process for fungi–algae complex. The results showed that pH was the key factor affecting formation of fungi–algae pellet, and pH could be controlled by adjusting glucose concentration and fungal spore number added. The best pelletization happened when adding 20 g/L glucose and approximately 1.2E8/L spores in BG-11 medium, under which almost 100 % of algal cells were captured onto the pellets with shorter retention time. The fungi–algae pellets can be easily harvested by simple filtration due to its large size (2–5 mm). The filtered fungi–algae pellets were reused as immobilized cells for treatment wastewaters and the nutrient removal rates of 100, 58.85, 89.83, and 62.53 % (for centrate) and 23.23, 44.68, 84.70, and 70.34 % (for diluted swine manure wastewater) for ammonium, total nitrogen, total phosphorus, and chemical oxygen demand, respectively, under both 1- and 2-day cultivations. The novel technology developed is highly promising compared with current algae harvesting and biological wastewater treatment technologies in the literature.

Keywords: Fungi pelletization; Microalgae algae harvesting; Municipal wastewater; Animal wastewater; Wastewater treatment

Antioxidant Effect of Azadirachta Indica on High Fat Diet Induced Diabetic Charles Foster Rats by Atul Shrivastava; Upma Chaturvedi; Ravi Sonkar; Ashok Kumar Khanna; J. K. Saxena; Gitika Bhatia (pp. 229-236).

Oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. Excessively high levels of free radicals cause damage to cellular proteins, membrane lipids and nucleic acids, and eventually cell death. The present study was designed to investigate the possible effect of Azadirachta indica leaf extract in high fat diet induced diabetic Charles Foster rats. The increased level of lipidperoxidation and altered levels of enzymatic (superoxide dismutase, glutathione peroxidase and catalase) and non-enzymatic (glutathione) antioxidants were seen in high fructose fed animals. The treatment with A. indica leaf extract significantly normalized the altered levels of lipid peroxidation and antioxidant status at 400 mg/kg b.w. dose. The A. indica leaf extract was also tested for in vitro inhibition of generation of superoxide anion and hydroxyl free radical in both enzymatic and non-enzymatic systems. The A. indica leaf extract was found to inhibit generation of superoxide anion and hydroxyl free radical significantly at 200 μg/ml concentration. Data of present study demonstrated that the A. indica leaf extract has both antidiabetic and antioxidant properties.

Keywords: Oxidative stress; Antioxidant; Superoxide dismutase; Glutathione peroxidase; Catalase; Glutathione; Lipid peroxidation; Azadirachta indica

Exploring the Cause of Oseltamivir Resistance Against Mutant H274Y Neuraminidase by Molecular Simulation Approach by V. Karthick; V. Shanthi; R. Rajasekaran; K. Ramanathan (pp. 237-249).

Oseltamivir (Tamiflu) is the preferred anti-viral drug employed to fight the flu virus in infected individuals. The principal target for this drug is a virus surface glycoprotein, neuraminidase (NA), which facilitates the release of nascent virus and thus spreads infections. Until recently, only a low prevalence of neuraminidase inhibitors (NAIs) resistance (<1 %) had been detected in circulating viruses. However, there have been reports of significant numbers of A (H1N1) influenza strains with a H274Y neuraminidase mutation that was highly resistant to the NAI, oseltamivir. In this study, we highlight the effect of point mutation-induced oseltamivir resistance in H1N1 subtype neuraminidases by molecular docking and molecular dynamics simulation approach. Our results suggested that wild-type NA could be more indispensable for the oseltamivir binding, as characterized by minimum number of H-bonds, high flexibility and largest binding affinity than mutant-type NA. This study throws light on the possible effects of drug-resistant mutations on the large functionally important collective motions in biological systems.

Keywords: Neuraminidase; Oseltamivir resistance; Molecular docking; Normal mode analysis; Molecular dynamic simulation

Selective Liquefaction of Wheat Straw in Phenol and Its Fractionation by Hongzhang Chen; Yuzhen Zhang; Shuangping Xie (pp. 250-258).

For the first time, a method of phenol-selective liquefaction is proposed for the fractionation and multilevel conversion of lignocellulose. Through phenol-selective liquefaction, lignin and hemicellulose are liquefied, with large amounts of cellulose retained in the unliquefied residues. Using a phenol/straw ratio of 3 and a sulfuric acid concentration of 3 %, large amounts of hemicellulose (≥85 %) and lignin (≥70 %) can be liquefied at 100 °C in 30 min, with a high quantity of cellulose (≥80 %) retained. Unliquefied residues from selective liquefaction have higher susceptibility for enzymatic attack. Enzymatic hydrolyzation of residues can be as high as 65 % in 48 h with 40.7 FPU/g of dry materials, which can then be used to prepare sugar platform intermediates. The liquefied products of wheat straw are then resinified with formaldehyde in the presence of NaOH as a catalyst and synthesized into phenol formaldehyde-type resins reaching up to GB/T 14732-2006 standards. Phenol selective liquefaction, a new technology for the fractionation of lignocellulose, achieves effective fractionation and multilevel conversion of straw components. Hence, it is an important tool to achieve full utilization of biomass and high value-added conversion of lignocellulose.

Keywords: Selective liquefaction; Fractionation; Biomass; Resin adhesive; Enzymatic hydrolysis

Immunogenicity of Japanese Encephalitis Virus Envelope Protein by Hyphantria cunea Nuclear Polyhedrosis Virus Vector in Guinea Pig by Hyung-Hoan Lee; Seung-Kuk Hong; Sang-Ho Yoon; Sung-Jae Jang; Young-Yil Bahk; Min-Dong Song; Pyo-Jam Park; Kwang-Ho Lee; Chan-Gil Kim; Bokyung Kim; Tae-Kyu Park; Hyun Kang (pp. 259-269).

Japanese encephalitis virus (JEV) is an important pathogen causing febrile syndrome, encephalitis, and death. Envelop (E) glycoprotein is the major target of inducing neutralizing antibodies and protective immunity in host. In this study, E glycoprotein of JEV was expressed in Spodoptera frugiperd 9 cells as a fusion protein containing a gX signal sequence of pseudorabies virus. This purified HcE recombinant protein was evaluated for their immunogenicity and protective efficacy in guinea pig. The survival rates of guinea pig immunized with HcE protein was significantly increased over that of JE vaccine. This result indicates helpful information for developing a subunit vaccine against JEV.

Keywords: Hyphantria cunea envelope (HcE); Japanese encephalitis virus (JEV); Envelop (E) protein; Protective immunity

Isolation, Screening, and Optimization of the Fermentation Conditions of Highly Cellulolytic Bacteria from the Hindgut of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae) by Ping Sheng; Shengwei Huang; Qi Wang; Ailing Wang; Hongyu Zhang (pp. 270-284).

From the hindgut contents of Holotrichia parallela, 93 cellulolytic bacterial isolates were isolated after enrichment in carboxymethyl cellulose medium. Among these isolates, a novel bacterium, designated HP207, with the highest endoglucanase productivity was selected for further study. This bacterium was identified as Pseudomonas sp. based on the results of the 16S ribosomal DNA analysis, morphological characteristics, and biochemical properties. The production of the endoglucanase was optimized by varying various physical culture conditions using a submerged fermentation method. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.432 U mL⁻¹ in bacterial cultures was obtained, higher than those of the most widely studied bacteria and fungi, which are the attractive candidates for the commercial producer of cellulase. And the crude endoglucanase enzyme was also highly thermostable; approximately 55 % of the original activity was maintained after pretreatment at 70 °C for 1 h. Thus, from the present study, the bacterium can be added up to the database of cellulolytic bacteria.

Keywords: Holotrichia parallela ; Cellulolytic bacteria; Pseudomonas sp. HP207; Endoglucanase

Effect of Acid, Steam Explosion, and Size Reduction Pretreatments on Bio-oil Production from Sweetgum, Switchgrass, and Corn Stover by Hui Wang; Radhakrishnan Srinivasan; Fei Yu; Philip Steele; Qi Li; Brian Mitchell; Aditya Samala (pp. 285-297).

Bio-oil produced from biomass by fast pyrolysis has the potential to be a valuable substitute for fossil fuels. In a recent work on pinewood, we found that pretreatment alters the structure and chemical composition of biomass, which influence fast pyrolysis. In this study, we evaluated dilute acid, steam explosion, and size reduction pretreatments on sweetgum, switchgrass, and corn stover feedstocks. Bio-oils were produced from untreated and pretreated feedstocks in an auger reactor at 450 °C. The bio-oil’s physical properties of pH, water content, acid value, density, and viscosity were measured. The chemical characteristics of the bio-oils were determined by gas chromatography–mass spectrometry. The results showed that bio-oil yield and composition were influenced by the pretreatment method and feedstock type. Bio-oil yields of 52, 33, and 35 wt% were obtained from medium-sized (0.68–1.532 mm) untreated sweetgum, switchgrass, and corn stover, respectively, which were higher than the yields from other sizes. Bio-oil yields of 56, 46, and 51 wt% were obtained from 1 % H₂SO₄-treated medium-sized sweetgum, switchgrass, and corn stover, respectively, which were higher than the yields from untreated and steam explosion treatments.

Keywords: Pretreatment; Biomass; Pyrolysis; Sweetgum; Corn stover; Switchgrass

A Study on the Stability and Enzymatic Activity of Yeast Alcohol Dehydrogenase in Presence of the Self-Assembling Block Copolymer Poloxamer 407 by Stefania Pucciarelli; Giulia Bonacucina; Franco Bernabucci; Marco Cespi; Giovanna Mencarelli; Giusi Serena De Fronzo; Paolo Natalini; Giovanni Filippo Palmieri (pp. 298-313).

Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH 9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH 7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.

Keywords: Poloxamer 407; Self-assembly; Alcohol dehydrogenase; Thermal stability; Enzyme catalysis

Environmental Degradation of Microbial Polyhydroxyalkanoates and Oil Palm-Based Composites by Y. S. Salim; A. Sharon; S. Vigneswari; M. N. Mohamad Ibrahim; A. A. Amirul (pp. 314-326).

This paper investigates the degradation of polyhydroxyalkanoates and its biofiber composites in both soil and lake environment. Time-dependent changes in the weight loss of films were monitored. The rate of degradation of poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-23 mol% 4HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-9 mol% 3HV-co-19 mol% 4HB)] were investigated. The rate of degradation in the lake is higher compared to that in the soil. The highest rate of degradation in lake environment (15.6 % w/w week⁻¹) was observed with P(3HB-co-3HV-co-4HB) terpolymer. Additionally, the rate of degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-38 mol% 3HV)] was compared to PHBV biofiber composites containing compatibilizers and empty fruit bunch (EFB). Here, composites with 30 % EFB displayed the highest rate of degradation both in the lake (25.6 % w/w week⁻¹) and soil (15.6 % w/w week⁻¹) environment.

Keywords: Polyhydroxyalkanoates (PHAs); Biodegradation; Biocomposites; Environment

Effect of Gold Nanosphere Surface Chemistry on Protein Adsorption and Cell Uptake In Vitro by Amrita Mukhopadhyay; Christin Grabinski; A. R. M. Nabiul Afrooz; Navid B. Saleh; Saber Hussain (pp. 327-337).

Gold nanoparticles exhibit unique spectral properties that make them ideal for biosensing, imaging, drug delivery, and other therapeutic applications. Interaction of gold nanoparticles within biological environments is dependent on surface characteristics, which may rely on particular capping agents. In this study, gold nanospheres (GNS) synthesized with different capping agents—specifically citric acid (CA) and tannic acid (TA)—were compared for serum protein adsorption and cellular uptake into a lung epithelial cell line (A549). Both GNS samples exhibited noticeable protein adsorption based on surface charge data after exposure to serum proteins. Light scattering measurements revealed that GNS-CA-protein composites were smaller and less dense compared to GNS-TA-protein composites. The cell uptake characteristics of these nanoparticles were also different. GNS-CA formed large clusters and elicited high uptake, while GNS-TA were taken up as discrete particles, possibly through nonendosomal mechanisms. These results indicate that the capping agents used for GNS synthesis result in unique biological interactions.

Keywords: Gold nanoparticles; Tannic acid; Citric acid; Surface chemistry; Stabilizing agent; Cell uptake; Protein adsorption

Elimination of Acetate Production to Improve Ethanol Yield During Continuous Synthesis Gas Fermentation by Engineered Biocatalyst Clostridium sp. MTEtOH550 by Vel Berzin; Michael Kiriukhin; Michael Tyurin (pp. 338-347).

Acetogen strain Clostridum sp. MT653 produced acetate 273 mM (p < 0.005) and ethanol 250 mM (p < 0.005) from synthesis gas blend mixture of 64 % CO and 36 % H₂. Clostridum sp. MT653 was metabolically engineered to the biocatalyst strain Clostridium sp. MTEtOH550. The biocatalyst increased ethanol yield to 590 mM with no acetate production during single-stage continuous syngas fermentation due to expression of synthetic adh cloned in a multi-copy number expression vector. The acetate production was eliminated by inactivation of the pta gene in Clostridium sp. MTEtOH550. Gene introduction and gene elimination were achieved only using Syngas Biofuels Energy, Inc. electroporation generator. The electrotransformation efficiencies were 8.0 ± 0.2 × 10⁶ per microgram of transforming DNA of the expression vector at cell viability ~15 %. The frequency of suicidal vector integration to inactivate pta was ~10⁻⁵ per the number of recipient cells. This is the first report on elimination of acetate production and overexpression of synthetic adh gene to engineer acetogen biocatalyst for selective biofuel ethanol production during continuous syngas fermentation.

Keywords: Acetogens; PTA inactivation; Synthetic ADH; Ethanol production; Syngas fermentation; Electrotransformation

Role of Vitamins B-3 and C in the Fashioning of Granules in UASB Reactor Sludge by Tasneem Abbasi; R. Sanjeevi; Manoj Makhija; S. A. Abbasi (pp. 348-357).

Whereas a myriad of possible factors have been reported which effect the formation of granules in a upflow anaerobic sludge blanket (UASB) reactor and influence their properties, there is no study on the effect of vitamins on the granulation of UASB reactor sludge. The present study was undertaken to bridge this gap. It was seen that vitamins helped in better granule formation—as reflected by favorable size distribution, sludge volume index, and settling velocity—compared to controls. The vitamin-spiked reactors also achieved >85 % COD removal efficiency in half the number of days the unspiked reactors took. The vitamin supplements were effective at concentrations ≤ 1 mg/l. Hence, their use in expediting granule formation as also in developing better-quality granules appears economically viable.

Keywords: UASB; Sludge granules; Vitamin B3; Vitamin C; Wastewater

Kinetic Studies on the Product Inhibition of Enzymatic Lignocellulose Hydrolysis by Yelian Miao; Jie Yu Chen; Xuejian Jiang; Zhou Huang (pp. 358-366).

In order to understand the product inhibition of enzymatic lignocellulose hydrolysis, the enzymatic hydrolysis of pretreated rice straw was carried out over an enzyme loading range of 2 to 30 FPU/g substrate, and the inhibition of enzymatic hydrolysis was analyzed kinetically based on the reducing sugars produced. It was shown that glucose, xylose, and arabinose were the main reducing sugar components contained in the hydrolysate. The mass ratio of glucose, xylose, and arabinose to the total reducing sugars was almost constant at 52.0 %, 29.7 % and 18.8 %, respectively, in the enzyme loading range. The reducing sugars exerted competitive inhibitory interferences to the enzymatic hydrolysis. Glucose, xylose, and arabinose had a dissociation constant of 1.24, 0.54 and 0.33 g/l, respectively. The inhibitory interferences by reducing sugars were superimposed on the enzymatic hydrolysis. The enzymatic hydrolysis could be improved by the removal of the produced reducing sugars from hydrolysate.

Keywords: Biomass; Enzymatic hydrolysis; Saccharification; Product inhibition; Kinetics

Purification and Characterization of Peroxidase from Papaya (Carica papaya) Fruit by Veda P. Pandey; Swati Singh; Rupinder Singh; Upendra N. Dwivedi (pp. 367-376).

Ripening of papaya fruit was found to be characterized with a decrease in peroxidase activity and its transcript. This peroxidase was purified to homogeneity through successive steps of ammonium sulfate fractionation, ion exchange and molecular exclusion chromatography. The peroxidase was purified 30.22-folds with overall recovery of 44.37 % and specific activity of 68.59. Purified peroxidase was found to be a heterotrimer of ~240 kDa, containing two subunits each of 85 and one of 70 kDa. Purified enzyme exhibited pH and temperature optima of 7.0 and 40 °C, respectively. K _m values for substrates o-dianicidin, guaiacol and ascorbic acid were found to be 0.125, 0.8 and 5.2 mM, respectively. K _m for H₂O₂ was found to be 0.25 mM. Salicylic acid was found to activate peroxidase up to 50 μM concentration, beyond which it acted as inhibitor. Ca²⁺ and Mg²⁺ activated peroxidase while sodium azide, SDS, and Triton X-100 were found to inhibit peroxidase.

Keywords: Fruit ripening; Peroxidase; Real-time PCR; Papaya; Salicylic acid

Modeling of Pretreatment Condition of Extrusion-Pretreated Prairie Cordgrass and Corn Stover with Poly (Oxyethylen)₂₀ Sorbitan Monolaurate by Anahita Dehkhoda Eckard; Kasiviswanathan Muthukumarappan; William Gibbons (pp. 377-393).

Extrusion processing has shown potential to be used as a pretreatment method for second-generation bioethanol production. Furthermore, surfactants have been shown to reduce enzyme deactivation and increase the efficiency of hydrolysis. Therefore, a sequential pretreatment technique was developed for corn stover (CS) and prairie cordgrass (PCG) in which a single screw extruder was used for the first pretreatment according to a previously optimized condition using 70–180 °C for feed, barrel, and die zones with 65–155 rpm screw speed. The second pretreatment was optimized in this study at 45–55 °C, 1–4 h, 0.15–0.6 g Tween 20/g glucan according to response surface methodology. Optimization of surfactant pretreatment facilitated the estimation of interaction and higher-order effects for major factors involved in surfactant treatment (temperature, time, surfactant loading). Using 8.6 FPU/g glucan cellulase, the optimum conditions found by fitting appropriate quadratic models to the data increased glucose and xylose yield by 27.5 and 33 % for CS and by 21.5 and 27 % for PCG, respectively. Tween 20 concentrations and pretreatment temperature were the most significant factors affecting sugar yield (p value <0.05). Studies of SDS concentration at and beyond critical micelle concentration (5.2–100 mM) demonstrated a decrease in sugar yield compared to control.

Keywords: Enzymatic hydrolysis; Lignocellulose; Pretreatment; SDS; Tween 20; Cellulase; Micelles

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #2)