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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.159, #3)

Environmentally Benign Bioprocesses for Energy and Chemicals Production by Jian-He Xu; Jie Bao; Qing Yang (pp. 589-590).

Screening of Oleaginous Yeast Strains Tolerant to Lignocellulose Degradation Compounds by Xi Chen; Zihui Li; Xiaoxi Zhang; Fengxian Hu; Dewey D. Y. Ryu; Jie Bao (pp. 591-604).

High cost of triacylglycerol lipid feedstock is the major barrier for commercial production of biodiesel. The fermentation of oleaginous yeasts for lipid production using lignocellulose biomass provides a practical option with high economic competitiveness. In this paper, the typical oleaginous yeast strains were screened under the pressure of lignocellulose degradation compounds for selection of the optimal strains tolerant to lignocellulose. The inhibitory effect of lignocellulose degradation products on the oleaginous yeast fermentation was carefully investigated. Preliminary screening was carried out in the minimum nutritious medium without adding any expensive complex ingredients then was carried out in the lignocellulosic hydrolysate pretreated by dilute sulfuric acid. Seven typical lignocellulose degradation products formed in various pretreatment and hydrolysis processing were selected as the model inhibitors, including three organic acids, two furan compounds, and two phenol derivatives. The inhibition of the degradation compounds on the cell growth and lipid productivity of the selected oleaginous yeasts were examined. Acetic acid, formic acid, furfural, and vanillin were found to be the strong inhibitors for the fermentation of oleaginous yeasts, while levulinic acid, 5-hydroxymethylfurfural, and hydroxybenzaldehyde were relatively weak inhibitors. Trichosporon cutaneum 2.1374 was found to be the most adopted strain to the lignocellulose degradation compounds.

Keywords: Triacylglycerol lipid; Oleaginous yeasts; Trichosporon cutaneum ; Screening; Lignocellulose degradation compounds

Efficient 1,3-propanediol Production by Fed-Batch Culture of Klebsiella Pneumoniae: The Role of pH Fluctuation by Xiao-Jun Ji; He Huang; Jian-Guo Zhu; Nan Hu; Shuang Li (pp. 605-613).

The fermentative production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under different fed-batch strategies was investigated. pH-stat fed-batch strategies proved to be not effective for economical 1,3-PD production for the existence of relatively high concentration of byproducts and residual glycerol at the end of the fermentation. However, in the pH-stat fed-batch strategy, an important phenomenon was observed that the yields of two main byproducts, 2,3-butanediol and lactic acid, were closely related to pH value. The dominant byproduct was 2,3-butanediol at a pH value of 5.0 to 6.5 but changed to be lactic acid at a pH value of 7.1 to 8.0. Based on the analysis of the phenomenon, a self-protection mechanism in K. pneumoniae, namely that the growing K. pneumoniae cells switch the metabolic pathways responding to environmental pH changes, was proposed. Thus a kind of feeding strategy was further applied during which the pH value was fluctuated between 6.3 and 7.3 periodically by feeding glycerol–ammonia mixture and sulphuric acid to make the metabolic pathways of 2,3-butanediol and lactic acid sub-active under the periodical low or high pH stress. At last, efficient 1,3-PD production was fulfilled under this fed-batch strategy, and the best results were achieved leading to 70 g/l 1,3-PD with a yield of 0.70 mol/mol glycerol and productivity of 0.97 g/l/h, while the two main byproducts and residual glycerol were under low concentrations.

Keywords: 1,3-propanediol; Klebsiella pneumoniae ; Glycerol; Fed-batch; pH fluctuation

Recombinant Glutamine Synthetase (GS) from C. glutamicum Existed as Both Hexamers & Dedocamers and C-terminal His-tag Enhanced Inclusion Bodies Formation in E. coli by Ye Li; Guang Yang; Xing Huang; Bo Ye; Ming Liu; Zhanglin Lin; Chun Li; Zhu-an Cao (pp. 614-622).

In order to investigate the effect of his-tag on glutamine synthetase (GS, EC 6.3.1.2) from Corynebacterium glutamicum, recombinant Escherichia coli strains overexpressing GSIM, HGSIM (GS fused with N-terminal his-tag), GSIMH (GS fused with C-terminal his-tag), and HGSIMH (GS fused with N-terminal & C-terminal his-tags) were constructed, respectively. Under similar expression conditions, GSIM and HGSIM were partially solubly expressed; no soluble GSIMH and HGSIMH were observed, based on the result of SDS-PAGE. Gel filtration of purified soluble HGSIM showed that hexamers and dedocamers coexisted in the quaternary structure of GS from C. glutamicum. Combined this result with the analysis of two GS crystal structure models, we hypothesized that C-terminal residues participated in GS folding after translation on ribosome. After the folding process, C-terminal residues were released again and exposed to solvent. Fused C-terminal his-tag interrupted the GS to fold into its correct conformation so that inclusion bodies formed.

Keywords: Corynebacterium glutamicum ; Glutamine synthetase; His-tag; Inclusion bodies; Quaternary structure

Biodegradation of Mixed Phenolic Compounds Under High Salt Conditions and Salinity Fluctuations by Arthrobacter sp. W1 by Ping Wang; Yuanyuan Qu; Jiti Zhou (pp. 623-633).

High salt concentration and salinity fluctuations in wastewater challenge the efficiency of microbial strains used for cleanup of pollutants. In this study, it was investigated that the new isolated Arthrobacter sp. W1 degraded mixed phenolic compounds under complex salt conditions. The results showed that Arthrobacter sp. W1 was able to utilize various phenolic compounds as carbon source under high salt conditions. It can degrade phenol and p-cresol mixture at 10% NaCl, although rates of degradation and cell growth were lower compared to 5% NaCl. The presence of trace p-cresol significantly inhibited phenol biodegradation. When salinity fluctuations were between 1% and 10% NaCl, strain W1 was able to degrade substrates and survived. It was also suggested that the presence of salts (i.e., NaCl, KCl, Na₂SO₄, and K₂SO₄) had almost no effects on the microbial growth and biodegradation process. Therefore, Arthrobacter sp. W1 would be a promising candidate for bioremediation of phenolic compounds under complex salt conditions.

Keywords: Biodegradation; Phenolic compounds; Salinity; Arthrobacter sp.

Glycerol-Assisted Hydrophobic Interaction Chromatography Improving Refolding of Recombinant Human Granulocyte Colony-Stimulating Factor by Fangwei Wang; Yongdong Liu; Guanghui Ma; Zhiguo Su (pp. 634-641).

Efficient refolding of recombinant proteins in the forms of inclusion bodies at higher concentration remains challenging. Here, we report a strategy of a dual-gradient hydrophobic interaction chromatography (HIC) mode to refold recombinant human granulocyte colony-stimulating factor from its inclusion bodies at high protein concentration. The strategy was taken to meet the demand of dynamic refolding proceeding by gradually decrease the denaturant (guanidine-HCl) concentration and gradually increase the hydrophilicity of media (column of Poros PE 20) with glycerol as additive to provide a mild refolding surroundings. Compared with dilution method, this dual-gradient HIC process gave about 8.5-fold of increase in specific activity and 30% increase in soluble protein recovery. Furthermore, much higher protein concentration could be obtained at the same time.

Keywords: Hydrophobic interaction chromatography; Refolding; Glycerol; Recombinant human granulocyte colony-stimulating factor

Improvement of Steroid Biotransformation with Hydroxypropyl-β-Cyclodextrin Induced Complexation by Liting Zhang; Min Wang; Yanbing Shen; Yinhu Ma; Jianmei Luo (pp. 642-654).

The inclusion complexes induced by cyclodextrins and its derivates have been shown previously to enhance the biotransformation of hydrophobic compounds. Using hydroxypropyl-β-cyclodextrin (HP-β-CD; 20% w/v), the water solubility of cortisone acetate increased from 0.039 to 7.382 g L⁻¹ at 32 °C. The solubilization effect of HP-β-CD was far superior to dimethylformamide (DMF) and ethanol. The dissolution rate also significantly increased in the presence of HP-β-CD. The enzymatic stability of Δ¹-dehydrogenase from Arthrobacter simplex TCCC 11037 was not influenced by the increasing concentrations of HP-β-CD contrary to the organic cosolvents which negatively influenced in the order DMF > ethanol. The activity inhibition effect caused by HP-β-CD was not so conspicuous as ethanol and DMF. Inactivation constants of ethanol, DMF, and HP-β-CD were 5.832, 4.541, and 1.216, respectively. The inactivation energy (E _a) was in the order of HP-β-CD (55.1 kJ mol⁻¹) > ethanol (39.9 kJ mol⁻¹) > DMF (37.1 kJ mol⁻¹).

Keywords: Hydroxypropyl-β-cyclodextrin; Δ¹-Dehydrogenase; Enzymatic activity; Enzymatic stability; Thermal stability

Enhanced Production of Spinosad in Saccharopolyspora spinosa by Genome Shuffling by Z. H. Jin; B. Xu; S. Z. Lin; Q. C. Jin; P. L. Cen (pp. 655-663).

Spinosad (spinosyns A and D) is a mixture of secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. In this study, we applied genome shuffling of S. spinosa to achieve a rapid improvement of spinosad production. Ten strains with subtle improvements in spinosad production were obtained from the populations generated by the mutation with nitrosoguanidine and ultraviolet irradiation, and then they were subjected for recursive protoplast fusion. After four rounds of genome shuffling, a high yielding strain, designated as S. spinosa 4-7, was successfully isolated. Its production reached 547 mg/L, which was increased by 200.55% and 436.27% in comparison with that of the highest parent strain and the original strain, respectively. The subculture experiments indicated that the high producer of S. spinosa 4-7 was stable. Spinosad fermentation experiments by S. spinosa 4-7 were carried out in a 5-L fermentor, and its production of spinosad reached 428 mg/L after 168 h of fermentation.

Keywords: Genome shuffling; Protoplast fusion; Spinosad; Saccharopolyspora spinosa

Combined MBR with Photocatalysis/Ozonation for Bromoamine Acid Removal by Yuan-yuan Qu; Qing Yang; Ji-ti Zhou; Min Gou; Lin-lin Xing; Fang Ma (pp. 664-672).

A combined biological (augmented membrane bioreactor (MBR)) and photochemical (photocatalysis and ozonation) treatment has been proposed for bromoamine acid (BAA) removal in dyeing wastewater. It was demonstrated that the color and chemical oxygen demand removal in the sequential augmented MBR was about 90% and 50%, respectively. By ribosomal intergenic spacer analysis, it was found that the introduced strain QYY was maintained as the predominant species and the diversity of the system was relatively low throughout the operation. Photocatalysis and ozonation processes were efficient to treat the effluents from MBR with high color and total organic carbon removal more than 90% within 120 min. Therefore, the hybrid treatment system is a possible way to achieve the complete mineralization of BAA.

Keywords: Membrane bioreactor; Bioaugmentation; Bromoamine acid; Ribosomal intergenic spacer; Photocatalysis and ozonation

The pH Shift and Precursor Feeding Strategy in a Low-Toxicity FR-008/Candicidin Derivative CS103 Fermentation Bioprocess by a Mutant of Streptomyces sp. FR-008 by Xiangzhao Mao; Feng Wang; Jianguo Zhang; Shi Chen; Zixin Deng; Yaling Shen; Dongzhi Wei (pp. 673-686).

CS103, the novel derivative of polyene macrolides antibiotic FR-008/candicidin with lower toxicity has been isolated from the culture mycelia of the mutant of Streptomyces sp. FR-008, with targeted deletions of the fscP cytochrome P450 gene from its chromosome. To enhance biosynthesis of CS103, pH shift and precursor feeding strategy for fermentation process by the mutant of Streptomyces sp. FR-008 in a stirred tank bioreactor was developed. According to the process parameters analysis, the effectiveness of the strategy was examined and confirmed by experiments. A maximal CS103 concentration of 139.98 μg/mL was obtained, 2.05-fold higher than that in the pH-uncontrolled fermentation. Compared to other three cases as pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch cultures, the proposed “pH shift and precursor feeding strategy” effectively avoided the scarcity of the antibiotic precursor, increased the CS103 yield from biomass (Y _P/X) and substrate (Y _P/S) by 110.61% and 48.52%, respectively, and at the time the fermentation time was shortened from 120 to 96 h. The highest CS103 production rate (1.46 μg mL⁻¹ h⁻¹) of the pH shift and precursor feeding strategy was 284.21%, 97.30%, and 58.70% higher than that of pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch culture cases, respectively.

Keywords: Two-stage pH control strategy; Precursor feeding; Polyene macrolides antibiotic; Low-toxicity FR-008/candicidin; Fermentation

Characterization of a Newly Isolated Biphenyl-Degrading Bacterium, Dyella ginsengisoli LA-4 by Ang Li; Yuanyuan Qu; Jiti Zhou; Fang Ma (pp. 687-695).

A novel biphenyl-degrading bacterium, Dyella ginsengisoli LA-4 was isolated from activated sludge. This isolate could utilize biphenyl as sole source of carbon and energy. The resting cells of strain LA-4 could utilize 100 mg/L biphenyl within 20 h, and they were able to degrade 500 mg/L biphenyl within 40 h. The surfactant, Tween 80, could accelerate the biodegradation process. The increase of NaCl concentration inhibited the biphenyl degradation. No biphenyl degradation was detected when the NaCl concentration exceeds 10%. The effects of metal ions on biphenyl degradation were investigated. The results indicated that metal ions such as Cu²⁺, Mn²⁺, and Co²⁺ could completely inhibit the biodegradation of biphenyl, but Mg²⁺, Ca²⁺, and Zn²⁺ had no effects on the degradation of biphenyl. The removal rate was about 64% and 37% in the presence of Fe³⁺ and Ni²⁺, respectively. This study suggested that strain LA-4 could be widely used for bioremediation of soil and wastewater contaminated by biphenyl, NaCl, and metal ions.

Keywords: Dyella ginsengisoli ; Biphenyl; Metal ions; NaCl; Surfactant

Inhibition Performance of Lignocellulose Degradation Products on Industrial Cellulase Enzymes During Cellulose Hydrolysis by Xinyun Jing; Xiaoxi Zhang; Jie Bao (pp. 696-707).

This study examined the inhibition performance by the major lignocellulose degradation products, including organic acids, furan derivatives, lignin derivatives, and ethanol, on a broadly used commercial cellulase enzyme Spezyme CP (Genencor International, Rochester, NY, USA) to cellulose hydrolysis at both the well-mixing state (shaking flask) and the static state (test tube). The cellulase activity in the cellulase complex of Spezyme CP was assumed to be one single “cellulase”, and the apparent kinetic parameters of this cellulase enzyme were measured as an approximate index of the inhibitory effect to the industrial cellulase enzyme. The inhibition performance of these degradation products was compared and analyzed using the determined apparent kinetic parameters. All the degradation products strongly inhibit the cellulose hydrolysis by cellulase enzyme, and the inhibitions on cellulase were all competitive type. The order of the inhibition strength by the lignocellulose degradation products to cellulase is lignin derivatives > furan derivatives > organic acids > ethanol. This study gave a quantitative view to the enzymatic hydrolysis of lignocellulose under the inhibition performance of the lignocellulose degradation products and will help to understand the lignocellulose recalcitrance to enzyme hydrolysis.

Keywords: Inhibition; Lignocellulose degradation products; Cellulase enzyme; Hydrolysis; Apparent inhibition parameters

Development of Direct Competitive Enzyme-Linked Immunosorbent Assay for the Determination Cadmium Residue in Farm Produce by Gongliang Liu; Jufang Wang; Zhiyong Li; Shizhong Liang; Jianghui Liu; Xiaoning Wang (pp. 708-717).

Cadmium, a toxic heavy metal, poses a significant threat to human health. Currently, the methods for detecting cadmium residue in farm produce need expensive equipment, intensive labor, and much time to finish one detection. In this study, a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) based on a cadmium-chelate-specific monoclonal antibody has been developed. The DC-ELISA showed an IC₅₀ of 2.30 μg/L with a detection limit of 0.20 μg/L for cadmium. The assay has been demonstrated to be highly specific since the monoclonal antibody showed little or no cross-reactivity with all tested metal chelates which include Cd²⁺, Pb²⁺, Hg²⁺, Zn²⁺, Na⁺, Ca²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Cu²⁺, Al³⁺, Co²⁺, Cr²⁺, Ni²⁺, Sn², and K⁺. The assay showed that a mean recovery ranged from 100.47% to 103.86%, and the coefficients of variations for intra- and inter-assay were 1.73–7.14% and 3.63–6.81%, respectively. Then, several farm produces including wheat flour, apple juice, rice flour, and tea were analyzed for cadmium residue with DC-ELISA and graphite furnace atomic absorption spectroscopy (GFAAS). The correlation coefficient between the DC-ELISA and GFAAS was 0.99. It was demonstrated that the DC-ELISA can be used as a simple and economic method to detect and quantitate cadmium residue in farm produce.

Keywords: Cadmium; Hapten; Chelator; Monoclonal antibody; ELISA; Farm produce

Construction of Targeting Vector for Expressing Human GDNF in Cattle Mammary Gland by Xueming Zhang; Yingji Wu; Fenhua Luo; Huimin Su; Yin Bai; Yue Hou; Boyang Yu (pp. 718-727).

Glial cell line-derived neurotrophic factor (GDNF) is a type of neurotrophic factor with significant potential in treatment of Parkinson’s disease. Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The aim of this study was to construct a gene-targeting vector for the human gdnf gene knockin at the bovine beta-casein gene locus so that human GDNF protein can be produced in the mammary gland of the gene-targeted bovine. The constructed vector contains the 2.2 kb 5′ homologous arm and the 5.7 kb 3′ homologous arm. The human gdnf cDNA was located at the downstream of the 5′ homologous arm. The neo gene placed between the 5′ and 3′ homologous arms as positive selection marker gene. The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes, respectively. The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing. The results show that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, the plasmid DNA was transfected into human mammary tumor cell line Bcap-37 by lipofectamine. Reverse transcription polymerase chain reaction and Western-blotting analysis showed that the transfected cells produced human GDNF mRNA and protein. The results show that the constructed targeting vector pNRTCNbG has bioactivity to efficiently express GDNF in mammary gland cells. At the same time, it is first time to confirm that human mammary tumor cell line Bcap-37 is valid for bioactivity analysis of mammary gland specific expression vector.

Keywords: Targeting vector; Human GDNF; Bovine β-casein gene locus; Bcap-37 cell line

Identification and Characteristics of A Novel Salt-Tolerant Exiguobacterium sp. for Azo Dyes Decolorization by Liang Tan; Yuan-yuan Qu; Ji-ti Zhou; Ang Li; Min Gou (pp. 728-738).

A salt-tolerant bacterium was isolated from the surface soil of a pharmaceutical factory, which could efficiently decolorize azo dyes. The strain was identified as Exiguobacterium sp. according to its morphological characteristics and 16S rRNA gene sequence analysis. Decolorization of X-3B with resting cells of this strain, which were catalyzed by redox mediator (anthraquinone), was studied, and the conditions were optimized. For color removal and cells growth, the optimal inoculation amount, pH, temperature, salinity, and metal ions were 6% (v/v), 5.4–7.0, 30–40 °C, 15% (w/v) NaCl, and 1 mmol L⁻¹ Mg²⁺ or Ca²⁺, respectively. It was exhibited that decolorization process proceeded primarily by enzymatic reduction associated with a minor portion of bio-adsorption to inactivated microbial cells. Anthraquinone could really accelerate the decolorization of X-3B under the optimal conditions.

Keywords: Azo dye; Decolorization; Salt-tolerant; Redox mediator

Transient Expression of Minimum Linear Gene Cassettes in Onion Epidermal Cells Via Direct Transformation by Yun-Qing Cheng; Jun Yang; Feng-Ping Xu; Li-Jia An; Jian-Feng Liu; Zhi-Wen Chen (pp. 739-749).

A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5′-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca²⁺, surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 °C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.

Keywords: Minimum linear gene cassettes; Onion low epidermal; Transient gene expression; Gus gene; Transformation buffer; Cofactors

Passive Immunization of Crayfish (Procambius clarkiaii) with Chicken Egg Yolk Immunoglobulin (IgY) Against White Spot Syndrome Virus (WSSV) by Yanan Lu; Junjun Liu; Liji Jin; Xiaoyu Li; YuHong Zhen; Hongyu Xue; Qiuye Lin; Yongping Xu (pp. 750-758).

White spot syndrome virus (WSSV) is a major cause of mortality in shrimp lacking a true adaptive immune response. In this study, high activity egg yolk immunoglobulin (IgY) against WSSV for passive immunization of crustaceans was already prepared as crude and purified product, while an indirect enzyme-linked immunosorbent assay test was used for quality control of IgY activity. The effectiveness of IgY of intramuscular injection, oral administration, and immersion was investigated in crayfish (Procambius clarkiaii) against WSSV. The result showed that the groups treated with IgY from inactivated WSSV and DNA vaccine were, respectively, 20% and 80% mortality, which were significant difference in survival rates (P < 0.05) from the positive control groups. The groups in diet added 10% egg yolk powder and 1% IgY power showed 53.3% and 67.7% mortality, respectively, and the immersion showed 46.7% mortality, which have significantly different compared to the positive groups (P < 0.05). These results indicated passive immunization of specific IgY antibodies through intramuscular injection, oral administration, and immersion have effective to protect crayfish against WSSV. It is noteworthy that IgY as feed additive and immersion solution is useful and feasible methods in practical work. Thus, our results suggest that the passive immunization of crayfish with IgY against WSSV will have potential development to prevent and control WSSV in practical culture.

Keywords: WSSV; IgY; Inactivated vaccine; DNA vaccine; Passive immunization; Crayfish

Water Activity Dependence of Lipases in Non-aqueous Biocatalysis by Xiaole Xia; Chen Wang; Bo Yang; Yong-Hua Wang; Xiaoning Wang (pp. 759-767).

Eleven lipases are tested and it was found that lipases can be divided into three types according to water activity dependence. The first type is lipase that has low water activity dependence and works in a low water activity, its performance changes little with the change of water activity. The optimum water activity is 0.19 and Newlase F (Rhizopus niveus), lipase FAP-15 (Rhizopus oryzae) belong to this type. The second type is lipase that has medium water activity dependence and its performance changes with the change of water activity. Most lipases belong to this type and the optimum water activity in this type is about 0.60. The third type is lipase that has a high water activity dependence and works only in a high water activity (a w > 0.75). WGL (wheat germ) belongs to this type and the optimum water activity is 0.90. The relationship between enantioselectivity and water activity is also discussed and the enantioselectivity seems to be independent of water activity. And we also compared the two control methods of water activity, it was found that the method which add solid salt hydrates to the reaction mixture (method II) is more stable and effective throughout the reaction than the method that pre-equilibrate via the vapor phase (method I). The addition concentration of salt hydrates is also investigated and the optimum concentration is 1 g/l.

Keywords: Water activity dependence; Non-aqueous biocatalysis; Lipase; Kinetic resolution

Synthesis and Insecticidal Activity of New 4-Hydroxy-2H-1-benzopyran-2-one Derivatives by Meijing Wang; Feng Chen; Jian Guan; Jing Zhao; Jiufu Zhang; Renhe Zhao (pp. 768-777).

Several stable and storable anticoagulant rodenticides involving both merits of acute and chronic rodenticides have been synthesized (Holbrook et al. in Arch Intern Med 165:1095–1106, 2005; Baglin et al. in Br J Haematol 132:277–285, 2006). The structures of synthesized compounds were confirmed by IR, ¹H NMR. The compounds were also evaluated for their anticoagulant and acute biologic activity (Lipton et al., JAMA 252:3004–3005, 3).

Keywords: Anticoagulant rodenticide; 4-Hydroxy-2H-1-benzopyran-2-one; Cinnamoyl chloride; 4-Bromodiphenyl; Synthesis

Chitosan–Alginate Microcapsules for Oral Delivery of Egg Yolk Immunoglobulin (IgY): Effects of Chitosan Concentration by Xiao-Yu Li; Li-Ji Jin; Ya-Nan Lu; Yu-Hong Zhen; Shu-Ying Li; Lin-Hui Wang; Yong-Ping Xu (pp. 778-787).

In our previous study, chitosan–alginate microcapsules were developed to protect egg yolk immunoglobulin (IgY) from gastric inactivation. The present study was undertaken to determine the effect of chitosan concentration (0–0.8%; w/v) on various properties of the microcapsules in order to produce the optimum chitosan–alginate microcapsules for use in the oral delivery of IgY. The properties investigated included microcapsule morphology, loading capacity for IgY (expressed as the IgY loading percentage, w/w, of microcapsules), encapsulation efficiency (EE%), in vitro gastroresistance, and IgY release. IgY loading percentage and EE% were both highest at 0.2% (w/v) chitosan, and, above this level, further increases were not observed. The stability of IgY in simulated gastric fluid (pH 1.2) was significantly improved by encapsulation in alginate microcapsules (IgY retained 43.5% of its activity) and was further improved by including chitosan at any of the chitosan concentrations assessed (IgY retained an average of 69.4% activity) although there was no difference in protection of gastric inactivation among concentrations of chitosan varying from 0.05% to 0.8% (w/v). Higher chitosan concentrations (i.e., ≥0.2%; w/v) prolonged the release of IgY from the microcapsules during simulated intestinal fluid incubation (pH 6.8). However, above the 0.2% (w/v) level, no significant differences were observed. We conclude that the optimum chitosan concentration for microencapsulation is 0.2% (w/v).

Keywords: Alginate; Chitosan; Chitosan concentration; Egg yolk immunoglobulin (IgY); Gastroresistance; Microcapsules

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.159, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.159, #3)