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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #8)

Agrobacterium tumefaciens Mediated Transformation of ChiV Gene to Trichoderma harzianum by Liming Yang; Qian Yang; Kening Sun; Ye Tian; Hulun Li (pp. 937-945).

As a soil-borne filamentous fungus, Trichoderma harzianum exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. In this study, the vectors pBI121 and pCAMBIA1301 and cloning vector pUC18 were used to successfully construct expression vector pCA-GChiV for filamentous fungi transformation mediated by Agrobacterium tumefaciens.The ChiV gene was successfully transferred into the biocontrol fungus T. harzianum with an efficiency of 90–110 transformants per 10⁷ spores using A. tumefaciens-mediated transformation. Putative transformants were analyzed to test the transformation by the southern blot, and the expression of ChiV was detected by reverse transcription PCR. The transformants were co-cultured to assay antifungal activities with Rhizoctonia solani. The inhibition rates of the transformants and no ChiV gene transferred T. harzianum were 98.56% and 82.42%, respectively, on the fourth day.The results showed that the ChiV transformants had significantly higher inhibition activity.

Keywords: Trichoderma harzianum ; Biocontrol; Agrobacterium tumefaciens

An Effective and Simplified Fed-Batch Strategy for Improved 2,3-Butanediol Production by Klebsiella oxytoca by Zhi-Kui Nie; Xiao-Jun Ji; He Huang; Jun Du; Zhi-Yong Li; Liang Qu; Qi Zhang; Ping-Kai Ouyang (pp. 946-953).

Substrate concentration in 2,3-butanediol (2,3-BD) fermentation could not be controlled well in traditional feeding strategies, such as constant, impulse, and exponential feeding strategies. In the present study, fermentative 2,3-BD production by Klebsiella oxytoca was investigated under different batch and fed-batch strategies. The glucose-feedback fed-batch strategy was proved to be not effective for economical 2,3-BD production for the inability of timely feeding, leading that the bacteria reused 2,3-BD as carbon source for cell growth. Based on the phenomena that the byproducing acids caused the pH declining and the requirement of maintaining the pH at a proper level for both cell growth and 2,3-BD accumulation, an improved strategy of pH-stat fed-batch culture with glucose and sodium hydrate fed at the same time was established. Thus, the residual glucose concentration could be controlled through the adjustment of pH automatically. At last, efficient 2,3-BD production was fulfilled under this fed-batch strategy, and the highest 2,3-BD concentration, productivity, and yield were 127.9 g/l, 1.78 g/(l•h), and 0.48 g/g (2,3-BD/glucose), respectively, compared to 98.5 g/l, 1.37 g/(l•h), and 0.43 g/g obtained in glucose-feedback fed-batch strategy. This feeding strategy was simple and easy to operate and could be feasible for industrial 2,3-BD production in the future.

Keywords: 2,3-Butanediol; Klebsiella oxytoca ; pH-stat; Fed-batch

Vectors for Glucose-Dependent Protein Expression in Saccharomyces cerevisiae by Simone Thierfelder; Kai Ostermann; Andy Göbel; Gerhard Rödel (pp. 954-964).

Based on the p426 series of expression vectors developed by Mumberg et al. (Gene 156, 119–122, 1995), we have generated a set of plasmids that allow the glucose-dependent expression of target genes in the yeast, Saccharomyces cerevisiae. The ADH1 promoter in plasmid p426-ADH1 was replaced by the 1-kb 5′-region from either of the following genes: HXK1, YGR243, HXT4 and HXT7. Expression mediated by the respective 5′-regions was monitored with EGFP, yEGFP3-CLN2pest and TurboGFP as marker genes. Fluorescence is induced 2.7-fold using the HXK1, 2.3-fold using the YGR243-, 5-fold using the HXT7- and 12.6-fold using the HXT4 5′-regions upon depletion of glucose to a concentration of <0.5 g/l.

Keywords: Saccharomyces cerevisiae ; 5′-Regulatory region; Glucose limitation; Gene expression; Vector construction

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems by Călin-Aurel Drăgan; Frank T. Peters; Pierre Bour; Andrea E. Schwaninger; Stefanie M. Schaan; Ina Neunzig; Maria Widjaja; Josef Zapp; Thomas Kraemer; Hans H. Maurer; Matthias Bureik (pp. 965-980).

The growing need for the characterization of cytochrome P450 (P450) metabolites often necessitates their synthesis up to Gram-scale. This task may in principle be achieved by using various techniques including chemical synthesis, the use of laboratory animals, in vitro P450 systems or microbial biotransformation. However, these approaches are in many instances unfavorable due to low yields, laborious purification, costs of cofactors, or the formation of non-physiologic metabolites. The fission yeast Schizosaccharomyces pombe has previously been shown by others and us to be very well suited for the heterologous expression of human P450s. In this study, we demonstrate whole-cell biotransformation reactions carried out with fission yeast strains that coexpress human cytochrome P450 reductase (CPR) and one of the following P450 isoforms: CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4, respectively. These strains could successfully convert their respective standard substrates but showed different responses with respect to incubation pH, the presence of glucose, and temperature, respectively. In addition, the preparative of synthesis of 2.8 g of 4′-hydroxydiclofenac was achieved by whole-cell biotransformation of diclofenac using a CPR-CYP2C9 coexpressing fission yeast strain.

Keywords: Cytochrome P450; CYP; Diclofenac; Fission yeast; Hydroxydiclofenac; Phase 1 metabolism; Schizosaccharomyces pombe

Production of Recombinant Porcine Interferon alpha Using PHB–Intein-Mediated Protein Purification Strategy by Xuezhang Zhou; Zhenwei Song; Xiaoming Liu; Fang Jia; Yujiong Wang (pp. 981-993).

Interferons (IFNs) are involved in the pathogenesis and recovery of viral and other infectious diseases. Recombinant IFNs have been used as anti-infectious agents exhibiting a broad range of antiviral and immunomodulatory properties in both human and domestic animals. In this report, we describe a highly efficient and economical approach to purify porcine IFN alpha (PoIFNα) using polyhydroxybutyrate (PHB) as the affinity carrier and intein for self-cleaving removal of the affinity tag. Additionally, the conditions of protein expression and purification have been optimized. Our results suggested that culture medium containing 1.62% (w/v) of sodium lactate dramatically increases the accumulation of PHB binding protein in Escherichia coli cells. High yields of recombinant PoIFNα (30–35 mg/L, 97% purity by high-performance liquid chromatography) were obtained using intein-mediated self-cleaving conditions using a cleavage-inducing buffer with a pH of 6.5 at 20 °C for 24–36 h. The antiviral activity of the recovered recombinant PoIFNα was up to 1.4 × 10⁶ IU/mg of protein ascertained using recombinant human IFNα1 as a standard. This report also demonstrates that large-scale production of intein-mediated purification of highly pure and active recombinant PoIFNα is feasible for the purposes of experimental studies, veterinary clinic therapeutics, and swine infectious disease control.

Keywords: Intein; Poly-β-hydroxybutyrate; Porcine; IFN alpha; Purification

Proline-Specific Extracellular Aminopeptidase Purified from Streptomyces lavendulae by Arya Nandan; Ashok Pandey; Kesavan Madhavan Nampoothiri (pp. 994-1001).

Aminopeptidases catalyze the cleavage of specific amino acids from the amino terminus of protein or peptide substrates. A proline-specific aminopeptidase was purified to homogeneity from the culture-free extract of Streptomyces lavendulae ATCC 14162 in sequential steps comprising ammonium sulfate precipitation, ultra-filtration, and column chromatography on Q-sepharose and Sephadex G-100. The purified protein showed approximately 60 kDa in SDS-PAGE and was optimally active at pH 6.5 and 40 °C. Kinetic studies showed a K _m and V _max of 0.23 mM and 0.087 μmol/min, respectively, using Pro-p-NA, the substrate with maximum specificity. Enzyme activity was inhibited by PMSF and ions like Zn²⁺, Co²⁺, and Ni²⁺. However, unlike other aminopeptidases, the activity was enhanced in the presence of DTT, 1,10-phenanthroline, EDTA, amastatin, and bestatin. Ions like Ca²⁺, Mg²⁺, and Mn²⁺ also enhanced the activity.

Keywords: l-Proline aminopeptidase; l-Proline p-nitroanilide; Serine protease; Streptomyces lavendulae

Increased In Vitro Lysosomal Function in Oxidative Stress-Induced Cell Lines by Jihee Yoon; Seung Hyuck Bang; Jin-Soo Park; Suk-Tai Chang; Yang-Hoon Kim; Jiho Min (pp. 1002-1011).

Exposure of mammalian cells to oxidative stress alters lysosomal enzymes. Through cytochemical analysis of lysosomes with LysoTracker, we demonstrated that the number and fluorescent intensity of lysosome-like organelles in HeLa cells increased with exposure to hydrogen peroxide (H₂O₂), 6-hydroxydopamine (6-OHDA), and UVB irradiation. The lysosomes isolated from HeLa cells exposed to three oxidative stressors showed the enhanced antimicrobial activity against Escherichia coli. Further, when lysosomes that were isolated from HeLa cells exposed by oxidative stress were treated to normal HeLa cells, the viability of the HeLa cells was drastically reduced, suggesting increased in vitro lysosomal function (i.e., antimicrobial activity, apoptotic cell death). In addition, we also found that cathepsin B and D were implicated in increased in vitro lysosomal function when isolated from HeLa cells exposed by oxidative stress. Decrease in cathepsin B activity and increase in cathepsin D activity were observed in lysosomes isolated from HeLa cells after treatment with H₂O₂, 6-ODHA, or UVB, but cathepsin B and D were not the sole factors to induce cell death by in vitro lysosomal function. Therefore, these studies suggest a new approach to use lysosomes as antimicrobial agents and as new materials for treating cancer cell lines.

Keywords: Oxidative stress; Lysosomes; In vitro function; Antimicrobial activity; Apoptotic cell death

Screening and Identification of a Fungal β-Glucosidase and the Enzymatic Synthesis of Gentiooligosaccharide by Yongling Qin; Yunkai Zhang; Haiyan He; Jing Zhu; Guiguang Chen; Wei Li; Zhiqun Liang (pp. 1012-1019).

After screening with 0.1% esculoside and 0.03% FeCl₃, we identified from rotten wood a fungal isolate HML0366 that produces high amount of β-glucosidase. Phenotypic and rDNA internal transcribed spacer sequence analyses indicated that the isolate belongs to Aspergillus oryzae. The β-glucosidase produced by HML0366 had an activity of 128 U/g. high performance liquid chromatography analysis also demonstrated a high transglycosylation activity of the crude enzyme. The β-glucosidase was stable between pH 4–10 at 60 °C. A gentiobiose yield of 30.86 g/L was achieved within 72 h of the enzymatic reaction at pH 5 and 55 °C using 50% glucose as the substrate. For the first time, we report here the isolation of an A. oryzae strain producing β-glucosidase with high hydrolytic activities. The crude enzyme has a high transglycosylation activity, which enables the enzymatic synthesis of gentiooligosaccharides.

Keywords: β-Glucosidase; Transglycosylation; Aspergillus oryzae ; Gentiooligosaccharide

Investigation of Yeast Invertase Immobilization onto Cupric Ion-Chelated, Porous, and Biocompatible Poly(Hydroxyethyl Methacrylate-n-Vinyl Imidazole) Microspheres by Müfrettin Murat Sari (pp. 1020-1037).

Cupric ion-chelated poly(hydroxyethyl methacrylate-n-vinyl imidazole) (poly(HEMA-VIM)) microspheres prepared by suspension polymerization were investigated as a specific adsorbent for immobilization of yeast invertase in a batch system. They were characterized by scanning electron microscopy, surface area, and pore size measurements. They have spherical shape and porous structure. The specific surface area of the p(HEMA-VIM) spheres was found to be 81.2 m²/g with a size range of 70–120 μm in diameter, and the swelling ratio was 86.9%. Then, Cu(II) ion chelated on the microspheres (546 μmol Cu(II)/g), and they were used in the invertase adsorption. Maximum invertase adsorption was 51.2 mg/g at pH 4.5. Cu(II) chelation increases the tendency from Freundlich-type to Langmuir-type adsorption model. The optimum activity for both free and adsorbed invertase was observed at pH 4.5. The optimum temperature for the poly(HEMA-VIM)/Cu(II)-invertase system was found to be at 55 °C, 10 °C higher than that of the free enzyme at 45 °C. V _max values were determined as 342 and 304 U/mg enzyme, for free and adsorbed invertase, respectively. K _m values were found to be same for free and adsorbed invertase (20 mM). Thermal and pH stability and reusability of invertase increased with immobilization.

Keywords: Enzyme immobilization; Invertase; Poly(HEMA-VIM) microsphere; Metal chelating; Cu(II) chelation; n-Vinyl imidazole

Synthesis and Characterization of an Exopolysaccharide by Antarctic Yeast Strain Cryptococcus laurentii AL₁₀₀ by Kostantsa Pavlova; Snezhana Rusinova-Videva; Margarita Kuncheva; Maria Kratchanova; Mariana Gocheva; Stela Dimitrova (pp. 1038-1052).

An exopolysaccharide-producing Antarctic yeast strain was selected and identified as Cryptococcus laurentii AL₁₀₀. The physiological properties of the strain and its ability to utilize and biotransform different carbon sources (pentoses, hexoses, and oligosaccharides) into exopolysaccharide and biomass were investigated. Sucrose was chosen as a suitable and accessible carbon source. The biosynthetic capacity of the strain was studied in its dynamics at different sucrose concentrations (20, 30, 40, and 50 g/L) and temperatures (22 and 24 °C). The maximum biopolymer quantity of 6.4 g/L was obtained at 40 g/L of sucrose, 22 °C temperature and 96-h fermentation duration. The newly synthesized microbial carbohydrate was a heteropolysaccharide having the following monosaccharide composition: arabinose, 61.1%; mannose, 15.0%; glucose, 12.0%; galactose, 5.9%; and rhamnose, 2.8%. It was characterized by polydispersity of the polymer molecule, 60% of it having molecular mass of 4200 Da. The exopolysaccharide demonstrated good emulsifying and stabilizing properties with regard to oil/water emulsions and a pronounced synergistic effect with other hydrocolloids such as xanthan gum, guar gum, and alginate.

Keywords: Cryptococcus laurentii AL₁₀₀ ; Biosynthesis; Exopolysaccharide; Yeast

Granulation of Simultaneous Partial Nitrification and Anammox Biomass in One Single SBR System by Xiaoming Li; Yang Xiao; Dexiang Liao; Wei Zheng; Ting Yi; Qi Yang; Guangming Zeng (pp. 1053-1065).

The granulation of simultaneous partial nitrification and anaerobic ammonium oxidation (Anammox) was investigated in a single, oxygen-limited, sequencing batch reactor. In this research, the reactor was started anaerobically and fed using the synthetic medium described by Van de Graaf et al. to cultivate Anammox biomass after inoculation with methanogenic granular sludge. Subsequently, mixture gas (air and nitrogen gas) was supplied to the reactor and a nitrifying population developed. Research results indicated that autotrophic granules was cultivated successfully by controlling the dissolved oxygen in the reactor between 0.3 and 0.5 mg/L, and a total inorganic nitrogen removal efficiency of 63.7% was obtained with a higher nitrogen load increased by reducing HRT to 3 days. It was also seen that the Ca and P concentrations of the feeding medium are important factors that influence the autotrophic granules from process running. When the Ca and P concentrations were exceeded the necessary quantity, salt precipitation was observed, interfered with microbial activity, and caused a decrease of the nitrogen removal rate of the system. After diminishing adequately the Ca and P concentrations, salt precipitation was avoided and the activity of the system restored quickly. Moreover, visual indication and scanning election microscopy observation revealed the process of sludge evolution and inner structure of the granules.

Keywords: Partial nitrification; Anammox; SBR; Granules; Salt precipitation

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #8)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #8)