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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #2)

A Viral Suppressor P1/HC-Pro Increases the GFP Gene Expression in Agrobacterium-mediated Transient Assay by Pengda Ma; Jinying Liu; Hongxia He; Meiying Yang; Meina Li; Xiaojuan Zhu; Xingzhi Wang (pp. 243-252).

More than 20 post-transcriptional gene silencing (PTGS) suppressors have been found since HC-Pro, the first gene silencing suppressor, was found in 1998. The silencing suppressor strongly suggested that gene silencing functions as natural defense mechanisms against viruses. It also represented a valuable tool for the dissection of the gene silencing pathway. We have used P1/HC-Pro RNA silencing suppressor activity to increase green fluorescent protein (GFP) expression in tobacco using an Agrobacterium-mediated transient expression system. P1/HC-Pro stimulated GFP-gene expression but not dsGFP-gene expression was shown by RT-PCR, Northern and Western blot analysis. Expression of the gene silencing suppressor and the target gene provided a new strategy of heterogeneous gene expressing in plants. It may be of commercial significance to produce foreign proteins using plant bioreactors.

Keywords: Post-transcriptional gene silencing (PTGS); Suppressor; P1/HC-Pro; Agrobacterium-mediated transient expression system; GFP; Semi-quantitative reverse transcription-PCR

A Modified Method using TRIzol^® Reagent and Liquid Nitrogen Produces High-Quality RNA from Rat Pancreas by Dongmin Li; Wuchao Ren; Xuan Wang; Feimiao Wang; Yu Gao; Qilan Ning; Yan Han; Tianbao Song; Shemin Lu (pp. 253-261).

To establish an economical and reproducible method for the high-quality RNA extraction from pancreas, we isolated total RNA from rat pancreas with TRIzol® reagent and liquid nitrogen. In the initial stage, we optimized three influential factors, the way to homogenize pancreas, the time to collect the pancreatic tissue from animals, and the weight of the pancreatic tissue in 1 ml of TRIzol® reagent. The RNA quality was determined by detecting total RNA content and its absorbance at 260/280 nm wavelength, visualizing RNA in non-denatured agarose gel and performing RT-PCR of pancreas-specific genes. The A ₂₆₀/A ₂₈₀ ratio of the total RNA extracted by grinding 20–30 mg of rat pancreatic tissue removed from the rats in liquid nitrogen within 1 min and then immersed in 1 ml of the TRIzol® Reagent was 1.75–1.89, and the ratio of 28S/18S ribosomal RNA bands was more than 1.8. Furthermore, full length of Pdx1 open-reading frame was amplified with RNA extracted from the grinding group rather than from the conventional group. The RT-PCR products of pancreas-specific genes from both exocrine and endocrine parts of pancreas were successfully derived from the extracted RNA. The results suggested that we successfully provided an economical, fast, and reproducible method to obtain the high-quality and intact RNA from rat pancreas with TRIzol® Reagent and liquid nitrogen.

Keywords: Rat pancreas; RNA; Total RNA extraction; TRIzol® Reagent; Liquid nitrogen

Breakthrough Model of Recombinant Human-Like Collagen in Immobilized Metal Affinity Chromatography by Xiao-Jun Wang; Dai-Di Fan; Yan-E Luo (pp. 262-276).

The adsorption of recombinant human-like collagen by metal chelate media was investigated in a batch reactor and in a fixed-bed column. The adsorption equilibrium and kinetics had been studied by batch adsorption experiments. Equilibrium parameters and protein diffusivities were estimated by matching the models with the experimental data. Using the parameters of equilibrium and kinetics, various models, such as axial diffusion model, linear driving force model, and constant pattern model, were used to simulate the breakthrough curves on the columns. As a result, the most suitable isotherm was the Langmuir–Freundlich model, and the ionic strength had no effect on the adsorption capacity of chelate media. In addition, the pore diffusion model fitted very well to the kinetic data. The pore diffusivities decreased with increasing the initial protein concentration, however had little change with the ionic strength. The results also indicated that the models predict breakthrough curves reasonably well to the experimental data, especially at low initial protein concentration (0.3 mg ml⁻¹) and low flow rate (34 cm h⁻¹). By the results, we optimized the experimental conditions of a chromatographic process using immobilized metal affinity chromatography to purify recombinant human-like collagen.

Keywords: Recombinant human-like collagen; Breakthrough curves; Immobilized metal affinity chromatography; Kinetics; Optimize

The Transgenic BmN Cells with Polyhedrin Gene: A Potential Way to Improve the Recombinant Baculovirus Infection Per Os to Insect Larvae by Lu Chen; Weide Shen; Yan Wu; Bing Li; Chengliang Gong; Wenbing Wang (pp. 277-284).

The principle of baculovirus expression system is that substitute exogenous gene for polyhedrin (polh) gene, and the recombinant baculovirus lacks the ability to infect insect larvae by oral inoculation. In this study, we cloned the polh gene with immediate early gene 1 (ie1) promoter of Bombyx mori nucleopolyhedrovirus (BmNPV) into transposon pigA3GFP vector, transported it into BmN cells by lipofectamine and obtained the transgenic BmN cell line. The mRNA transcription of the polyhedrin gene was demonstrated by reverse transcription-polymerase chain reaction. Then the polh gene negative viruses (BmPAK6 and BmGFP), infected the transgenic BmN cells and Polyhedrin-like structures were observed in the infected cells. Subsequently, the viruses (vBmPAK6 and vBmGFP) from infected cells were used to orally inoculated the fifth instar larvae of B. mori, respectively. The results showed that B. mori larvae could be infected per os with the recombinant baculoviruses vBmPAK6 and vBmGFP, respectively. These results suggest that the products of polh gene expressed in the transgenic BmN cells could package the recombinant baculoviruses when the viruses infected the cells and raise the pathogenicity of the recombinant virus in orally infected B. mori larvae.

Keywords: Baculovirus; Polyhedrin; Transgenic; Bombyx mori ; Infection

Purification and Biochemical Characterization of Polyphenol Oxidases from Embryogenic and Nonembryogenic Cotton (Gossypium hirsutum L.) Cells by Tanoh Hilaire Kouakou; Yatty Justin Kouadio; Patrice Kouamé; Pierre Waffo-Téguo; Alain Décendit; Jean-Michel Mérillon (pp. 285-301).

Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM. The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature of 25 °C and was relatively stable at 20–30 °C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl₂, and FeCl₃ markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg²⁺, Ca²⁺, and Mn²⁺ and was moderately inhibited by Ba²⁺, Cu²⁺, and Zn²⁺. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000.

Keywords: Cell suspension; Characterization; Cotton; Gossypium hirsutum L.; Polyphenol oxidase; Purification

Environmental Effects on Transglutaminase Production and Cell Sporulation in Submerged Cultivation of Bacillus circulans by Claucia Fernanda Volken de Souza; Gilvane Souza de Matos; Simone Hickmann Flôres; Marco Antônio Záchia Ayub (pp. 302-312).

In this research, the effects of pH, temperature, and oxygen on growth kinetics of a newly isolated strain of Bacillus circulans from the Amazon and their correlations with transglutaminase (TGase) production and cell sporulation were investigated. Statistical experimental methods were used to optimize these parameters, while induction of sporulation was achieved by oxygen culture control. Full factorial composite experimental design and response surface methodology were experimentally tested. The model showed that temperature has a positive and significant effect on TGase production (P < 0.05) while pH and temperature, associated with anoxic conditions, have a marked effect on cell sporulation which is consistently linked with TGase production. The contour plot of results showed that the best culture conditions for TGase production of B. circulans were 30°C, initial pH 8.5, and the highest production was obtained in late-stationary culture phase with maximal specific enzyme activity of 655 U g⁻¹ of cells (0.37 U/mL). A correlation between enzyme production and cell sporulation, as mediated by oxygen culture conditions, was also demonstrated and, although demonstrated only for B. subtilis, it corroborates the molecular mechanisms involved in this process. It can be suggested that B. circulans BL32 is a strong biological system for the industrial production of TGases.

Keywords: Bacillus circulans BL32; Cell sporulation; Culture conditions optimization; Microbial transglutaminase; Submerged bacterial cultivation

High Soluble Expression of d-Amino Acid Oxidase in Escherichia coli Regulated by a Native Promoter by Yangqiu Liu; Qiang Li; Hongyu Zhu; Jichu Yang (pp. 313-322).

To express high-active soluble d-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (P_Hase), was constructed. A d-amino acid oxidase gene (dao) was ligated with the P_Hase and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl β-d-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m³ fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 °C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 °C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.

Keywords: d-Amino acid oxidase; Hydantoinase promoter; Constitutive expression; Ribosome binding site; Fermentation

Unfolding and Inactivation of Abalone (Haliotis diversicolor) Alkaline Phosphatase During Denaturation by Guanidine Hydrochloride by Jin Hua Liao; Qing Xi Chen; Qian Zhang; Yi Yang; Yan Shi (pp. 323-333).

Abalone, a kind of low poikilothermic invertebrate, is easily exposed to ocean environment stress. Since it is one of the important mariculture animals, the attention paid to the abalone study becomes increasing. Alkaline phosphatase (ALPase, EC 3.1.3.1) is a kind of zinc-contained metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. Unfolding and inactivation of ALPase from abalone (Haliotis diversicolor) during denaturation by guanidine hydrochloride (GuHCl) of different concentrations has first been studied. The kinetic theory of the substrate reaction by enzyme was described by Tsou, which was applied to the study on ALPase’s kinetic course of inactivation by GuHCl. The result showed that the inactivation of the enzyme by GuHCl was a slow, reversible reaction with fractional remaining activity. The microscopic rate constants were determined. The result, $$k_{ + 0} , > k_{ + 0}^prime $$ , showed that the enzyme was protected by the substrate to a certain extent during guanidine denaturation. The changes of conformation of the enzyme in different concentrations of GuHCl have been studied by means of measuring the fluorescence spectra. The results showed that the inactivation occurred before the noticeable conformational changes of the enzyme molecule as a whole can be detected, which suggests that the active site of the enzyme has more flexibility than the whole enzyme molecule. These studies will facilitate the understanding of physiological and biochemical features of the H. diversicolor and will also help in the understanding of the abalone immune system.

Keywords: Haliotis diversicolor ; Alkaline phosphatase; Denaturation and inactivation; Guanidine hydrochloride; Kinetics

Recombination System Based on Cre α Complementation and Leucine Zipper Fusions by Azadeh Seidi; Masayasu Mie; Eiry Kobatake (pp. 334-342).

In a previous study, a recombination system based on the α complementation of cre recombinase and protein transduction was established. This system relied on the transient expression of the inactive, self-excisable C-terminal (β) and the transduction of the N-terminal (α) cre fragments to cells as a purified protein. This recombination system potentially results in a less invasive and more controllable cre recombination in mammalian cells. In this study, we have employed a more efficient complementation triggering sequence using more than only the overlapping amino acids to help the α and β fragments reassociate. In order to increase the association efficiency of the complementing fragments of cre recombinase, we chose to use a fusion of cre fragments to a self-heterodimerizing pair of proteins to trigger their binding and thus increase the efficiency of the restored enzymatic activity. For this purpose, the leucine zipper motifs (bJun and bFos) of the AP-1 transcription were fused to cre fragments (α and β, respectively). This resulted in an increased reassociation efficiency of the fragments and a two times more efficient recombination system compared with the previous study.

Keywords: α complementation; Cre recombinase; Leucine zipper motif; Protein transduction; Reassociation efficiency

Cloning, Protein Sequence Clarification, and Substrate Specificity of a Leucine Dehydrogenase from Bacillus sphaericus ATCC4525 by Hongmei Li; Dunming Zhu; Brooke A. Hyatt; Fahad M. Malik; Edward R. Biehl; Ling Hua (pp. 343-351).

Although an X-ray model sequence of a leucine dehydrogenase from Bacillus sphaericus ATCC4525 was reported, the amino acid sequence of this enzyme has not been confirmed. In the current study, this leucine dehydrogenase gene was cloned, sequenced, and over-expressed in Escherichia coli, and the protein sequence has been clarified. This leucine dehydrogenase is not identical with that of B. sphaericus IFO3525 because there are 16 different amino acid residues between these two proteins. Since the information on the catalytic properties of leucine dehydrogenase from B. sphaericus ATCC4525 has been limited, the recombinant enzyme was purified as His-tagged protein and further studied. This enzyme showed activity toward aliphatic substrates for both oxidative deamination and reductive amination and is an effective catalyst for the asymmetric synthesis of α-amino acids from the corresponding α-ketoacids.

Keywords: Leucine dehydrogenase; Gene cloning; Bacillus sphaericus ; Reductive amination; Synthesis of α-amino acids

Hydrolytic Methods for the Quantification of Fructose Equivalents in Herbaceous Biomass by Stefanie K. Nguyen; Supaporn Sophonputtanaphoca; Eugene Kim; Michael H. Penner (pp. 352-361).

A low, but significant, fraction of the carbohydrate portion of herbaceous biomass may be composed of fructose/fructosyl-containing components (“fructose equivalents”); such carbohydrates include sucrose, fructooligosaccharides, and fructans. Standard methods used for the quantification of structural-carbohydrate-derived neutral monosaccharide equivalents in biomass are not particularly well suited for the quantification of fructose equivalents due to the inherent instability of fructose in conditions commonly used for hemicellulose/cellulose hydrolysis (>80% degradation of fructose standards treated at 4% sulfuric acid, 121°C, 1 h). Alternative time, temperature, and acid concentration combinations for fructan hydrolysis were considered using model fructans (inulin, β-2,1, and levan, β-2,6) and a grass seed straw (tall fescue, Festuca arundinacea) as representative feedstocks. The instability of fructose, relative to glucose and xylose, at higher acid/temperature combinations is demonstrated, all rates of fructose degradation being acid and temperature dependent. Fructans are shown to be completely hydrolyzed at acid concentrations well below that used for the structural carbohydrates, as low as 0.2%, at 121°C for 1 h. Lower temperatures are also shown to be effective, with corresponding adjustments in acid concentration and time. Thus, fructans can be effectively hydrolyzed under conditions where fructose degradation is maintained below 10%. Hydrolysis of the β-2,1 fructans at temperatures ≥50°C, at all conditions consistent with complete hydrolysis, appears to generate difructose dianhydrides. These same compounds were not detected upon hydrolysis of levan, sucrose, or straw components. It is suggested that fructan hydrolysis conditions be chosen such that hydrolysis goes to completion; fructose degradation is minimized, and difructose dianhydride production is accounted for.

Keywords: Fructose; Fructans; Fructooligosaccharides; Inulin; Levan; Lignocellulosics; Hydrolysis

Extraction of Co-Products from Biomass: Example of Thermal Degradation of Silymarin Compounds in Subcritical Water by Lijun Duan; Sunny N. Wallace; Abigail Engelberth; Justin K. Lovelady; Edgar C. Clausen; Jerry W. King; Danielle Julie Carrier (pp. 362-373).

In an effort to increase revenues from a given feedstock, valuable co-products could be extracted prior to biochemical or thermochemical conversion with subcritical water. Although subcritical water shows significant promise in replacing organic solvents as an extraction solvent, compound degradation has been observed at elevated extraction temperatures. First order thermal degradation kinetics from a model system, silymarin extracted from Silybum marianum, in water at pH 5.1 and 100, 120, 140, and 160 °C were investigated. Water pressure was maintained slightly above its vapor pressure. Silymarin is a mixture of taxifolin, silichristin, silidianin, silibinin, and isosilibinin. The degradation rate constants ranged from 0.0104 min⁻¹ at 100 °C for silichristin to a maximum of 0.0840 min⁻¹ at 160 °C for silybin B. Half-lives, calculated from the rate constants, ranged from a low of 6.2 min at 160 °C to a high of 58.3 min at 100 °C, both for silichristin. The respective activation energies for the compounds ranged from 37.2 kJ/gmole for silidianin to 45.2 kJ/gmole for silichristin. In extracting the silymarin with pure ethanol at 140 °C, no degradation was observed. However, when extracting with ethanol/water mixtures at and 140 °C, degradation increased exponentially as the concentration of water increased.

Keywords: Extraction; Kinetics; Silymarin; Subcritical water; Thermal degradation

Rapid Screening and Isolation of a Fungus for Sitosterol to Androstenedione Biotransformation by Alok Malaviya; James Gomes (pp. 374-386).

Androstenedione (AD) is a steroid intermediate used in the pharmaceutical industry for the production of several important anabolic drugs. An important route for producing AD is by the microbial transformation of sterols. Microbes capable of efficiently transforming sterols to AD are few and newer strains need to be isolated. Conventional procedures for screening and isolation are time consuming. A new procedure was used for screening and isolation of fungal microorganisms capable of biotransformation of sitosterol to androstenedione. In this procedure, Basic Alignment Search Tool (BLAST) and Position-Specific Iterative BLAST were employed to obtain a parent set of candidate microorganisms. The parent set was reduced using heuristics and constraints to obtain a manageable number of microorganisms that may be tested experimentally. For this work, screening of the entire NCBI database yielded a parent set containing 64 microorganisms. Among these, only two microorganisms, Aspergillus oryzae and Aspergillus nidulans FGSC A4, qualified to the experimental stage. Sitosterol biotransformation experiments were carried out using A. oryzae and the production of AD in culture medium was confirmed.

Keywords: Androstenedione; Biotransformation; BLAST; β-Sitosterol; Isolation; Screening

Stress Response Kinetics of Two Nisin Producer Strains of Lactococcus lactis spp. lactis by Ömer Şimşek; Sencer Buzrul; Nefise Akkoç; Hami Alpas; Mustafa Akçelik (pp. 387-397).

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.

Keywords: L. lactis spp. lactis ; Nisin production; Stress response; Survival; Growth

Rapid Determination of l-Glutamine using Engineered Escherichia coli Overexpressing Glutamine Synthetase by Ni-Ning Hong; Guang Yang; Jian Li; Yao-Ping Zhang; Ji-Lun Li (pp. 398-407).

A genetically engineered Escherichia coli was developed as the source of enzyme for rapidly quantifying glutamine. E. coli BL21 (DE3) cells overexpressing a glutamine synthetase from Bacillus subtilis were prepared as tube aliquots and used in a small volume of nontoxic mixture. The current method was compared to high performance liquid chromatography analysis, Sigma kit (GLN-1) and Mecke method. The method is applicable to a wide range of glutamine concentrations (0.05–2.5 mM) and correlates well to the detection results obtained from high performance liquid chromatography (Pearson correlation is 0.978 at the 0.01 level). Moreover, the whole assay procedure takes less than 15 min and uses nontoxic reagents, so it can be applied to monitor glutamine production and utilization conveniently.

Keywords: Escherichia coli ; Glutamine synthetase; l-glutamine; Rapid determination

Monopalmityloxy Shikimic Acid: Enzymatic Synthesis and Anticoagulation Activity Evaluation by Luhong Tang; Hong Xiang; Yang Sun; Liying Qiu; Dongqun Chen; Chao Deng; Wei Chen (pp. 408-415).

The monopalmityloxy shikimic acids have been synthesized from shikimic acid and palmitic acid catalyzed by Novozym 435 in 2-methyl-2-butanol. The anticoagulation activity in vivo via oral administration of monopalmityloxy shikimic acid has been evaluated through arteriovenous shunt model of rats and through the determination of thrombin time, prothrombin time, and activated partial thromboplastin time via rats. After reaction, the solid shikimic acid has been observed to dissolve in the reaction system completely. The subsequent high-performance liquid chromatography–mass spectroscopy analysis showed that the monopalmityloxy shikimic acids, as the only products, had been formed and the overall conversion rate was over 70%. The result showed that it had anti-thrombosis activity, could prolong the coagulating time and bleeding time in vivo, and lengthen the coagulating time in vitro. Compared with control group, the differences of the treatment group and aspirin group of rats are significant (P < 0.05) for prothrombin time and thrombin time, and very significant (P < 0.01) for activated partial thromboplastin time. It suggested that the product had the anticoagulation activity. The mechanism might be the co-action of the inhibition of intrinsic coagulation and the inhibition of extrinsic coagulation, and the inhibiting effect on intrinsic pathway is stronger than that on extrinsic pathway.

Keywords: Monopalmityloxy shikimic acid; Shikimic acid; Novozym 435; Anticoagulation; Anti-thrombosis activity

A New Stenotrophomonas maltophilia Strain Producing Laccase. Use in Decolorization of Synthetics Dyes by Said Galai; Ferid Limam; M. Nejib Marzouki (pp. 416-431).

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO₄ in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K _m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K _m = 700 μM), and pyrocatechol (K _m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.

Keywords: Stenotrophomonas maltophilia AAP56 ; Laccase; Decolorization; Synthetic dyes; Industrial effluents

Expression, Purification, and Characterization of Hepatitis B Virus Surface Antigens (HBsAg) in Yeast Pichia Pastoris by Rushi Liu; Qinlu Lin; Yi Sun; Xiangyang Lu; Yilan Qiu; Ye Li; Xiangrong Guo (pp. 432-444).

Prevention of the prevalence of HB depends upon the development of efficient diagnostic reagent and preventive vaccine. Pichia pastoris offers many advantages over the other expression systems in the production of recombinant HBsAg. In this study, we reported that the recombinant P. pastoris strains were cultured in shake flasks and then scaled up in a 5.0-l bioreactor: approximately 27 mg/l of the protein and the maximal cell OD at 600 nm of 310 were achieved in the bioreactor. The recombinant HBsAg was purified by three steps of purification procedures. SDS-PAGE showed that the purified recombinant HBsAg constituted only one homogeneous band of ~24 kDa. CsCl density gradient ultracentrifugation assay indicated that the density of the HBsAg was 1.2 mg/ml, which was in agreement with the natural HBsAg, the HBsAg expressed in Saccharomyces cerevisiae and in mammalian cells. Electron microscope observation revealed that the purified recombinant HBsAg was homogeneous 22-nm particles, suggesting the HBsAg expressed in P. pastoris was self-assembled to virus-like structures. Competitive ELISA indicated that P. pastoris-derived HBsAg possessed the excellent immunoreaction with anti-HBsAg. Animal immunization showed that the immunogenicity of P. pastoris-derived HBsAg was superior to that of S. cerevisiae-derived HBsAg. Together, our results demonstrated that the recombinant HBsAg expressed in P. pastoris could provide promising, inexpensive, and large-scale materials for the diagnostic reagent and vaccine to prevent HBV infection.

Keywords: Hepatitis B surface antigen; P. pastoris ; Recombinant HBsAg; Expression; Characterization

Intensification of Biocatalytical Processes by Synergistic Substrate Conversion. Fungal Peroxidase Catalyzed N-Hydroxy Derivative Oxidation in Presence of 10-Propyl Sulfonic Acid Phenoxazine by Juozas Kulys; Zilvinas Dapkunas; Robert Stupak (pp. 445-456).

Many industrial pollutants, xenobiotics, and industry-important compounds are known to be oxidized by peroxidases. It has been shown that highly efficient peroxidase substrates are able to enhance the oxidation of low reactive substrate by acting as mediators. To explore this effect, the oxidation of two N-hydroxy derivatives, i.e., N-hydroxy-N-phenyl-acetamide (HPA) and N-hydroxy-N-phenyl-carbamic acid methyl ester (HPCM) catalyzed by recombinant Coprinus cinereus (rCiP) peroxidase has been studied in presence of efficient substrate 3-(4a,10a-dihydro- phenoxazin-10-yl)-propane-1-sulfonic acid (PPSA) at pH 8.5. The bimolecular constant of PPSA cation radical reaction with HPA was estimated to be (2.5 ± 0.2)·10⁷ M⁻¹ s⁻¹ and for HPCM was even higher. The kinetic measurements show that rCiP-catalyzed oxidation of HPA and HPCM can increase up to 33,000 times and 5,500 times in the presence of equivalent concentration of high reactive substrate PPSA. The mathematical model of synergistic rCiP-catalyzed HPA–PPSA and HPCM–PPSA oxidation was proposed. Experimentally obtained rate constants were in good agreement with those calculated from the model confirming the synergistic scheme of the substrate oxidation. In order to explain the different reactivity of substrates, the docking of substrates in the active site of the enzyme was calculated. Molecular dynamic calculations show that the enzyme–substrate complexes are structurally stable. The high reactive PPSA exhibited higher affinity to enzyme active site than HPA and HPCM. Furthermore, the orientation of HPA and HPCM was not favorable for proton transfer to the distal histidine, and different substrate reactivity was explained by these diversities.

Keywords: Synergistic; Coprinus cinereus peroxidase; N-hydroxy derivative; 3-(4a,10a-dihydro- phenoxazin-10-yl)-propane-1-sulfonic acid; Kinetics; Docking modeling; Molecular dynamics

Biosorption of Lead, Copper, and Cadmium by Phanerochaete chrysosporium in Ternary Metal Mixtures: Statistical Analysis of Individual and Interaction Effects by K. Pakshirajan; T. Swaminathan (pp. 457-469).

Biosorption of three divalent metals, viz., lead, copper, and cadmium in ternary aqueous mixtures was studied using Phanerochaete chrysosporium in batch shake flasks. The mixtures were prepared containing the metals at their either varying optimum or equal initial concentration combinations in aqueous solution of pH optimum to each of the metals. Following were the optimum initial concentration ranges of the metals in mixture: lead, 60–100 mg/L; copper, 20–60 mg/L; and cadmium, 5–15 mg/L. And, for varying these optimum concentration levels of the metals, a 2³ full factorial design of experiments was employed. The results revealed that an increase in lead and cadmium concentrations helped in their better biosorption by the fungus, but an increase in initial copper concentration slightly diminished its removal. Statistical analysis of the results in the form of analysis of variance and Student t test gave a clear interpretation on the roles of both the individual metals and their interactions in the uptake of metals from mixture. Compared to the uptake of metals when presented individually, lead biosorption in mixture was found to be enhanced to a degree as high as 99%; on the other hand, copper and cadmium removals from mixtures were inhibited to the extent of 100% and 98%, respectively. However, this extent of inhibition or enhancement in the metal removals compared to the individual removals was less in mixtures containing all equal concentrations of the metals.

Keywords: Biosorption; Phanerochaete chrysosporium ; Heavy metals; Ternary metal mixture; Full factorial design of experiments; ANOVA

Callus Induction and Plant Regeneration from Different Explants of Actinidia deliciosa by Filiz Akbaş; Çiğdem Işikalan; Süreyya Namli (pp. 470-475).

In this study, an efficient procedure was developed for callus induction and regeneration of kiwifruit (Actinidia deliciosa) using different organs of shoots developed under in vitro conditions. Effects of explants source and media (M₁, 1.0 mg l⁻¹ BA + 2.0 mg l⁻¹ 2,4-D–M₂, 1.0 mg l⁻¹ NAA + 2.0 mg l⁻¹ 2,4-D) on initiation of callus were examined in order to obtain callus for organogenesis. The best callus for plant regeneration was obtained from leaf explants on Murashige and Skoog’s medium (MS) supplemented with M₂. Formation of callus from leaf of kiwifruit (A. deliciosa) was cultured in MS medium containing different concentration of N⁶-benzylaminopurin (BA; 0.0, 1.0, 2.0, 4.0, 6.0, 8.0 mg l⁻¹) for callus proliferation and plant regeneration. Although the first shoot formation was appeared in medium containing 6.0 and 8.0 mg l⁻¹ BA, the best shoots formation was obtained in medium with 4.0 mg l⁻¹ BA.

Keywords: Kiwifruit; Callus; Organogenesis; In vitro

Red Yeast Rice Fermentation by Selected Monascus sp. with Deep-Red Color, Lovastatin Production but No Citrinin, and Effect of Temperature-Shift Cultivation on Lovastatin Production by Masatoshi Tsukahara; Naoya Shinzato; Yasutomo Tamaki; Tomoyuki Namihira; Toru Matsui (pp. 476-482).

Monascus pilosus NBRC4520 was selected for functional fermented food inoculation for its high lovastatin and low citrinin production with a deep-red color. For koji (mold rice) with high lovastatin production, separation of the growth phase and lovastatin production phase by shifting the temperature from 30 to 23 °C increased lovastatin production by nearly 20 times compared to temperature-constant cultivation. In addition, citrinin was not produced even in the lovastatin production phase, although the pigment was increased. With temperature-shift cultivation, 225 μg lovastatin/g dry koji was produced in 14 days without citrinin.

Keywords: Monascus; Lovastatin; Temperature-shift cultivation; Pigment; Citrinin

Production of Cordycepin and Mycelia by Submerged Fermentation of Cordyceps militaris in Mixture Natural Culture by Chun-yan Xie; Zhen-xin Gu; Gong-jian Fan; Fei-rong Gu; Yong-bin Han; Zhi-gang Chen (pp. 483-492).

The submerged fermentation of Cordyceps militaris for cordycepin production and mycelial growth was investigated in this study. Three natural materials of brown rice paste (BRP), beerwort (B), and soybean meal juice (SMJ) were used for fermentation of C. militaris in shaking flasks. The effects of the ratio of three natural materials on dry mycelium weight (DMW) and on cordycepin yield (CY) were analyzed. D-Optional mixture design was used to optimize the ratio of these materials. Compared with the signal culture, the higher mycelial growth and cordycepin production were obtained in mixture. The analysis of Design Expert 6.0 indicated that BRP, B, and SMJ very significantly influenced (P < 0.001) DMW and CY of C. militaris, respectively. The highest DMW (18.96 g/l) and CY (2.17 mg/g) were both obtained at a ratio of 53:6:42. The experiments’ results indicated that the above mixture of these natural materials by D-optional mixture design can be used as a proper medium for the growth of mycelium and the production of cordycepin.

Keywords: Cordyceps militaris ; Submerged cultivation; Medium; D-optimal mixture design

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #2)