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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #2)

The Effect of Temperature on the Structure and Function of a Cellulose-Degrading Microbial Community by Yucai Lü; Ning Li; Dachun Gong; Xiaofen Wang; Zongjun Cui (pp. 219-233).

The purpose of this study was to investigate the effect of temperature on the structure and straw degradation capability of a microbial community grown from wheat straw compost. Two cellulolytic microbial communities, WDC1 and WDC2, were obtained from compost. The communities had been cultured under 50 and 60 °C by continuous enrichment, respectively. The wheat straw degradation capabilities were 45.69 % (WDC1) and 59.5 % (WDC2). By changing the culture temperatures, two new stable communities were obtained: WDC1-6N (WDC1, cultivated at 60 °C for eight generations) and WDC2-5N (WDC2, cultivated at 50 °C for eight generations). The wheat straw degradation capabilities for the new communities were 59.75 and 52.60 %, respectively. The results showed that compared to 50 °C, the wheat straw degradation capability of the communities cultured at 60 °C was stronger. Sequencing of selected denaturing gradient gel electrophoresis (DGGE) bands and analysis of DGGE profiles indicated that the WDC2 structure was significantly different from the structure of WDC1. This was so even though the two communities were enriched from the same compost. With the change of culture temperature, the community structures underwent significant transitions. Included communities were thermophilic, anaerobic bacteria, and any cellulolytic bacteria (e.g., Clostridium thermocellum) that were active and abundant at conditions under 60 °C. These results have the potential to significantly aid in the enrichment of a cellulose-degrading community from the environment and to enhance the community capability to conduct straw biotransformation.

Keywords: Cellulose degradation; Wheat straw; Microbial community; Cultivation temperature; Community structure

Bromelain Enzyme from Pineapple: In Vitro Activity Study under Different Micropropagation Conditions by Jaci Lima Vilanova Neta; Ana da Silva Lédo; Aloisio André Bonfim Lima; José Carlos Curvelo Santana; Nadjma Souza Leite; Denise Santos Ruzene; Daniel Pereira Silva; Roberto Rodrigues de Souza (pp. 234-246).

The aim of this work was to evaluate the activity of bromelain in pineapple plants (Ananas comosus var. Comosus), Pérola cultivar, produced in vitro in different culture conditions. This enzyme, besides its pharmacological effects, is also employed in food industries, such as breweries and meat processing. In this work, the enzymatic activity was evaluated in the tissues of leaves and stems of plants grown in culture medium without plant growth regulator. The most significant levels of bromelain were observed in leaf tissue after 4 months of culture in vitro in medium with a filter paper bridge, followed by medium gelled by the agar. The results of this study, regarding the different structures of the pineapple (leaves and stems) in vitro showed that the activity of bromelain varied depending on the culture conditions, the time and structure of which was quantified, ensuring a viable strategy in the production of seedlings with high levels of bromelain in subsequent phases of micropropagation.

Keywords: Bromelain; Ananas comosus var. Comosus ; Pineapple; Secondary metabolic; Culture in vitro

Arginine Residues in the C-terminal and their Relationship with the Analgesic Activity of the Toxin from the Chinese Scorpion Buthus martensii Karsch (BmK AGP-SYPU1) by Yu Wang; Yong-Bo Song; Guang-Zhao Yang; Yong Cui; Yong-Shan Zhao; Yan-Feng Liu; Yan Ma; Chun-Fu Wu; Jing-Hai Zhang (pp. 247-255).

In this study, we investigated the functional role of arginines in the C-terminal (65–67) of BmK AGP-SYPU1, an analgesic peptide from the Chinese scorpion Buthus martensii Karsch. Using site-directed mutagenesis, arginines at the C-terminal (65–66) were deleted or added to the C-terminal (67). The genes for three mutants of BmK AGP-SYPU1 were obtained by PCR. An analgesic activity assay was used to evaluate the role of arginine residues in the analgesic activity. The three-dimensional structure of BmK AGP-SYPU1 was established by homology modeling. As a result, we showed that the arginines in the C-terminal are crucial for the analgesic activity and may be located at analgesic functional sites. Our work has implications for further modification of scorpion toxins to obtain new analgesic peptides with enhanced activity.

Keywords: BmK AGP-SYPU1; C-terminal positively charged residues; Arginine; Site-directed mutagenesis; Analgesic activity

Heat Shock Treatment Improves Trametes versicolor Laccase Production by Feng Wang; Chen Guo; Tao Wei; Tian Zhang; Chun-Zhao Liu (pp. 256-265).

An efficient heat shock strategy has been developed to improve laccase production in submerged Trametes versicolor cultures. The optimized heat shock strategy consists of subjecting T. versicolor mycelial pellets to three heat shock treatments at 45 °C for 45 min, starting at culture day 0, with a 24-h interval between treatments. Laccase production increased by more than 1.6-fold relative to the control in both flasks and a 5-L bioreactor because the expression of the laccase gene was enhanced by heat shock induction. The present work demonstrates that heat shock induction is a promising method because it both improves fungal laccase production and has a good potential in industrial application.

Keywords: Heat shock; Trametes versicolor ; Laccase; mRNA

Improvement of Bioethanol Productivity of Immobilized Saccharomyces Bayanus with Using Sodium Alginate-Graft-Poly(N-Vinyl-2-Pyrrolidone) Matrix by Murat İnal; Mustafa Yiğitoğlu (pp. 266-278).

In this study, immobilization conditions and bioethanol production characteristics of immobilized Saccharomyces bayanus were investigated into sodium alginate-graft-poly(N-vinyl-2-pyrrolidone; NaAlg-g-PVP) matrix. The matrix that crosslinked with calcium clorid was used for immobilization of S. bayanus. Bioethanol productivity of the NaAlg-g-PVP matrix was found to increase from 4.21 to 4.84 gL⁻¹ h⁻¹ when compared with the convential sodium alginate matrix. The production of bioethanol was affected by initial glucose concentration and percentage of immobilized cell beads in fermentation medium. Bioethanol productivity was increased from 3.62 to 4.84 gL⁻¹ h⁻¹ while the glucose concentration increasing from 50 to 100 gL⁻¹. Due to the increase in percentage from 10 to 20 % of immobilized cell beads in the fermentation medium, bioethanol productivity was increased from 4.84 to 8.68 gL⁻¹ h⁻¹. The cell immobilized NaAlg-g-PVP beads were protected 92 % of initial activity after six repeated fermentation.

Keywords: Sodium alginate; N-vinyl-2-pyrrolidone; Graft copolymerization; Bioethanol production; Immobilization; Saccharomyces bayanus

Adsorption Equilibrium, Kinetics and Thermodynamics of α-Amylase on Poly(DVB-VIM)-Cu⁺² Magnetic Metal-Chelate Affinity Sorbent by Bilgen Osman; Ali Kara; Emel Demirbel; Senay Kök; Necati Beşirli (pp. 279-294).

Designing an immobilised metal ion affinity process on large-scale demands that a thorough understanding be developed regarding the adsorption behaviour of proteins on metal-loaded gels and the characteristic adsorption parameters to be evaluated. In view of this requirement, interaction of α-amylase as a model protein with newly synthesised magnetic-poly(divinylbenzene-1-vinylimidazole) [m-poly(DVB-VIM)] microbeads (average diameter, 53–212 μm) was investigated. The m-poly(DVB-VIM) microbeads were prepared by copolymerising of divinylbenzene (DVB) with 1-vinylimidazole (VIM). The m-poly(DVB-VIM) microbeads were characterised by N₂ adsorption/desorption isotherms, electron spin resonance, elemental analysis, scanning electron microscope and swelling studies. Cu²⁺ ions were chelated on the m-poly(DVB–VIM) beads and used in adsorption of α-amylase in a batch system. The maximum α-amylase adsorption capacity of the m-poly(DVB–VIM)–Cu²⁺ beads was determined as 10.84 mg/g at pH 6.0, 25 °C. The adsorption data were analyzed using three isotherm models, which are the Langmuir, Freundlich and Dubinin–Radushkevich isotherm models. The pseudo-first-order, pseudo-second-order, modified Ritchie’s-second-order and intraparticle diffusion models were used to test dynamic experimental data. The study of temperature effect was quantified by calculating various thermodynamic parameters such as Gibbs free energy, enthalpy and entropy changes.

Keywords: Immobilised metal ion affinity; IMA; Protein adsorption; Magnetic beads; α-Amylase

Magnetic Bead Cellulose as a Suitable Support for Immobilization of α-Chymotrypsin by Petr Prikryl; Jiri Lenfeld; Daniel Horak; Marie Ticha; Zdenka Kucerova (pp. 295-305).

Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol–gel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of α-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. α-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.

Keywords: Enzyme immobilization; Human gastric juice; Immobilized α-chymotrypsin; Magnetic bead cellulose; Porcine pepsin A

Apple Hypanthium Firmness: New Insights from Comparative Proteomics by Claudius Marondedze; Ludivine A. Thomas (pp. 306-326).

Fruit firmness constitutes an important textural property and is one of the key parameters for estimating ripening and shelf life, which has a major impact on commercialization. In order to decipher the mechanisms related to firmness of apples (Malus × domestica Borkh.), two-dimensional gel electrophoresis (2-DE) was used to compare the total proteome of high and low firmness phenotypes from apple hypanthia of a ‘Golden Delicious’ × ‘Dietrich’ population. A total of 36 differentially regulated protein spots were positively identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and then validated against the Malus expressed sequence tags (EST) database. The findings of this study indicated a lower expression of ethylene biosynthesis related proteins in the high firmness phenotype, which could be linked to the slowing down of the ripening and softening processes. The reduced accumulation of proteins involved in ethylene biosynthesis juxtaposed to the upregulation of a transposase and a GTP-binding protein in the high firmness phenotype. The results also showed higher expression of cytoskeleton proteins in the high firmness phenotype compared to the low firmness phenotype, which play a role in maintaining cell structure and possibly fruit integrity. Finally, a number of proteins involved in detoxification and defense were expressed in fruit hypanthium. This proteomic study provides a contribution towards a better understanding of regulatory networks involved in fruit hypanthium firmness and/or softening, which could be instrumental in the development of improved fruit quality.

Keywords: Cytoskeleton; Ethylene biosynthesis; Fruit firmness; Hypanthium; Proteomics; Two-dimensional electrophoresis

Characterization of a New Cold-adapted Lipase from Pseudomonas sp. TK-3 by Daisuke Tanaka; Satoru Yoneda; Yoko Yamashiro; Akihiro Sakatoku; Takuro Kayashima; Kasumi Yamakawa; Shogo Nakamura (pp. 327-338).

A psychrotrophic Pseudomonas sp. TK-3 was isolated from dirty and cool stream water in Toyama, Japan from which we cloned and characterized the bacterial lipase LipTK-3. The sequenced DNA fragment contains an open reading frame of 1,428 bp that encoded a protein of 476 amino acids with an estimated molecular mass of 50,132 Da. The lipase showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 20–25 °C, lower than in most other subfamily Ι.3 lipases. The lipase exhibited about 30 % of maximal activity at 5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by EDTA and was highly dependent on Ca²⁺. Tricaprylin and p-nitrophenyl caprylate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipTK-3 also showed high activity towards natural substrates including edible vegetable oils and animal fats. Furthermore, LipTK-3 was very active and stable in the presence of several detergents, metal ions, and organic solvents. This cold-adapted lipase may prove useful for future applications.

Keywords: Pseudomonas sp.; Cold-adapted lipase; Characterization

Agrobacterium-Mediated Transformation in Alpinia galanga (Linn.) Willd. for Enhanced Acetoxychavicol Acetate Production by Kiranmayee Rao; Bhuvaneswari Chodisetti; Lakshmi Narasu Mangamoori; Archana Giri (pp. 339-347).

Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A 4, 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A 4 transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A 4. The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.

Keywords: Alpinia galanga ; Acetoxychavicol acetate; Agrobacterium rhizogenes ; Hairy roots; HPLC

Mass Cultivation of Microalgae on Animal Wastewater: a Sequential Two-Stage Cultivation Process for Energy Crop and Omega-3-Rich Animal Feed Production by Wenguang Zhou; Bing Hu; Yecong Li; Min Min; Michael Mohr; Zhenyi Du; Paul Chen; Roger Ruan (pp. 348-363).

In this study, 97 microalgal strains purchased from algae bank and 50 microalgal strains isolated from local waters in Minnesota were screened for their adaptability growing on a 20-fold diluted digested swine manure wastewater (DSMW). A pool of candidate strains well adapted to the DSMW was established through a high-throughput screening process. Two top-performing facultative heterotrophic strains with high growth rate (0.536 day⁻¹ for UMN 271 and 0.433 day⁻¹ for UMN 231) and one strain with high omega-3 unsaturated fatty acid (EPA, 3.75 % of total fatty acids for UMN 231) were selected. Subsequently, a sequential two-stage mixo-photoautotrophic culture strategy was developed for biofuel and animal feed production as well as simultaneous swine wastewater treatment using above two strains. The maximal biomass concentration and lipid content at the first and second stages reached 2.03 g/L and 23.0 %, and 0.83 g/L and 19.0 % for UMN 271 and UMN 231, respectively. The maximal nutrient removals for total phosphorus and ammonia after second-stage cultivation were 100 and 89.46 %, respectively. The experiments showed that this sequential two-stage cultivation process has great potential for economically viable and environmentally friendly production of both renewable biofuel and high-value animal feed and at the same time for animal wastewater treatment.

Keywords: Animal feed; Swine manure wastewater; Energy crop; Facultative heterotrophic microalgae; Mixo-photoautotrophic cultivation; Omega-3 fatty acid

Lipase-Catalyzed Preparation of Diacylglycerol-Enriched Oil from High-Acid Rice Bran Oil in Solvent-Free System by Zhihua Song; Yuanfa Liu; Qingzhe Jin; Lei Li; Xingguo Wang; Jianhua Huang; Ruijie Liu (pp. 364-374).

The ability of immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) to catalyze the reaction of high-acid rice bran oil (RBO) and monoglyceride (MG) for diacylglycerol-enriched rice bran oil (RBO-DG) preparation was investigated. The effects of substrate ratio, reaction temperature, time, and enzyme load on the respective content of free fatty acid (FFA) and DG in the final RBO-DG products was investigated. Enzyme screening on the reaction was also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (50–70 °C), the enzyme load (2–6 %; relative to the weight of total substrates), and the reaction time (4–8 h) on the respective content of FFA and DG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values. The optimum preparation conditions were as follows: MG/RBO, 0.25; temperature, 56 °C; enzyme load, 4.77 %; and reaction time, 5.75 h. Under the suggested conditions, the respective content of FFA and DG was 0.28 and 27.98 %, respectively. Repeated reaction tests indicated that Lipozyme RM IM could be used nine times under the optimum conditions with 90 % of its original catalytic activity still retained.

Keywords: Diacylglycerol-enriched rice bran oil (RBO-DG); Enzymatic catalyzed; Response surface methodology (RSM)

Production of XynX, a Large Multimodular Protein of Clostridium thermocellum, by Protease-Deficient Bacillus subtilis Strains by Nguyen Dinh Phuong; Yu Seok Jeong; Thangaswamy Selvaraj; Sung Kyum Kim; Yong Ho Kim; Kyung Hwa Jung; Jungho Kim; Han Dae Yun; Sui-Lam Wong; Jung-Kul Lee; Hoon Kim (pp. 375-382).

XynX of Clostridium thermocellum is a large, multimodular xylanase of 116 kDa. An Escherichia coli transformant carrying the entire xynX produced three active truncated xylanase species of 105, 85, and 64 kDa intracellularly. The Bacillus subtilis WB700 transformant with the xynX, a strain deficient in seven proteases including Vpr, secreted two active truncated xylanase species of 65 and 44 kDa. The B. subtilis WB800 transformant with xynX, a strain deficient in eight proteases including Vpr and WprA, secreted more active enzymes, 8.46 U ml⁻¹, mostly in the form of 105 and 85 kDa, than the WB700 transformant, 6.93 U ml⁻¹. This indicates that the additional deletion of wprA enabled the WB800 to secrete XynX in its intact form. B. subtilis WB800 produced more total enzyme activity than E. coli (1,692 ± 274 U vs. 141.9 ± 27.1 U), and, more importantly, secreted almost all the enzyme activity. The results suggest the potential use of B. subtilis WB800 as a host system for the production of large multimodular proteins.

Keywords: Clostridium thermocellum XynX; Protease-deficient Bacillus subtilis WB800; Expression of multimodular enzyme; Truncated enzyme; Intact form

Identification and Expression of an APETALA2-Like Gene from Nelumbo nucifera by Zhaolei Liu; Chunsun Gu; Fadi Chen; Jiafu Jiang; Yinghao Yang; Peiling Li; Sumei Chen; Zhen Zhang (pp. 383-391).

Arabidopsis transcription factor APETALA2 (AP2) controls multiple aspects of plant growth and development, including seed development, stem cell maintenance, and specification of floral organ identity. Based on sequence similar of Arabidopsis AP2 and its homologues genes from other plant species, degenerate RT-PCR and rapid amplification of cDNA ends assay were used to clone AP2 genes from lotus (Nelumbo nucifera). A 2,048-bp cDNA fragment was obtained, which contains a 1,536-bp open reading frame encoding a protein of 511 amino acids. The protein contains two AP2 domains that are conserved in AP2 proteins from other plant species, thus was named as N. nucifera APETALA2 (NnAP2). Quantitative RT-PCR revealed that NnAP2 gene was expressed in flowers, roots, leaves, and stems of N. nucifera, with flowers which have the highest transcript levels. Further analysis showed that in all five lotus cultivars examined, including “Zhongguogudailian,” “Yaoniangyujiao,” “Jinxia,” “Hongtailian,” and “Yiliangqianban,” petals always have the highest expression levels when compared with the other four flower organs, though the number of petals in these cultivars ranged from simple to thousands. However, NnAP2 expression level in four nonsimple petal flower cultivars was higher than that in the simple petal flower cultivar Zhongguogudailian, indicating that NnAP2 may play a role in specification of petal identity during the evolutionary process of the ancient species N. nucifera.

Keywords: APETALA2; Nelumbo nucifera ; Expression; Flowers

Bifidobacterium longum l-Arabinose Isomerase—Overexpression in Lactococcus lactis, Purification, and Characterization by Noora Salonen; Antti Nyyssölä; Kalle Salonen; Ossi Turunen (pp. 392-405).

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum l-AI was characterized using d-galactose and l-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0–6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K _m value was 120 mM for l-arabinose and 590 mM for d-galactose. The V _max was 42 U mg⁻¹ with l-arabinose and 7.7 U mg⁻¹ with d-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg²⁺, Mn²⁺). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum l-AI as the catalyst at 35 °C, equilibrium yields of 36 % d-tagatose and 11 % l-ribulose with 1.67 M d-galactose and l-arabinose, respectively, as the substrates were reached.

Keywords: l-Arabinose isomerase; Bifidobacterium longum ; d-Tagatose; Protein production; Lactococcus lactis

Effect of Pretreatment Severity on Accumulation of Major Degradation Products from Dilute Acid Pretreated Corn Stover and Subsequent Inhibition of Enzymatic Hydrolysis of Cellulose by Byung-Hwan Um; G. Peter van Walsum (pp. 406-420).

The concept of reaction severity, which combines residence time and temperature, is often used in the pulp and paper and biorefining industries. The influence of corn stover pretreatment severity on yield of sugar and major degradation products and subsequent effects on enzymatic cellulose hydrolysis was investigated. The pretreatment residence time and temperature, combined into the severity factor (Log R _o), were varied with constant acid concentration. With increasing severity, increasing concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) coincided with decreasing yields of oligosaccharides. With further increase in severity factor, the concentrations of furans decreased, while the formation of formic acid and lactic acid increased. For example, from severity 3.87 to 4.32, xylose decreased from 6.39 to 5.26 mg/mL, while furfural increased from 1.04 to 1.33 mg/mL; as the severity was further increased to 4.42, furfural diminished to 1.23 mg/mL as formate rose from 0.62 to 1.83 mg/mL. The effects of dilute acid hydrolyzate, acetic acid, and lignin, in particular, on enzymatic hydrolysis were investigated with a rapid microassay method. The microplate method gave considerable time and cost savings compared to the traditional assay protocol, and it is applicable to a broad range of lignocellulosic substrates.

Keywords: Dilute acid; Pretreatment; Accelerated Solvent Extraction (ASE); Severity; Enzymatic hydrolysis

Enzymatic Conversion of Xylan Residues from Dilute Acid-Pretreated Corn Stover by Joseph Shekiro; Erik M. Kuhn; Michael J. Selig; Nicholas J. Nagle; Stephen R. Decker; Richard T. Elander (pp. 421-433).

Enzymatic conversion of oligomeric xylose and insoluble xylan remaining after effective pretreatment offers significant potential to improve xylan-to-xylose yields while minimizing yields of degredation products and fermentation inhibitors. In this work, a commercial enzyme cocktail is demonstrated to convert up to 70 % of xylo-oligomers found in dilute acid-pretreated hydrolyzate liquor at varying levels of dilution when supplemented with accessory enzymes targeting common side chains. Commercial enzyme cocktails are also shown to convert roughly 80 % of insoluble xylan remaining after effective high-solids, dilute acid pretreatment.

Keywords: Cellulosic ethanol; Xylan; Xylose; Xylanase; Lignocellulosic biomass; Enzyme conversion; Xylo-oligomer; Xylo-oligomer conversion

Three-Stage Extraction of Gelatines from Tendons of Abattoir Cattle: 2—Properties of Gelatines by Pavel Mokrejs; Svatopluk Sukop; Petr Svoboda (pp. 434-445).

The subject of our previous paper (part 1) was three-stage extraction of gelatines from short cattle tendons. In this paper, we studied influence of extraction conditions on quality of produced gelatines—protein content, ash content, rigidity of gelatine gels, viscosity, and distribution of molecular weights. Ash content of gelatines ranged in limits 3.5–10.5 %. Rigidity of the gels was influenced by extraction conditions and by elastin content in gelatines; highest-quality gels display rigidity of 213 Bloom. Viscosity of gelatines ranged from 2.47 to 12.11 mPa s. Molecular weights of gelatines display a proportion of fractions from 20 to 36 kDa, a quite high proportion of fractions from approximately 50 to 100 kDa, in some cases even fractions above 200 kDa appear. It was found that transition temperature and melting temperature of gelatines extracted from tendons are in accord with data on gelatines obtained by traditional extraction techniques from skins and bones. With respect to efficiency of the whole extraction process and to quality of extracted gelatine, we may recommend extraction conditions as follows: in the 1st processing stage, shaking degreased starting material with water in ratio 1:10 at 25 °C for 5 h; in the 2nd stage, treating the swelled material with 5 % (w/w) added proteolytic enzyme at 40 °C for 25 h; in 3rd stage, extracting gelatine while boiling for 16 min.

Keywords: Cattle tendons; Gelatine; Gel strength; Molecular weight distribution; Thermal analysis; Properties

Cloning, Sequence, and Association Analysis of Porcine OSAP Gene by Yu Lei; Liu Yonggang (pp. 446-454).

Ovary-specific acidic protein (OSAP) is an important reproduction-related gene. In this study, we cloned the full-length cDNA sequence of porcine OSAP gene through the rapid amplification of cDNA ends method. The porcine OSAP gene encodes a protein of 238 amino acids which shares high homology with the OSAP of five species: rhesus monkey (72 %), human (70 %), cattle (68 %), horse (68 %), and dog (61 %). This gene is structured in four exons and three introns as revealed by computer-assisted analysis. PCR-RFLP was established to detect the GU373669:c.854A>T substitution of porcine OSAP gene mRNA and association of this mutation with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations. Results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White sows and Landrace sows (P < 0.05). Therefore, OSAP gene could be a useful candidate gene in selection for increasing litter size in pigs. These data serve as a foundation for further insight into this novel porcine gene.

Keywords: Pig; OSAP; Polymorphism; Reproductive traits

Adaptation of Glycolysis and Growth to Acetate in Sporolactobacillus sp. Y2-8 by Dong Liu; Yong Chen; An Li; Ting Zhao; Tao Zhou; Bingbing Li; Jingjing Xie; Xiaochun Chen; Jianxin Bai; Yanan Hu; Bingfang He; Hanjie Ying (pp. 455-463).

Exogenous addition of a low concentration of acetate (2 g/L sodium acetate) effectively decreased acetic acid excretion and lowered the ATP content in Sporolactobacillus sp. Y2-8 without any growth defect although the acetate could not be utilized at an initial glucose concentration of 150 g/L. This induced an enhanced glycolytic flux with increased specific activities of hexokinase and phosphofructokinase, probably to compensate for the lowered efficiency of ATP production. However, with increasing concentrations (5 g/L sodium acetate), acetate was utilized first before being produced again, causing a growth lag at the transition. Glucose consumption was also reduced at high acetate concentrations, resulting in decreased d-lactic acid production. These results demonstrate that acetate plays a significant role in regulating glycolysis and growth of Sporolactobacillus.

Keywords: Glycolytic flux; Sporolactobacillus ; Acetate accumulation; ATP content; d-lactic acid

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #2)