Biotechnology and Applied Biochemistry (Biotechnol Appl Biochem)

Publications in this journal

• Article: Expression, purification, and lipolytic activity of recombinant human serum albumin fusion proteins with one domain of human growth hormone in Pichia pastoris.

  Furong Wang, Min Wu, Wenhui Liu, Qi Shen, Hongying Sun, Shuqing Chen
  [show abstract] [hide abstract]
  ABSTRACT: Human growth hormone (hGH) can mobilize lipid and inhibit the synthesis of triglycerides. However, it is not a potentially useful drug for treating obesity because it has many other actions resulting in several side effects. Here, we report a novel approach to develop the lipolytic function of hGH. The amino terminus of hGH was replaced by an inactive protein so that the actions unrelated to lipolytic function would be avoided. The fusion genes encoding human serum albumin (HSA) and lipolytic domain of hGH were constructed and expressed in Pichia pastoris. The recombinant proteins were purified and characterized by SDS-PAGE and Western blot. The preliminary stability tests demonstrated that HSA-hGH166-191 and HSA-hGH177-191 were stable at different pH levels after four days at 37°C. Lipolytic activity assay revealed that fusion proteins could increase the amounts of glycerol released from the isolated adipocytes. The HSA fusion proteins constructed in this work can be further developed as antiobesity agents.
  Biotechnology and Applied Biochemistry 06/2013;
• Article: In silico design and construction of metal binding hybrid proteins for specific removal of cadmium based on CS3 pili display on the surface of Escherichia coli.

  Vajiheh Eskandari, Bagher Yakhchali, Mehdi Sadeghi, Ali Asghar Karkhane
  [show abstract] [hide abstract]
  ABSTRACT: In this study, through a combination of bioinformatics and genetic engineering procedures, high-affinity metal binding peptides were designed and expressed on the surface of Escherichia coli for selective Cd(2+) adsorption. Putative cadmium binding motifs were identified by searches against the Prosite database and permissive sites in the major subunit (CstH) of the enterotoxigenic Escherichia coli pili were predicted based on the data derived from modeling of 3-D structures, secondary structure prediction and assignment, inspection of protein hydropathy and exposed regions and also protein interaction sites. The metal binding motifs were inserted into one permissive site of the CstH (amino acid 38) with the aid of SOEing PCR technique. The capacity and selectivity of the recombinant bacteria displaying hybrid pili to adsorb cadmium were evaluated with the atomic absorption procedure. The levels of Cd(2+) accumulation in the recombinant E. coli strains were 13.9 and 11.33 -fold higher than those in the control strain. Cd(2+) was selectively uptaken from a solution containing equal concentrations of four metals, resulting in more than 90% of the total adsorbed metals being Cd(2+) , showing a relatively high affinity for Cd(2+) over other co-existing metal ions. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 06/2013;
• Article: Experimental characterization of next generation expanded bed adsorbents for capture of a recombinant protein expressed in high cell density yeast fermentation.

  William Kelly, Phillip Garcia, Stefanie McDermott, Peter Mullen, Guy Kamguia, Gerard Jones, Antonio Ubiera, Kent Göklen
  [show abstract] [hide abstract]
  ABSTRACT: Expanded bed adsorption (EBA) can be particularly useful in protein recovery from high cell density fermentation broth where conventional methods for harvest and clarification, such as continuous centrifugation and depth filtration, demand long processing times and are associated with high costs. In this work the use of next-generation high particle density EBA adsorbents, including two mixed mode resins, for the direct capture of a recombinant protein expressed in yeast at high cell densities is evaluated. Using classical experimental approaches and under different conditions (pH, salt etc..), Langmuir isotherm parameters for these resins are obtained along with pore diffusivity values. Additional batch adsorption studies with Fastline® MabDirect, the resin that demonstrated the highest static binding capacity for the recombinant protein of interest under the conditions evaluated in this study, indicate competitive binding of non-target proteins and approximately a 30% reduction in equilibrium binding capacity to 50 mg/ml settled bed in the presence of a 5-10% cell concentration. Packed bed dynamic binding capacities for the MabDirect resin (25 - 40 mg/ml packed bed) were significantly higher than for the Fastline® HSA resin and for the MabDirect MM resin in expanded bed mode (5-10 mg/ml settled bed). Bed expansion behavior for the mMabDirect MM resin along with process yield and eluate purity are identified as a function of linear velocity and cell density; demonstrating process feasibility for pilot scale use. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 06/2013;
• Article: Isolation and enzymatic properties of a nonspecific Acid phosphatase from Vigna aconitifolia seeds.

  Asha Anand, Pramod Kumar Srivastava
  [show abstract] [hide abstract]
  ABSTRACT: Acid phosphatase (EC 3.1.3.2) from Vigna aconitifolia seeds was purified to apparent homogeneity by using ammonium sulphate fractionation and cation-exchange chromatography (CM-cellulose). The enzyme was 228 fold purified with 14.6% recovery. Analytical gel filtration chromatography on Sephadex G-200 column showed that Mr of native enzyme was 58 kDa and denaturing polyacrylamide gel electrophoresis demonstrated that it was made up of two subunits of 24 and ̴ 27 kDa. The enzyme showed its optimum activity at pH 5.0 and 60 ⁰C. It exhibited broad substrate specificity and showed higher specificity constant for p-NPP, Na β-Naphthyl phosphate and AMP. Cu(+2) , Mo(+6) , Fe(+3) , phosphate and fluoride ions were reported as strong inhibitors for the enzyme. Active site study for the enzyme demonstrated that tryptophan and aspartic acid may be important for the catalysis. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 06/2013;
• Article: Hydrogen-producing Escherichia coli strains, overexpressing lactose permease: FT-IR analysis of the lactose-induced stress.

  Mara Grube, Ilze Dimanta, Marita Gavare, Inese Strazdina, Janis Liepins, Talis Juhna, Uldis Kalnenieks
  [show abstract] [hide abstract]
  ABSTRACT: Lactose permease gene (lacY) was overexpressed in the septuple knock-out mutant of Escherichia coli, previously engineered for hydrogen production from glucose. It was expected that raising the lactose transporter activity would elevate the intracellular lactose concentration, inactivate the lactose repressor, induce the lactose operon, and in result, stimulate the overall lactose consumption and conversion. However, overexpression of the lactose transporter caused a considerable growth delay of the recombinant strain on lactose, resembling to some extent the 'lactose killing' phenomenon, described by Dykhuizen and Hartl [1]. Therefore, the recombinant strain was subjected to selection on lactose-containing media. Selection on plates with 3% lactose yielded a strain with a decreased content of the recombinant plasmid, yet with an improved ability to grow and produce hydrogen on lactose. Macromolecular analysis of its biomass by means of FT-IR spectroscopy demonstrated that increase of the cellular polysaccharide content might contribute to the adaptation of E. coli to lactose stress. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Human Umblical Cord Derived Mesenchymal Stem Cells Can Secret Insulin In Vitro and In Vivo.

  Zahra Boroujeni, Ahmad Aleyasin
  [show abstract] [hide abstract]
  ABSTRACT: Diabetes mellitus is characterized by autoimmune destruction of pancreatic beta cells, leading to decrease insulin production. Differentiation of mesenchymal stem cells (MSCs) into insulin producing cells offers novel ways to diabetes treatment. MSCs can be isolated from the human umbilical cord tissue and differentiate into insulin-secreting cells. Human umbilical cord derived stem cells (hUDSCs) were obtained after birth, selected by plastic adhesion, and characterized by flow cytometric analysis. Human UDSCs transduced with Non-integrated lentivirus harboring PDX1 (Non-integrated LV-PDX1) and cultured in differentiation medium in 21 days. Pancreatic duodenum homeobox protein-1 (PDX1) is a transcription factor in pancreatic development. Significant expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2), somatostatin were detected by quantitative RT-PCR (P < 0.05). PDX1 and insulin proteins were shown by immunocytochemistry analysis. The insulin secretion of hUDSCs (PDX1+) in the high-glucose medium was 1.8 μU/ml. They were used for treatment of diabetic rats and could decrease the blood glucose level from 400 mg/dL to the normal level in 4 days. In conclusion, our results demonstrated that hUDSCs are able to differentiate into insulin producing cells by transduction with non-integrated LV-PDX1. This hUDSCs (PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Strain and temperature dependent changes of fatty acid composition in Wickerhamomyces anomalus and Blastobotrys adeninivorans.

  Matilda Olstorpe, Jana Pickova, Anders Kiessling, Volkmar Passoth
  [show abstract] [hide abstract]
  ABSTRACT: The fatty acid (FA) profiles of two strains of the yeasts Wickerhamomyces anomalus and Blastobotrys (Arxula) adeninivorans at cultivation temperatures from 15°C-30°C were characterised. Beside of the common even numbered C16 and C18 FAs, substantial proportions of the uneven numbered C17:1 were found in both species. C18:3 (n-3) (alpha linolenic acid) made up to 3% of the total FAs in all strains. Considerable strain differences occurred, both with regard to the presence of single FAs and parameters like double binding index (DBI) and C16:C18 ratio. W. anomalus J121 formed C18:1 (n-5) (up to 10.9% of the total FAs) but no C18:1 (n-7), while in W. anomalus VKM160 no C18:1 (n-5) was found but up to 14.6% C18:1 (n-7). Similarly, B. adeninivorans CBS 8244 formed exclusively C18:1 (n-7) (maximum 9%) and CBS 7377 C18:1 (n-5) (maximum 12.6%). W. anomalus J121 had the lowest DBI (0.72) at 15°C, the highest (0.92) at 20°C, and then the values decreased with increasing temperatures. In W. anomalus VKM 160 and both B. adeninivorans-strains, DBI was highest at 15°C and decreased with increasing temperatures. In J121, C16:C18 ratio was highest at 15°C to decrease at higher temperatures, while in the other strains an opposite trend was observed. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Mutational analysis of conserved regions harbouring catalytic triad residues of the levansucrase protein encoded by lsc-3 (lsc3) gene of Pseudomonas syringae pv. tomato DC3000.

  Karin Mardo, Triinu Visnapuu, Heiki Vija, Triin Elmi, Tiina Alamäe
  [show abstract] [hide abstract]
  ABSTRACT: Levansucrase encoded by lsc3 (lsc-3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, eighteen single amino acid mutants of thirteen positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219 and Glu303 were proved as members of catalytic triad. Respective alanine replacement mutants were practically inactive with their kcat values reduced up to ∼130 000 times. Additionally, requirement of Trp61, Gln301 and Arg304, located in conserved sequence blocks around the catalytic triad positions, for the catalysis was shown. The catalytic significance of position equivalent to Arg304 was shown for levansucrases for the first time. Replacement of Gln301 specifically affected polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225 and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. One mg of Lsc3 synthesized in 20 h reaction with 1200 mM sucrose 15.4 g of FOS with degree of polymerization from 3 to 7. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 litre of induced culture of recombinant Escherichia coli. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: An effective protein extraction method for two-dimensional electrophoresis in the anticancer herb (Andrographis paniculata Nees.).

  Daryush Talei, Alireza Valdiani, Mohd Puad Abdullah
  [show abstract] [hide abstract]
  ABSTRACT: Proteomic analysis of plants relies on high yields of pure protein. In plants, protein extraction and purification present a great challenge due to accumulation of a large amount of interfering substances including polysaccharides, polyphenols and secondary metabolites. Therefore, it is necessary to modify the extraction protocols. A study was conducted to compare four protein extraction and precipitation methods for proteomic analysis. The results showed significant differences in protein contents among the four methods. The chloroform-TCA-acetone method using HEPES buffer provided the best results in terms of protein contents, pellets, spot resolution and intensity, unique spots detected. An overall of 83 qualitative or quantitative significant differential spots were found among the four methods. Based on the 2D gel map, the method is expected to benefit the development of high-level proteomic and biochemical studies in Andrographis paniculata which may also be applied to other recalcitrant medicinal plant tissues. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Recombinant l-phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent.

  Olga O Babich, Vadim S Pokrovsky, Natalia Yu Anisimova, Nikolai N Sokolov, Alexander Yu Prosekov
  [show abstract] [hide abstract]
  ABSTRACT: The recombinant producer strain expressing Rhodosporidium toruloides l-phenylalanine ammonia lyase (PAL) has been obtained, and a purification procedure of PAL has been developed. The purified enzyme, PAL, has the following biochemical and catalytic characteristics: Km for l-Phe of 0.49 mM, pH optimum at 8.5, and temperature optimum at 50°C. PAL exhibited a significant cytotoxic effect toward the following cell lines: MCF7 (IC50 = 1.97 U/mL), DU145 (IC50 = 7.3 U/mL), which are comparable with E. coli l-asparaginase type-II cytotoxicity in vitro. Administration of PAL (200-400 U/kg) to L5178y-bearing mice for five times (a total dose of 1000-2000 U/kg) was well tolerated and showed the increase of life span (ILS) = 12-16%, P < 0.05. Data obtained suggest that PAL from R. toruloides has a potential for cancer treatment.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Influence of ammonium salts on the lipase/esterase activity assay using p-nitrophenyl esters as substrates.

  Hong De Yan, Yin Jun Zhang, Hong Cai Liu, Jian Yong Zheng, Zhao Wang
  [show abstract] [hide abstract]
  ABSTRACT: p-Nitrophenyl esters with a short-chain carboxylic group, such as p-nitrophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB), could be effectively hydrolyzed by ammonium salts. p-Nitrophenyl esters were usually used as substrates to assay the lipase/esterase activity. Ammonium sulfate precipitation was often used to purify proteins, and some ammonium salts were usually used as nitrogen sources or inorganic salts for the lipase/esterase production. To study the effect of ammonium salts on the assay of the lipase/esterase activity, the contributing factors of hydrolysis of p-NPA/p-NPB catalyzed by ammonium salts were investigated. The lipase activities were compared in the presence and absence of ammonium sulfate. The hydrolysis reaction could be catalyzed under neutral and alkaline circumstances. The hydrolysis rate increased with the increase in the reaction temperature or the concentration of ammonium ion. When p-NPA was employed as the substrate for the analysis of the lipase/esterase activity, the effect of ammonium sulfate on the analysis could be neutralized by setting a control when the concentration of ammonium sulfate was less than 40% saturation. However, when the concentration of ammonium sulfate increased from 40% to 100% saturation, the enzyme activities decreased about 13-40%, which could not be ignored for accurate analysis of the enzyme activity.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Growth, metabolic activity, and productivity of immobilized and freely suspended CHO cells in perfusion culture.

  Ali Hilal-Alnaqbi, Alan Y C Hu, Zhibing Zhang, Mohamed Al-Rubeai
  [show abstract] [hide abstract]
  ABSTRACT: Chinese hamster ovary (CHO) cells producing β-galactosidase (β-gal) were successfully cultured on silicone-based porous microcarriers (ImmobaSil FS) in a 1 L stirred-tank perfusion bioreactor. We studied the growth, metabolism, and productivity of free and immobilized cells to understand cellular activity in immobilized conditions. CHO cells attached to ImmobaSil FS significantly better than to other microcarriers. Scanning electron microscope images showed that the CHO cells thoroughly colonized the porous surfaces of the ImmobaSil FS, exhibiting a spherical morphology with microvilli that extended to anchorage cells on the silicone surface. In perfusion culture, the concentration of the attached cells reached 8 × 10(8) cells/mL of carrier, whereas those that remained freely suspended reached 2 × 10(7) cells/mL medium. The β-gal concentration reached more than 5 unit/mL in perfusion culture, more than fivefold that of batch culture. The maximum concentration per microcarrier was proportional to the initial cell density. The specific growth rate, the specific β-gal production rate, the percentage of S phase, and the oxygen uptake rate were all relatively lower for immobilized cells than freely suspended cells in the same bioreactor, indicating that not only do cells survive and grow to a greater extent in a free suspension state, but they are also metabolically more active than viable cells inside the pores of the microcarriers.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Crosslinked Gelatin Microspheres with Continuously Tunable Degradation Profiles for Renal Tissue Regeneration.

  Monica A Serban, Toyin Knight, Richard G Payne, Joydeep Basu, Elias A Rivera, Neil Robbins, Darell McCoy, Craig Halberstadt, Deepak Jain, Timothy A Bertram
  [show abstract] [hide abstract]
  ABSTRACT: Collagen and gelatin-based biomaterials are widely used in tissue engineering applications. Various methods have been reported for the crosslinking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). In this study, a carbodiimide-based crosslinking method, also used in the production of FDA-approved products, was employed to obtain differentially crosslinked gelatin beads. The colorimetric determination of the in vitro enzymatic susceptibility of the beads indicated that the resistance to degradation linearly correlated with the concentration of carbodiimide used for the crosslinking reaction. This result was also confirmed in vivo by the histological evaluation of the residence time of orthotopically injected cell-seeded beads. These data would indicate that the production of gelatin-based microbeads with tunable degradation profiles may be applicable towards development of products that catalyze regeneration of kidney and other solid organs. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Enhanced stability of newly isolated trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization.

  Hemraj S Nandanwar, Rakesh M Vohra, Gurinder S Hoondal
  [show abstract] [hide abstract]
  ABSTRACT: Newly isolated and partially purified trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 was immobilized by covalent coupling to a well-known support material, Eupergit® C. Approximately 80% enzyme activity yield was achieved with ≈61% binding of a soluble protein from a solution containing 5 mg/mL protein. The immobilized preparation was found to be quite unstable due to a poor multisubunit covalent interaction of trimeric enzyme. Additional cross-linking with polyaldehyde-dextran was done to sustain the biotechnological application of immobilized enzyme. The temperature and pH optima of immobilized enzyme were increased by 10°C and 0.5 unit, respectively. The enzyme was significantly stabilized and retained ≈93% enzyme activity when incubated at 60°C for 60 Min, whereas free enzyme lost ≈50% activity. It was recycled nine times with ≈100% conversion efficiency when batch experiments were carried out at 35°C, pH 7.5, for the 180 Min cycle, using 5% N-carbamoyl-l-methionine as the substrate. The half-life of the immobilized preparation was determined as 23 cycles and is significant. Approximately 50% of enzyme activity was retained even after 5 months of storage at 4°C, whereas free enzyme lost complete enzyme activity. Hence, we could enhance the stability of l-methionine-N-carbamoylase to make it a potential biocatalyst for biotechnological production of α-amino acids.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Effect of cross-linkers in fabrication of carrageenan-alginate matrices for tissue engineering application.

  Saet Byul Ki, Deepti Singh, Seong Cheol Kim, Son Tae Won, Sung Soo Han
  [show abstract] [hide abstract]
  ABSTRACT: The three-dimensional (3-D) scaffold serves as a structural substrate and as a niche for cell proliferation to ensure tissue regeneration. Ideal scaffolds should have porous structures with high pore interconnectivity to allow cell adherence, differentiation and proliferation besides suitable mechanical strength and biodegradability without inflicting any immune response. Cross-linker is one of major factors that affect to mechanical and biological properties of scaffolds. In this study, different chemical cross-linker effects on scaffold architecture were examined. Porous 3D scaffolds based on carrageenan and alginate (CA) were successfully fabricated by freeze-drying technique and using various cross-linkers like glutaraldehyde (GA), genipin, ethyl (dimethylamino propyl) carbodiimide/ N-Hydroxysuccinimide (EDC/NHS). The chemical cross-linker effects on CA scaffold was characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetry (TG). Human fibroblast cell line (L929) was seeded into the fabricated scaffold and the cell proliferation was assessed by MTT and live/dead assay. Overall results suggested the potential cross-linkers for ideal CA biomaterial could be EDC/NHS among other agents tested as the scaffold CAEN was found to be porous, interconnected, physically and mechanically stable. Comparing to matrices with other cross-linkers higher cell attachment, better cellular response, higher metabolic activity could be observed in scaffold synthesized using EDC/NHS as cross-linker. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Attachment of alginate microcapsules onto plasma-treated PDMS sheet for retrieval after transplantation.

  Soojeong Shin, Jeong Eun Shin, Young Je Yoo
  [show abstract] [hide abstract]
  ABSTRACT: Although transplantation of microencapsulated islets has been proposed as a therapy for the treatment of diabetes mellitus, limited retrievability of the cells has impeded its medical usage. To achieve retrieval of microencapsulated islets, capsules were attached to polydimethylsiloxane(PDMS) with a biocompatible adhesive. Because the hydrophobic nature of the PDMS surface prevents attachment, surface modification is essential. Alginate microcapsules were attached to modified PDMS sheets, and the mechanical stability of the resulting constructs was determined. Acrylic acid (AA) and acrylamide (AM) mixtures were grafted on the surfaces of PDMS sheets using a two-step oxygen plasma treatment (TSPT). TSPT-PDMS was characterized according to water contact angle and zeta-potential measurements. The contact angle was altered by changing the ratio of AM to AA to generate hydrophilic surface. Evaluation of the surface charge at pH 2, 7, and 12 confirmed the presence of polar groups on the modified surface. Microcapsules were attached to TSPT-PDMS using Histoacryl® and shown to be in a mono-layered and half-exposed state. The shear stress resistance of alginate capsules attached to the PDMS sheet indicates the possibility of transplantation of encapsulated cells without scattering in vivo. This method is applicable to retrieve microencapsulated porcine islets when required. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Amylase enzyme from Bacillus subtilis S8-18: A potential desizing agent from marine environment.

  Balu Jancy Kalpana, Muthukrishnan Sindhulakshmi, Shunmugiah Karutha Pandian
  [show abstract] [hide abstract]
  ABSTRACT: The present study is aimed at developing an economical medium for the production of α-amylase from Bacillus subtilis S8-18, a marine sediment isolate from Palk Bay, with various agricultural by-products which are cheap and rich in starch. These products included wheat bran, wheat husk, rice bran, rice husk and potato peel and used to replace soluble starch present in the LB broth (synthetic medium). The rice husk was found to be the best to influence enzyme production significantly (61186 IU mL(-1) ) when compared to the yield of 30026 IU mL(-1) obtained by commercial starch. Hence, LB broth containing rice husk was termed as economical medium. Besides, the effect of various nutritional and physiological factors on enzyme production was also investigated. Furthermore, the desizing efficiency of α-amylases produced by synthetic and economical medium was evaluated through various assays like reducing sugar estimation, weight loss assay, drop absorbency assay, SEM and FTIR analyses. In addition, a commercial α-amylase from B. subtilis was also used in desizing analyses for comparative purpose. It revealed that the α-amylase from economical medium was highly effective in desizing the cotton fabrics as that of the commercial enzyme and much superior to the enzyme produced through synthetic medium. This article is protected by copyright. All rights reserved.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Functional expression of amyloidogenic human stefins A and B in Pichia pastoris using codon optimization.

  Kosuke Nakamura, Yuki Maeda, Kensuke Morimoto, Shigeru Katayama, Kazunari Kondo, Soichiro Nakamura
  [show abstract] [hide abstract]
  ABSTRACT: Complementary DNAs encoding human stefins A (HSA) and B (HSB) were synthesized using Pichia-preferred codons by overlap extension PCR. The full-length genes were ligated downstream of the glyceraldehyde-3-phosphate dehydrogenase promoter in the Pichia expression vector pGAPZαC and successfully expressed in Pichia pastoris strain X-33. Functional recombinant HSA and HSB proteins were purified from culture medium at yields of 121.3 ± 13.5 (n = 3) and 95.4 ± 4.1 (n = 3) mg/L, respectively. Using this expression strategy, we demonstrated that high levels of bioactive recombinant HSA and HSB can be produced by fermentation in P. pastoris.
  Biotechnology and Applied Biochemistry 05/2013;
• Article: Optimization of neutral protease production from Bacillus subtilis: Using agroindustrial residues as substrates and response surface methodology.

  Ming-Jun Zhu, Jing-Rong Cheng, Hong-Tu Chen, Mao-Cheng Deng, Wen-Hua Xie
  [show abstract] [hide abstract]
  ABSTRACT: Statistically based experimental designs were applied to optimize the fermentation medium and cultural conditions for the maximization of neutral protease using three agroindustrial residues (cassava pulp, soybean meal, and wheat bran) and Bacillus subtilis DES-59. The Plackett-Burman design was used to evaluate the effects of variables such as the concentration of substrates, initial pH, shaker's rotating speed, temperature, inoculum size, and incubation time. Among the eight parameters, three significant variables (cassava pulp, soybean meal, and inoculum size) were selected for the optimization study, in which a central composite design was used to optimize the concentrations of cassava pulp and soybean meal and inoculum size and investigate the interactive effects of the three variables. The optimal parameters obtained from response surface methodology are 37.78 g/L of cassava pulp, 15 g/L of soybean meal, and 6.5% (v/v) of inoculum size, respectively, resulting in a maximum neutral protease activity of 4107 ± 122 U/mL.
  Biotechnology and Applied Biochemistry 05/2013;