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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.162, #7)

Value-Added Production of Nisin from Soy Whey by Debjani Mitra; Anthony L. Pometto III; Samir K. Khanal; Bishnu Karki; Byron F. Brehm-Stecher; J. (Hans) van Leeuwen (pp. 1819-1833).

The objective of this study was to evaluate the potential of low/negative value soy whey (SW) as an alternative, inexpensive fermentation substrate to culture Lactococcus lactis subsp. lactis for nisin production. Initially, a microtiter plate assay using a Bioscreen C Microbiology Plate Reader was used for rapid optimization of culture conditions. Various treatments were examined in efforts to optimize nisin production from SW, including different methods for SW sterilization, ultrasonication of soy flake slurries for possible nutrient release, comparison of diluted and undiluted SW, and supplementation of SW with nutrients. In subsequent flask-based experiments, dry bacterial mass and nisin yields obtained from SW were 2.18 g/L and 619 mg/L, respectively, as compared to 2.17 g/L and 672 mg/L from a complex medium, de Man–Rogosa–Sharpe broth. Ultrasonication of soybean flake slurries (10% solid content) in water prior to production of SW resulted in ∼2% increase in biomass yields and ∼1% decrease in nisin yields. Nutrient supplementation to SW resulted in ∼3% and ∼7% increase in cell and nisin yields, respectively. This proof-of-concept study demonstrates the potential for use of a low/negative value liquid waste stream from soybean processing for production of a high-value fermentation end product.

Keywords: Soy whey; Nisin; Value-added production; Lactococcus lactis ; Bioscreen; Fermentation

Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in Bombyx mori Chrysalides by Soledad Chazarra; Salvador Aznar-Cervantes; Luis Sánchez-del-Campo; Juan Cabezas-Herrera; Wu Xiaofeng; José Luis Cenis; José Neptuno Rodríguez-López (pp. 1834-1846).

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.

Keywords: Dihydrofolate reductase; Bombyx mori ; Chrysalides; Baculovirus; Protein expression

Ammonia Fiber Expansion (AFEX) Pretreatment, Enzymatic Hydrolysis, and Fermentation on Empty Palm Fruit Bunch Fiber (EPFBF) for Cellulosic Ethanol Production by Ming J. Lau; Ming W. Lau; Christa Gunawan; Bruce E. Dale (pp. 1847-1857).

Empty palm fruit bunch fiber (EPFBF), a readily available cellulosic biomass from palm processing facilities, is investigated as a potential carbohydrate source for cellulosic ethanol production. This feedstock was pretreated using ammonia fiber expansion (AFEX) and enzymatically hydrolyzed. The best tested AFEX conditions were at 135 °C, 45 min retention time, water to dry biomass loading of 1:1 (weight ratio), and ammonia to dry biomass loading of 1:1 (weight ratio). The particle size of the pretreated biomass was reduced post-AFEX. The optimized enzyme formulation consists of Accellerase (84 μL/g biomass), Multifect Xylanase (31 μL/g biomass), and Multifect Pectinase (24 μL/g biomass). This mixture achieved close to 90% of the total maximum yield within 72 h of enzymatic hydrolysis. Fermentation on the water extract of this biomass affirms that nutrients solely from the pretreated EPFBF can support yeast growth for complete glucose fermentation. These results suggest that AFEX-treated EPFBF can be used for cellulosic biofuels production because biomass recalcitrance has been overcome without reducing the fermentability of the pretreated materials.

Keywords: Cellulosic ethanol; Palm fiber; AFEX; Saccharomyces cerevisiae ; Biofuel; Pretreatment

Molecular Cloning, Expression, and Characterization of Cathepsin L from Mud Loach (Misgurnus mizolepis) by Sang Jung Ahn; Ji Hea Sung; Na Young Kim; A Ram Lee; Soo Jin Jeon; Jung-Soo Lee; Joong Kyun Kim; Joon Ki Chung; Hyung Ho Lee (pp. 1858-1871).

Cathepsin L is an important protease in the initiation of protein degradation and one of the most powerful endopeptidases. In this study, we cloned mud loach (Misgurnus mizolepis) cathepsin L (MlCtL) cDNA, and the pro-mature enzyme of MlCtL (proMlCtL) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4 T-1 vector. The recombinant proMlCtL was overexpressed in E. coli DH5αMCR as a 62-kDa protein. Its activity was quantified by measuring the cleavage of synthetic fluorogenic peptide substrates, and the protease activity of proMlCtL was also demonstrated by gelatin zymography. Antipain and leupeptin were shown to inhibit the protease activity of proMlCtL. Our results suggest that the structural features and evolutionary relationship of the mud loach cathepsin L gene were similar to that of the other mammalian cathepsin Ls; however, the proMlCtL protein was more stable at neutral and alkaline pH. The optimum temperature for the proMlCtL enzyme was found to be 40 °C. In addition, proMlCtL activity was dependent upon the presence of several metal ions and detergents.

Keywords: Cysteine protease; Cathepsin L; Glutathione S-transferase (GST)-fusion protein; Mud loach (Misgurnus mizolepis); Zymography

Ball Milling Pretreatment of Corn Stover for Enhancing the Efficiency of Enzymatic Hydrolysis by Zengxiang Lin; He Huang; Hongman Zhang; Lin Zhang; Lishi Yan; Jingwen Chen (pp. 1872-1880).

Ethanol can be produced from lignocellulosic biomass with the usage of ball milling pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields from lignocellulosic feed stocks are critical parameters for ethanol production process. The research results from this paper indicated that the yields of glucose and xylose were improved by adding any of the following dilute chemical reagents: H₂SO₄, HCl, HNO₃, CH₃COOH, HCOOH, H₃PO₄, and NaOH, KOH, Ca(OH)₂, NH₃·H₂O in the ball milling pretreatment of corn stover. The optimal enzymatic hydrolysis efficiencies were obtained under the conditions of ball milling in the alkali medium that was due to delignification. The data also demonstrated that ball milling pretreatment was a robust process. From the microscope image of ball milling-pretreated corn stover, it could be observed that the particle size of material was decreased and the fiber structure was more loosely organized. Meanwhile, the results indicate that the treatment effect of wet milling is better than that of dry milling. The optimum parameters for the milling process were ball speed of 350 r/min, solid/liquid ratio of 1:10, raw material particle size with 0.5 mm, and number of balls of 20 (steel ball, Φ = 10 mm), grinding for 30 min. In comparison with water milling process, alkaline milling treatment could increase the enzymatic hydrolysis efficiency of corn stover by 110%; and through the digestion process with the combination of xylanase and cellulase mixture, the hydrolysis efficiency could increase by 160%.

Keywords: Lignocellulosics; Ball milling; Pretreatment; Enzymatic hydrolysis; Corn stover

Esterification Activity of Novel Fungal and Yeast Lipases by Elisandra Rigo; André E. Polloni; Daniela Remonatto; Francieli Arbter; Silvana Menoncin; J. Vladimir Oliveira; Débora de Oliveira; Helen Treichel; Susana J. Kalil; Jorge L. Ninow; Marco Di Luccio (pp. 1881-1888).

The main objective of this work was the isolation and screening of microorganisms with potential for producing lipases for the synthesis of fatty esters as well as evaluating the specificity of the enzymes produced, using different alcohols (methanol, ethanol, n-propanol, and butanol) and fatty acids (oleic and lauric acids) as substrates. Promising biocatalysts for organic synthesis were obtained in this work. The isolated strains 69F and 161Y showed ability to efficiently catalyze the reaction for production of n-propyl oleate. Other strains can also be considered of potential interest, as 74F, 111Y, and 186Y. The future development of production using different substrates could result in cheap crude lipase of high importance to industrial applicability.

Keywords: Lipase; Microorganism screening; Synthesis activity; Esterification

Cellulases and Xylanases Production by Penicillium echinulatum Grown on Sugar Cane Bagasse in Solid-State Fermentation by Marli Camassola; Aldo J. P. Dillon (pp. 1889-1900).

To investigate the production of cellulases and xylanases from Penicillium echinulatum 9A02S1, solid-state fermentation (SSF) was performed by using different ratios of sugar cane bagasse (SCB) and wheat bran (WB). The greatest filter paper activity obtained was 45.82 ± 1.88 U gdm⁻¹ in a culture containing 6SCB/4WB on the third day. The greatest β-glucosidase activities were 40.13 ± 5.10 U gdm⁻¹ obtained on the third day for the 0SCB/10WB culture and 29.17 ± 1.06 U gdm⁻¹ for the 2SCB/8WB culture. For endoglucanase, the greatest activities were 290.47 ± 43.57 and 276.84 ± 15.47 U gdm⁻¹, for the culture 6SCB/4WB on the fourth and fifth days of cultivation, respectively. The greatest xylanase activities were found on the third day for the cultures 6SCB/4WB (36.38 ± 5.38 U gdm⁻¹) and 4SCB/6WB (37.87 ± 2.26 U gdm⁻¹). In conclusion, the results presented in this article showed that it was possible to obtain large amounts of cellulases and xylanases enzymes using low-cost substrates, such as SCB and WB.

Keywords: Solid-state fermentation; Cellulases; Xylanases; Sugar cane bagasse; Second-generation ethanol

Bacterially Expressed Double-Stranded RNAs against Hot-Spot Sequences of Tobacco Mosaic Virus or Potato Virus Y Genome Have Different Ability to Protect Tobacco from Viral Infection by Zhao-Nan Sun; Guo-Hua Yin; Yun-Zhi Song; Hai-Long An; Chang-Xiang Zhu; Fu-Jiang Wen (pp. 1901-1914).

Posttranscriptional gene silencing, also known as RNA interference, involves degradation of homologous mRNA sequences in organisms. In plants, posttranscriptional gene silencing is part of a defense mechanism against virus infection, and double-stranded RNA is the pivotal factor that induces gene silencing. In this paper, we got seven hairpin RNAs (hpRNAs) constructs against different hot-spot sequences of Tobacco mosaic virus (TMV) or Potato virus Y (PVY) genome. After expression in Escherichia coli HT115, we extracted the seven hpRNAs for the test in tobacco against TMV or PVY infection. The data suggest that different hpRNAs against different hot-spot sequences of TMV or PVY genome had different ability to protect tobacco plants from viral infection. The resistance to TMV conferred by the hpRNA against the TMV movement protein was stronger than other TMV hpRNAs; the resistance to PVY conferred by the hpRNA against the PVY nuclear inclusion b was better than that induced by any other PVY hpRNAs. Northern blotting of siRNA showed that the resistance was indeed an RNA-mediated virus resistance.

Keywords: Tobacco mosaic virus; Potato virus Y; RNA interference; hpRNAs; Hot-spot sequences

Integration of Succinic Acid and Ethanol Production With Potential Application in a Corn or Barley Biorefinery by Nhuan P. Nghiem; Kevin B. Hicks; David B. Johnston (pp. 1915-1928).

Production of succinic acid from glucose by Escherichia coli strain AFP184 was studied in a batch fermentor. The bases used for pH control included NaOH, KOH, NH₄OH, and Na₂CO₃. The yield of succinic acid without and with carbon dioxide supplied by an adjacent ethanol fermentor using either corn or barley as feedstock was examined. The carbon dioxide gas from the ethanol fermentor was sparged directly into the liquid media in the succinic acid fermentor without any pretreatment. Without the CO₂ supplement, the highest succinic acid yield was observed with Na₂CO₃, followed by NH₄OH, and lowest with the other two bases. When the CO₂ produced in the ethanol fermentation was sparged into the media in the succinic acid fermentor, no improvement of succinic acid yield was observed with Na₂CO₃. However, several-fold increases in succinic acid yield were observed with the other bases, with NH₄OH giving the highest yield increase. The yield of succinic acid with CO₂ supplement from the ethanol fermentor when NH₄OH was used for pH control was equal to that obtained when Na₂CO₃ was used, with or without CO₂ supplementation. The benefit of sparging CO₂ from ethanol fermentation on the yield of succinic acid demonstrated the feasibility of integration of succinic acid fermentation with ethanol fermentation in a biorefinery for production of fuels and industrial chemicals.

Keywords: Succinic acid; Escherichia coli AFP184; CO₂ fixation; Ethanol fermentation; Biorefinery

Rapid In Vitro Production of Cloned Plants of Uraria picta (Jacq.) DC—A Rare Medicinal Herb in Long-Term Culture by Santosh Kumar Rai; Meena Sharma; Madhu Jain; Abhishek Awasthi; Dharmendra Kumar Purshottam; Narayanan Kuttanpillai Nair; Ashok Kumar Sharma (pp. 1929-1937).

An efficient in vitro process for rapid production of cloned plants of Uraria picta has been developed employing nodal stem segments taken from field-grown plants. Explants showed bud-break followed by regeneration of shoots with restricted growth within 12 days on modified Murashige and Skoog’s medium supplemented with 0.25 mg l^–1 each of 6-benzylaminopurine and indole-3-acetic acid and 25 mg l^-1 adenine sulfate. Normal growth of shoots with good proliferation rate was achieved by reducing the concentrations of 6-benzylaminopurine and indole-3-acetic acid to 0.1 mg l^-1 each and incorporating 0.5 mg l^-1 gibberellic acid in the medium in which, on an average, 19.6 shoots per explant were produced. Further, during successive subcultures, increased concentrations of adenine sulfate (50 mg l^-l) and gibberellic acid (2 mg l^-l) along with the addition of 20 mg l^-l dl-tryptophan were found conducive to control the problem of necrosis of shoots. In this treatment, several “crops” of shoots were obtained from single culture by repeated subculturing of basal portion of stalk in long-term. Isolated shoots rooted 100% in 0.25 mg l^-1 indole-3-butyric acid. In vitro-raised plants after hardening in inorganic salt solution grew normally in soil and came to flowering. Genetic fidelity of in vitro-raised plants was ascertained by rapid amplified polymorphic DNA (RAPD) markers. Also, quantitative estimation of two isoflavonones in their root extracts further confirmed true-to-type nature of plantlets.

Keywords: Conservation; Genetic fidelity; In vitro cloning; Nodal stem segments; Rare medicinal herb

A Novel α-Glucosidase Inhibitor Protein from the Rhizomes of Zingiber ottensii Valeton by Nathachai Tiengburanatam; Apaporn Boonmee; Polkit Sangvanich; Aphichart Karnchanatat (pp. 1938-1951).

The objective of this study was to investigate a new protein with α-glucosidase inhibitory activity from the rhizomes of Zingiber ottensii. With a simple salting-out technique followed by single-step anion-exchange purification, the protein was successfully purified from the rhizomes. This protein was found to have three likely sub-unit types, 32.5, 15.2, and 13.8 kDa, as revealed by native and reducing SDS-PAGE analysis. Determination of the kinetics of the inhibition of α-glucosidase from Saccharomyces cerevisiae by standard enzymatic methods indicated the maximum percent inhibition; IC₅₀ and K i of this protein were 77.5%, 30.15 μg/ml, and 140 μmol, while the K m and V max were 2.35 μmol and 0.11 mM/min, respectively. The inhibitory action was pH-independent within the pH range 2–10, but was potentially affected by buffer salts, and was relatively temperature-stable between 4–35 °C, with a maximum activity at 65 °C. The amino acid sequence of an internal fragment of this purified Z. ottensii rhizomal protein had a similarity to the sequence from the plant cysteine proteinase family. Although this α-glucosidase inhibitory protein was purified from Z. ottensii rhizomes and preliminarily characterized, further studies are needed prior to firm applications being envisaged.

Keywords: α-Glucosidase inhibitor; Zingiber ottensii

Effect of Initial Cell Concentration on Ethanol Production by Flocculent Saccharomyces cerevisiae with Xylose-Fermenting Ability by Akinori Matsushika; Shigeki Sawayama (pp. 1952-1960).

Different initial cell concentrations of a recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated for their effects on xylose fermentation and glucose–xylose cofermentation. A high initial cell concentration greatly increased both the substrate utilization and ethanol production rates. During xylose fermentation, the highest rates of xylose consumption (2.58 g/L h) and ethanol production (0.83 g/L h) were obtained at an initial cell concentration of 13.1 g/L. During cofermentation, the highest rates of glucose consumption (14.4 g/L h), xylose consumption (2.79 g/L h), and ethanol production (6.68 g/L h) were obtained at an initial cell concentration of 12.7 g/L. However, a high initial cell density had no positive effect on the maximum ethanol concentration and ethanol yield mainly due to the increased amount of by-products including xylitol. The ethanol yield remained almost constant (0.34 g/g) throughout xylose fermentation (initial cell concentration range, 1.81–13.1 g/L), while it was slightly lower at high initial cell concentrations (9.87 and 12.7 g/L) during cofermentation. The determination of the appropriate initial cell concentration is necessary for the improvement of substrate utilization and ethanol yield.

Keywords: Recombinant Saccharomyces cerevisiae ; Xylose; Ethanol; Cofermentation; Cell concentration

Gene Expression in Secondary Metabolism and Metabolic Switching Phase of Phanerochaete chrysosporium by Jin-Ming Wu; Yi-zheng Zhang (pp. 1961-1977).

Ligninolytic enzymes are well-known to play the crucial roles in lignin biodegradation and have potential applications in industrial processes. The filamentous white-rot fungus, Phanerochaete chrysosporium, has been widely used as a model organism for studying these ligninolytic enzymes that are able to degrade the lignin during the secondary metabolism. To study the gene expression in secondary metabolism and metabolic switching phase of P. chrysosporium, we constructed a metabolic-switching phase suppression subtractive hybridization (SSH) cDNA library and a secondary metabolic phase SSH cDNA library to compare their mRNA expression profiles. We isolated the genes that are specially expressed and subsequently identified four genes that specially expressed during metabolic-switching phase while 22 genes in secondary metabolic phase. Accordingly, these specially expressed genes might play key roles in different metabolic stages, which would offer more new insights into the shift from nitrogen to lignin metabolism.

Keywords: Ligninolytic enzymes; Phanerochaete chrysosporium ; Metabolic-switching phase; Secondary metabolic phase; cDNA library

Oil Accumulation via Heterotrophic/Mixotrophic Chlorella protothecoides by Tamarys Heredia-Arroyo; Wei Wei; Bo Hu (pp. 1978-1995).

Microalgal oil is a potential energy source because it can be easily converted to fatty acid methyl ester or hydrocarbon type of diesel, and it is produced with relatively higher productivity compared with oil from plants and animals. Heterotrophic growth of microalgae is superior due to its high final product concentration; however, the cost of the raw materials is unacceptable if sugar is utilized as the carbon source. The aim of this study is to optimize the lipid accumulation of Chlorella protothecoides by using carbon sources other than glucose in heterotrophic and mixotrophic cultures. Different factors such as different carbon sources, carbon to nitrogen ratio, initial pH level, salinity, and rotational speed are studied in affecting the cell growth and the oil accumulation. Our experiments revealed that the heterotrophic and mixotrophic cultures of C. protothecoides grew better than autotrophic cultures. C. protothecoides can grow on glycerol or acetate, as well as on glucose. Several stress factors were confirmed or discovered to significantly increase the lipid content of microalgae cells. The replacement of glycerol and acetate as carbon sources for microalgae cultivations provides potential for waste utilization: glycerol from biodiesel industry and acetate from biohydrogen production.

Keywords: Microalgae oil accumulation; Chlorella; Glycerol; Acetate; Bioenergy

Monitoring of Cell Viability and Proliferation in Hydrogel-Encapsulated System by Resazurin Assay by Jing Xiao; Ying Zhang; Jianzheng Wang; Weiting Yu; Wei Wang; Xiaojun Ma (pp. 1996-2007).

Cell microencapsulation is a promising approach for cell implantation, cell-based gene therapy and large-scale cell culture. For better quality control, it is important to accurately measure the microencapsulated cell viability and proliferation in the culture. A number of assays have been used for this purpose, but limitations arise. In this study, we investigated the feasibility and reliability of resazurin as a cell growth indicator in microencapsulated culture system. According to the experiment data, there was a reversible, time- and dose-dependent growth inhibition as observed for resazurin application in encapsulated cells. A positive relationship was observed between reduction of resazurin and CHO cell number in microcapsule. Moreover, the resazurin assay provided an equivalent result to the commonly used MTT method in determining CHO cell proliferation in APA microcapsule with no notable influence on cell distribution and organization pattern. In conclusion, resazurin assay is offered as a simple, rapid and non-invasive method for in vitro microencapsulated cell viability and proliferation measurement.

Keywords: Microencapsulated cell; APA; Resazurin; MTT; Non-invasive monitoring of cell proliferation

Obtaining Mannanoligosaccharide Preparations by Means of the Mechanoenzymatic Hydrolysis of Yeast Biomass by Alexey Leonidovich Bychkov; Kirill G. Korolev; Oleg I. Lomovsky (pp. 2008-2014).

A new method of obtaining biologically active mannanoligosaccharides is proposed. It involves mechanical activation of the enzymatic hydrolysis of components forming the supramolecular structure of the cell wall. Processes that take place during mechanical treatment and enzymatic hydrolysis of yeast biomass and lead to an increase in the availability of mannanoligosaccharides of the yeast cell walls are investigated. The efficiency of the use of mechanoenzymatic approach to obtaining mannanoligosaccharide preparations is evaluated.

Keywords: Mechanoenzymatic hydrolysis; Yeast biomass; Enzyme; Mannanoligosaccharide

Lipase Immobilized on the Hydrophobic Polytetrafluoroethene Membrane with Nonwoven Fabric and Its Application in Intensifying Synthesis of Butyl Oleate by Shu-Guang Wang; Wei-Dong Zhang; Zheng Li; Zhong-Qi Ren; Hong-Xia Liu (pp. 2015-2026).

The synthesis of butyl oleate was studied in this paper with immobilized lipase. Five types of membrane were used as support to immobilize Rhizopus arrhizus lipase by following a procedure combining filtration and protein cross-linking. Results showed that hydrophobic polytetrafluoroethene membrane with nonwoven fabric (HO-PTFE-NF) was the favorite choice in terms of higher protein loading, activity, and specific activity of immobilized lipase. The factors including solvent polarity, lipase dosage, concentration, and molar ratio of substrate and temperature were found to have significant influence on conversion. Results showed that hexane (logP = 3.53) was a favorable solvent for the biosynthesis of butyl oleate in our studies. The optimal conditions were experimentally determined of 50 U immobilized lipase, molar ratio of oleic acid to butanol of 1.0, substrate concentration of 0.12 mol/L, temperature of 37 °C, and reaction time of 2 h. The conversion was beyond 91% and decreased slightly after 18 cycles. Lipase immobilization can improve the conversion and the repeated use of immobilized lipase relative to free lipase.

Keywords: Lipase immobilization; Butyl oleate; Conversion

High-Level Expression and Efficient Purification of Bioactive Swollenin in Aspergillus oryzae by Meihua Wang; Jin Cai; Lei Huang; Zhengbin Lv; Yaozhou Zhang; Zhinan Xu (pp. 2027-2036).

The bioactivity of swollenin is beneficial to cellulose decomposition by cellulase despite the lack of hydrolytic activity itself. In order to improve the productivity of swollenin, the effects of culture conditions on the expression level in recombinant Aspergillus oryzae were investigated systematically. With regard to the bioactivity of swollenin, glycerin and peanut meal were the optimal carbon or nitrogen source, respectively. The highest level production of swollenin (50 mg L⁻¹) was attained after 88 h cultivation with the initial pH of 5.6 in the culture medium. Then the soluble swollenin was effectively purified from the cultural supernatant by ammonium sulfate precipitation and cationic exchange chromatography with recovery yield of 53.2%. The purified swollenin was fully bioactive due to its strong synergistic activity with cellulose.

Keywords: Aspergillus oryzae ; Swollenin; Purification; Soluble expression; Synergistic activity

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris by Keivan Majidzadeh-A; Vahid Khalaj; Davami Fatemeh; Hemayatkar Mahdi; Barkhordari Farzaneh; Adeli Ahmad; Fereidoun Mahboudi (pp. 2037-2048).

Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS–PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

Keywords: Human tissue plasminogen activator; t-PA; Pichia pastoris ; Cloning; Expression; Zymography; Amidolytic assay; Densitometry

A Highly Thermostable Alkaline Cellulase-Free Xylanase from Thermoalkalophilic Bacillus sp. JB 99 Suitable for Paper and Pulp Industry: Purification and Characterization by Dengeti Shrinivas; Gunashekaran Savitha; Kumar Raviranjan; Gajanan Ramchandra Naik (pp. 2049-2057).

A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0–10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K _m and V _max of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min⁻¹ mg⁻¹, respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

Keywords: Bacillus sp. JB 99; Thermoalkalophilic; Xylanase; Purification; Glycosyl hydrolases

Study of Starch Degradation by Yeasts During Fermentation for Using in Animal Feed by Santiago Alonso; Maria Arévalo-Villena; Juan Úbeda; Ana Briones (pp. 2058-2066).

Sixteen amylolytic yeasts, drawn from culture collections or isolated from different environments, were used in the present study. Experiments were performed to evaluate the starch degradation due to exocellular activity. The culture media of growth was optimized to find the maximum activity, and some strains produced an extracellular amylolytic enzyme when cultured in presence of starch in the medium. On the other hand, one yeast showed activity in cell extract when cultured under varying conditions in a bench bioreactor; this strain would thus be suitable for use as an animal feed supplement, both as a source of proteins and nucleic acids and in terms of its amylolytic activity.

Keywords: Starch degradation; Yeast fermentation; Bioreactor scale-up; Amylolytic activity; Animal feed

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Petiole Explants of Cassia angustifolia Vahl.—a Potential Medicinal Plant by Iram Siddique; M. Anis; I. M. Aref (pp. 2067-2074).

An effective protocol was developed for in vitro regeneration of the Cassia angustifolia via indirect organogenesis from petiole explants excised from 21-day-old axenic seedlings. Organogenic callus were induced on Murashige and Skoog (MS) medium supplemented with 5.0 µM 2,4-dichlorophenoxy acetic acid and 2.5 µM thidiazuron (TDZ). Adventitious shoot regeneration was achieved on MS medium supplemented with 5.0 µM TDZ as it induced 8.5 ± 0.98 shoots in 85% cultures. The number of shoots and shoot length was significantly enhanced when cultures were subcultured on auxin–cytokinin-containing medium. The highest number of shoots (12.5 ± 1.10) and shoot length (4.3 ± 0.20 cm) was recorded on MS medium supplemented with 5.0 µM TDZ and 1.5 µM indole-3-acetic acid. Regenerated shoots were rooted best on MS medium supplemented with 10.0 µM indole-3-butyric acid followed by their transfer to liquid MS filter paper bridge medium. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 70% survival rate. The plants showed normal morphological characteristics similar to the field grown plants.

Keywords: Adventitious; Indole-3-butyric acid; Petiole; Pulse treatment; Sennosides; Shoot bud

Improved Synthesis of (S)-1-Phenyl-2-Propanol in High Concentration with Coupled Whole Cells of Rhodococcus erythropolis and Bacillus subtilis on Preparative Scale by Jian-Zhong Jin; He Li; Jie Zhang (pp. 2075-2086).

Bioreduction of 1-phenyl-2-propanone to prepare (S)-1-phenyl-2-propanol, a useful pharmaceutical intermediate, was performed with growing cells of Rhodococcus erythropolis JX-021, giving 14 mM (1.9 g/L) product in 99% e.e. at 5 h in the catalysis of 15 mM substrate. The reduction stopped afterwards due to strong inhibition of substrate and formed product, a problem that is often encountered in biotransformation. While the substrate inhibition was solved by stepwise feeding, product inhibition was tackled by different methods: repeated removal of the product by centrifugation, by absorption with Amberlite XAD-7 resin, and by the use of dodecanol as the second phase gave the final product in 58, 68, and 61 mM in the catalysis of 80 mM substrate, respectively. The inhibition was caused by the partial permeabilization of cell membrane of R. erythropolis JX-021, and addition of NADPH or glucose 6-phosphate to such cell culture retained the reduction activity. Therefore, higher productivity in the reduction of 1 with resting cells of R. erythropolis JX-021 was achieved through cofactor regeneration and recycling by the addition of glucose and permeabilized cells of Bacillus subtilis BGSC 1A1 containing a glucose dehydrogenase, giving the product in 62 mM without addition of cofactor and 78 mM with the addition of 0.01 mM NADP⁺ in the catalysis of 120 mM substrate. The product e.e. retained 99% during the process which showed industrial possibility.

Keywords: Reductase; (S)-1-phenyl-2-propanol; Amphetaminil; Coupled microorganisms; NADPH regeneration; Preparative scale

Effects of Surfactant and Salt Species in Reverse Micellar Forward Extraction Efficiency of Isoflavones with Enriched Protein from Soy Flour by Xiaoyan Zhao; Zhiyi Wei; Fangling Du; Junqing Zhu (pp. 2087-2097).

Suitability of reverse micelles of anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT) and sodium dodecyl sulfate (SDS), cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) and nonionic surfactant polyoxyethylene p-t-octylphenol (TritonX-100) in organic solvent isooctane for extraction of soy isoflavone-enriching proteins was investigated. The results showed that the order of combined isoflavone contents was SDS>CTAB>Triton X-100>AOT, while the order of protein recovery was SDS>AOT>TritonX-100>CTAB. As compared with ACN-HCl extraction, the total amount of isoflavones was lower than reverse micellar extraction. Ion strength was one of the important conditions to control extraction of isoflavone-enriching proteins with AOT reversed micelles. For the six salt systems, KNO₃, KCl, MgCl₂, CaCl₂, NaCl, and Na₂SO₄, extracted fraction of isoflavone-enriching proteins was measured. Salt solutions greatly influenced the extraction efficiency of isoflavones in an order of KNO₃>MgCl₂>CaCl₂>KCl>NaCl>Na₂SO₄, while protein in an order of MgCl₂>CaCl₂>NaCl>KNO₃>Na₂SO₄>KCl.

Keywords: Reverse micelle; Extraction; Soybean flour; Protein; Isoflavone

Expression, One-Step Purification, and Immobilization of HaloTag^TM Fusion Proteins on Chloroalkane-Functionalized Magnetic Beads by Hassan Motejadded; Bertolt Kranz; Sonja Berensmeier; Matthias Franzreb; Josef Altenbuchner (pp. 2098-2110).

The presented work introduces a novel method to immobilize enzymes either purified or directly out of a crude extract onto magnetic particles in the micrometer range. This method is based on the creation of a fusion protein consisting of the enzyme of choice and a mutant dehalogenase. The dehalogenase gene is commercially available from the company Promega under the name HaloTag^TM. When the fusion protein is contacted with magnetic beads having chemically synthesized, chloroalkane ligands on their surface, the dehalogenase and the ligand undergo a covalent coupling leading to stable and spatially defined immobilization. The principle was proved with a lipase fused to the HaloTag^TM gene and magnetic poly(methyl)methacrylate beads as carriers. The solubility of the tagged lipase was strongly increased by fusion of the malE gene at the N-terminal end of the HaloTag^TM lipase gene. This tripartite protein was purified on amylose resin and used for immobilization. About 13 µg protein could be immobilized per 1 mg of beads within a few minutes. Due to the defined binding site, no activity loss was observed in the course of the immobilization. The resulting enzyme carrier was tested with the same beads up to six times for lipase activity over a storage period of 36 days at 8 °C. No loss of activity was found during this time.

Keywords: Enzyme immobilization; HaloTag™; Fusion protein; Magnetic bead; Dehalogenase; Expression

Trichoderma harzianum IOC-4038: A Promising Strain for the Production of a Cellulolytic Complex with Significant β-Glucosidase Activity from Sugarcane Bagasse Cellulignin by Aline Machado de Castro; Kelly Cristina Nascimento Rodrigues Pedro; Juliana Cunha da Cruz; Marcela Costa Ferreira; Selma Gomes Ferreira Leite; Nei Pereira Jr (pp. 2111-2122).

Sugarcane bagasse is an agroindustrial residue generated in large amounts in Brazil. This biomass can be used for the production of cellulases, aiming at their use in second-generation processes for bioethanol production. Therefore, this work reports the ability of a fungal strain, Trichoderma harzianum IOC-4038, to produce cellulases on a novel material, xylan free and cellulose rich, generated from sugarcane bagasse, named partially delignified cellulignin. The extract produced by T. harzianum under submerged conditions reached 745, 97, and 559 U L⁻¹ of β-glucosidase, FPase, and endoglucanase activities, respectively. The partial characterization of this enzyme complex indicated, using a dual analysis, that the optimal pH values for the biocatalysis ranged from 4.9 to 5.2 and optimal temperatures were between 47 and 54 °C, depending on the activity studied. Thermal stability analyses revealed no significant decrease in activity at 37 °C during 23 h of incubation. When compared to model strains, Aspergillus niger ATCC-16404 and Trichoderma reesei RutC30, T. harzianum fermentation was faster and its extract showed a better balanced enzyme complex, with adequate characteristics for its application in simultaneous saccharification and fermentation processes.

Keywords: Sugarcane bagasse; Cellulase; β-glucosidase; Cellulignin; Endoglucanase; Trichoderma harzianum ; Aspergillus niger ; Trichoderma reesei

Erratum to: α-Amylase: An Ideal Representative of Thermostable Enzymes by Om Prakash; Nivedita Jaiswal (pp. 2123-2124).

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.162, #7)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.162, #7)