Applied Biochemistry and Biotechnology (v.162, #2)

α-Mannosidases from the Digestive Fluid of Rhynchophorus Palmarum Larvae as Novel Biocatalysts for Transmannosylation Reactions by Ehuié M. Bédikou; Martial F. Koné; Amédée P. Ahi; Jean T. Gonnety; Betty M. Faulet; Lucien P. Kouamé; Sébastien L. Niamké (307-320).
Two biological fluids, namely hemolymph and digestive fluid from the larval stage of Rhynchophorus palmarum Linneaus, a serious pest in agroecosystem exploiting oil palm, were screened for hydrolytic activities, by the use of synthetic and natural glycoside substrates. Several exo and endoglycosidase activities were observed but, the interesting α-mannosidase activity (0.41 ± 0.04 UI) had attracted our attention. So, we have previously demonstrated that this activity harbours four distinctive α-mannosidase isoforms named RpltM, RplM1, RplM2 and RplM3. We have extended this work to determine the ability of these enzymes to catalyze synthesis reactions. Finally, we have revealed that, α-mannosidases from the digestive fluid of R. palmarum larvae catalyze transmannosylation reactions. The stability of the enzymes and the optimization of the transfer product yield were studied as functions of pH, enzyme unit, starting concentration of donor or acceptor and time. It was shown that, in experimental optimum conditions, average yields of 12.34 ± 0.75, 12.15 ± 0.79, 5.59 ± 0.35 and 8.43 ± 0.50% were obtained for the α-mannosidases RpltM, RplM1, RplM2 and RplM3, respectively. On the basis of this work, α-mannosidases from the digestive fluid of Rhynchophorus palmarum larvae appear to be a valuable tool for the preparation of neoglycoconjugates.
Keywords: α-Mannosidases; Transmannosylation reactions; Oil palm weevil larvae; Rhynchophorus palmarum ; Coleoptera

A Law of Mutation: Power Decay of Small Insertions and Small Deletions Associated with Human Diseases by Jia Zhang; Li Xiao; Yufang Yin; Pierre Sirois; Hanlin Gao; Kai Li (321-328).
Indels in evolutionary studies are rapidly decayed obeying a power law. The present study analyzed the length distribution of small insertions and deletions associated with human diseases and confirmed that the decay pattern of these small mutations is similar to that of indels when the mutation datasets are large enough. The describable decay pattern of somatic mutations may have application in the evaluation of varied penetrance of different mutations and in association study of gene mutation with carcinogenesis.
Keywords: Indels; Cancer; Length distribution

A keratinolytic enzyme (KerA1) secreted by a newly isolated Bacillus pumilus strain A1 cultivated in medium containing chicken feather meal was purified and characterized, and the gene was isolated and sequenced. The molecular mass of the purified enzyme was estimated to be 34,000 Da by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the purified keratinase were 9.0 and 60 °C, respectively, using keratin as a substrate. KerA1 showed a high stability towards nonionic surfactants. It was found to be relatively stable toward the strong anionic surfactant (SDS). The deduced amino acid sequence of the keratinase KerA1 differs from both the organic solvent tolerant protease of B. pumilus 115b and the dehairing protease of B. pumilus UN-31-C-42 by one and nine amino acids, respectively. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers and for applications in detergent formulations.
Keywords: Bacillus pumilus ; Keratinase; Purification; Molecular characterization

Chitin is a polysaccharide found in abundance in the shell of crustaceans. In this study, the protease from Bacillus cereus SV1 was applied for chitin extraction from shrimp waste material of Metapenaeus monoceros. A high level of deproteinization 88.8% ± 0.4 was recorded with an E/S ratio of 20. The demineralization was completely achieved within 6 h at room temperature in HCl 1.25 M, and the residual content of calcium in chitin was below 0.01%. 13C CP/MAS-NMR spectral analysis of chitin prepared by the enzymatic deproteinization of shrimp wastes was found to be similar to that obtained by alkaline treatment and to the commercial α-chitin. The degree of N-acetylation, calculated from the spectrum, was 89.5%. Chitin obtained by treatment with crude protease from B. cereus was converted to chitosan by N-deacetylation, and the antibacterial activity of chitosan solution against different bacteria was investigated. Results showed that chitosan solution at 50 mg/mL markedly inhibited the growth of most Gram-negative and Gram-positive bacteria tested. Furthermore, the antioxidant potential of the protein hydrolysates obtained during enzymatic isolation of chitin was evaluated using various in vitro assays. All the samples exerted remarkable antioxidant activities. These results suggest that enzymatic deproteinization of the shrimp shell wastes, using B. cereus SV1 protease, could be applicable to the chitin production process.
Keywords: Shrimp waste; Enzymatic deproteinization; Hydrolysates; Chitin; 13C CP/MAS-NMR; Chitosan

Potential of Crocus sativus (saffron) and its Constituent, Crocin, as Hypolipidemic and Antioxidant in Rats by Syed Mohammed Basheeruddin Asdaq; Mohammed Naseeruddin Inamdar (358-372).
The aim of the present study was to evaluate the hypolipidemic and antioxidant potential of saffron and its active constituent, crocin, in hyperlipidemic rats. The animals fed either with normal fat diet or high fat diet were administered orally saffron (25, 50, and 100 mg/kg) or crocin (4.84, 9.69, and 19.38 mg/kg) in their respective groups for five consecutive days. Biochemical estimations of triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), alkaline phosphatase (ALP), aspartate transaminase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), glutathione peroxidase enzyme activity (GSHPx), total glutathione (GSH), and oxidized glutathione (GSSG) in serum and superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive species (TBARS), ferric reducing/antioxidant power (FRAP), and total sulfhydryl (SH) groups in liver tissue homogenate were carried out. Both saffron and crocin were effective in decreasing the elevated levels of TG, TC, ALP, AST, ALT, MDA, GSHPx, GSH, and GSSG in serum and increasing SOD, CAT, FRAP, and SH values in liver tissue with reduction in TBARS. The saffron was found to be superior to crocin indicating the involvement of other potential constituents of saffron apart from crocin for its synergistic behavior of quenching the free radicals and ameliorating the damages of hyperlipidemia.
Keywords: Antioxidant; Biochemical estimations; Crocin; Hypolipidemic; Saffron

Purification and Characterization of a Low Molecular Weight Endo-xylanase from Mushroom Termitomyces clypeatus by Dhananjay Soren; Mohanlal Jana; Subhabrata Sengupta; Anil K. Ghosh (373-389).
A low molecular weight endo-xylanase (EC was purified from an edible mushroom Termitomyces clypeatus grown in submerged medium with oat spelt xylan. Xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. Its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kDa. The enzyme was found to be most active at 50°C and pH 5.0, being most stable at pH 6.5. The Km for oat spelt xylan was determined to be 10.4 mg/ml. The specificities of the enzyme was observed to be highly specific towards oat spelt xylan and was inhibited by mercuric chloride (HgCl2), N-bromosuccinimide, and trans-1,2-diaminocyclohexane-N′,N′,N′,N′-tetraacetic acid strongly. The inhibitory action of N-bromosuccinimide on enzyme confirmed the presence of one tryptophan residue in its substrate-binding site. Amino acid analysis for xylanase showed the presence of high amount of hydrophobic serine, glycine, threonine, and alanine residues. The N-terminal sequencing study for the previously purified and characterized 56 kDa xylanolytic amyloglucosidase reveal the presence of 33.30% identity with glucoamylase chain A from Aspergillus awamori. The N-terminal sequence analysis of the present 12 kDa enzyme showed highest similarity (72.22% identity) towards xylanase from Neurospora crassa.
Keywords: Endo-xylanase; Termitomyces clypeatus ; N-terminal sequencing

Erratum to: Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment by Xun He; Yelian Miao; Xuejian Jiang; Zidong Xu; Pingkai Ouyang (390-390).

Thermodynamic Feasibility of Enzymatic Reduction of Carbon Dioxide to Methanol by F. Suhan Baskaya; Xueyan Zhao; Michael C. Flickinger; Ping Wang (391-398).
Production of valuable chemicals from CO2 is highly desired for the purpose of controlling CO2 emission. Toward that, enzymatic reduction of CO2 for the production of methanol appeared to be especially promising. That has been achieved by reversing the biological metabolic reaction pathways. However, hitherto, there has been little discussion on the thermodynamic feasibility of reversing such biological pathways. The reported yields of methanol have been generally very low under regular reaction conditions preferred by naturally evolved enzymes. The current work examines the sequential enzymatic conversion of CO2 into methanol from a thermodynamic point of view with a focus on factors that control the reaction equilibrium. Our analysis showed that the enzymatic conversion of carbon dioxide is highly sensitive to the pH value of the reaction solution and, by conducting the reactions at low pHs (such as pH 6 or 5) and ionic strength, it is possible to shift the biological methanol metabolic reaction equilibrium constants significantly (by a factor of several orders of magnitude) to favor the synthesis of methanol.
Keywords: Carbon dioxide; Enzymatic biocatalysis; Reduction; Sequestration; Methanol; Thermodynamics

Construction of a Novel Expression System in Klebsiella pneumoniae and its Application for 1,3-Propanediol Production by Zheng Ma; Zhiming Rao; Bin Zhuge; Huiying Fang; Xiangru Liao; Jian Zhuge (399-407).
A novel expression system of Klebsiella pneumoniae was developed in order to improve 1,3-propanediol (1,3-PD) production using a K. pneumoniae–Escherichia coli shuttle vector pET28a consisting of the kanamycin-resistance gene promoter Pkan. The recombinant plasmid pETPkan-cat carrying the chloramphenicol acetyltransferase gene cat as selectable marker was constructed to test the availability of the promoter Pkan in K. pneumoniae. The results showed that the chloramphenicol acetyltransferase was apparently expressed in K. pneumoniae, and the recombinant strain had a high-level resistance to chloramphenicol, suggesting that the promoter Pkan was efficient in K. pneumoniae. Then, the expression system was applied to the expression of 1,3-PD oxidoreductase in K. pneumoniae. The enzyme was over-expressed, and the recombinant K. pneumoniae showed a nearly 3.0-fold decrease in peak level of the intermediary metabolite 3-hydroxypropionaldehyde and an increase of 16.5% in yield of 1,3-PD with respect to the wild-type strain. From these results, the first reported expression system has paved the way for improvement of 1,3-PD production and will be available and efficient for other heterologous gene expression in K. pneumoniae.
Keywords: K. pneumoniae ; 1,3-Propanediol; Expression system; Constitutive promoter

Comparison of the potential for laccase and Mn-oxidizing peroxidases synthesis by ten strains of Ganoderma lucidum, originating from different worldwide areas, during solid-state fermentation of selected plant raw materials was the aim of this study. The great intraspecific variability in the production of analyzed enzymes as well as the dependence of the enzyme activity on plant raw materials were reported. The strain HAI 957 was the best laccase producer in the presence of corn stem, as a unique carbon source (129.46 U/L). The highest level of Mn-dependent peroxidase activity was noted after wheat straw fermentation by G. lucidum HAI 246 (78.64 U/L), while the maximal versatile peroxidase production (59.72 U/L) was observed in strain HAI 957 in the medium with oak sawdust.
Keywords: Ganoderma lucidum ; Laccase; Mn-dependent peroxidase; Versatile peroxidase; Plant raw materials

Microwave Exposure Affecting Reproductive System in Male Rats by Kavindra Kumar Kesari; Jitendra Behari (416-428).
The object of present study is to investigate the effects of 50 GHz microwave frequency electromagnetic fields on reproductive system of male rats. Male rats of Wistar strain were used in the study. Animals 60 days old were divided into two groups—group I sham exposed and group II experimental (microwave exposed). During exposure, rats were confined in Plexiglas cages with drilled ventilation holes for 2 h a day for 45 days continuously at a specified specific absorption rate of 8.0 × 10−4 W/kg. After the last exposure, the rats were sacrificed immediately and sperms were collected. Antioxidant enzyme (superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase), histone kinase, apoptosis, and cell cycle were analyzed in sperm cells. Result shows a significant decrease in the level of sperm GPx and SOD activity (p ≤ 0.05), whereas catalase shows significant increase in exposed group of sperm samples as compared with control (p < 0.02). We observed a statistically significant decrease in mean activity of histone kinase as compared to the control (p < 0.016). The percentage of cells dividing in a spermatogenesis was estimated by analyzing DNA per cell by flow cytometry. The percentage of apoptosis in electromagnetic field exposed group shows increased ratio as compared to sham exposed (p < 0.004). There were no significant differences in the G0/G1 phase; however, a significant decrease (p < 0.026) in S phase was obtained. Results also indicate a decrease in percentage of G2/M transition phase of cell cycle in exposed group as compared to sham exposed (p < 0.019). We conclude that these radiations may have a significant effect on reproductive system of male rats, which may be an indication of male infertility.
Keywords: Microwave radiation; Histone kinase; Cell cycle; Flow cytometer; Antioxidant

Two xylanases from the crude culture filtrate of Penicillium sclerotiorum were purified to homogeneity by a rapid and efficient procedure, using ion-exchange and molecular exclusion chromatography. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 23.9 and 33.1 kDa for xylanase I and II, respectively. The native enzymes’ molecular masses of 23.8 and 30.8 kDa were estimated for xylanase I and II, respectively, by molecular exclusion chromatography. Both enzymes are glycoproteins with optimum temperature and pH of 50 °C and pH 2.5 for xylanase I and 55 °C and pH 4.5 for xylanase II. The reducing agents β-mercaptoethanol and dithio-treitol enhanced xylanase activities, while the ions Hg2+ and Cu2+ as well the detergent SDS were strong inhibitors of both enzymes, but xylanase II was stimulated when incubated with Mn2+. The K m value of xylanase I for birchwood xylan and for oat spelt xylan were 6.5 and 2.6 mg mL−1, respectively, whereas the K m values of xylanase II for these substrates were 26.61 and 23.45 mg mL−1. The hydrolysis of oat spelt xylan by xylanase I released xylobiose and larger xylooligosaccharides while xylooligosaccharides with a decreasing polymerization degree up to xylotriose were observed by the action of xylanase II. The present study is among the first works to examine and describe an extracellular, highly acidophilic xylanase, with an unusual optimum pH at 2.5. Previously, only one work described a xylanase with optimum pH 2.0. This novel xylanase showed interesting characteristics for biotechnological process such as feed and food industries.
Keywords: Penicillium sclerotiorum ; Xylanase; Enzyme purification; Enzyme characterization

Engineering of a Bacillus α-Amylase with Improved Thermostability and Calcium Independency by Marzieh Ghollasi; Khosro Khajeh; Hossein Naderi-Manesh; Atiyeh Ghasemi (444-459).
Successful industrial use of amylases requires that they are sufficiently stable and active at application conditions, e.g., at high temperature in starch-liquefaction process. In the present study, site-directed mutagenesis was used to enhance the thermal stability and calcium independency of a mesophilic α-amylase from Bacillus megaterium WHO. Mutations (A53S and H58I) were designed at the calcium-binding site based on the sequence alignment. Kinetic and thermostability parameters of the mutants were analyzed and compared with that of the wild type. In the presence of calcium, the affinity of the enzymes (wild type and mutants) toward starch was increased. In comparison to the wild type, calcium ion had more effect on the catalytic efficiency, k cat/K m, and half-life (at 60 °C) of A53S mutant. In A53S, the dependence of half-life on calcium concentration showed that the enhanced calcium binding is likely to be responsible for the increased stability. In contrast, calcium-independent mutant (H58I) possessed high thermostability. In addition, thermodynamic parameters of amylolytic reaction exhibited an increase in the activation energy and the entropy of the system. Kinetics of irreversible thermal inactivation suggests that the activation energy increased by 1.4-fold in the most stable variant.
Keywords: α-Amylase; Bacillus megaterium ; Calcium; Site-directed mutagenesis; Thermostability

Activation of Nanoparticles by Biosorption for E. coli Detection in Milk and Apple Juice by Ghinwa Naja; Pierre Bouvrette; Julie Champagne; Roland Brousseau; John H.T. Luong (460-475).
Two types of silver nanoparticles were activated by specific sorption of biomolecules for the detection of Escherichia coli. The capture of this bacterium was performed using polyclonal antibodies (anti-E. coli) biosorbed onto nanospheres or nanorice through a protein-A layer. The bacterial detection was achieved using surface enhancement Raman scattering in order to compare the performance of these two nanoparticles. The activated silver nanospheres showed a better performance mainly due to the dimension of these nanoparticles. The detection limit has been established using the automated Raman mapping system. The technique was capable of detecting 103 cells/mL in milk and apple juice without any pre-enrichment. With an overall assay time less than 1 h, the process could be easily adapted to detect other pathogens by selecting the pertinent antibody. Furthermore, PCR was used for the DNA verification to assess whether the selected bacterial strain was identical before and after detection.
Keywords: Contaminated milk and apple juice; Escherichia coli; Nanoparticles; Detection limit; Bacterial capture; Biosorption

Isolation, Purification, and Properties of a Novel Small Heat Shock Protein from the Hyperthermophile Sulfolobus solfataricus by Yonghua Wang; Xun Xu; Zhenzhen Wen; Wencheng Li; Bo Yang; Chris Whiteley (476-485).
The isolation, purification, and properties of a putative small heat shock protein (sHsp), named SsHSP14.1, from the hyperthermophilic archaeon Sulfolobus solfataricus have been investigated. The sHsp was successfully expressed and purified from Escherichia coli. In vivo chaperone function of SsHSP14.1 for preventing aggregation of proteins during heating was investigated. It was found that recombinant SsHSP14.1 with a molecular mass of 17.8 kDa prevented E. coli proteins from aggregating in vivo at 50 °C. This result suggested that SsHSP14.1 confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures. In vitro, the purified SsHSP14.1 protein was able to prevent Candida antarctica lipase B from aggregation for up to 60 min at 80 °C. Moreover, the SsHSP14.1 enhanced thermostability of bromelain extending its half-life at 55 °C by 67%.
Keywords: Small heat shock protein; Purification; Chaperone-like activity; Thermostability; Protein aggregation

Production and Characterization of Cellulose by Acetobacter sp. V6 Using a Cost-Effective Molasses–Corn Steep Liquor Medium by Ho-Il Jung; O-Mi Lee; Jin-Ha Jeong; Young-Dong Jeon; Ki-Hyun Park; Hong-Sung Kim; Won-Gun An; Hong-Joo Son (486-497).
In order to reduce of the manufacturing cost of bacterial cellulose (BC), BC production by Acetobacter sp. V6 was investigated in shaking culture using molasses and corn steep liquor (CSL) as the sole carbon and nitrogen sources, respectively. The highest BC production was obtained with Ca3(PO4)2-treated molasses. Maximum BC yield (2.21 ± 0.04 g/l) was obtained at 5% (w/v) total sugar in molasses. In improved medium containing molasses and CSL, BC production was observed in the medium after 1 day of incubation and increased rapidly thereafter with maximum yield (3.12 ± 0.03 g/l) at 8 days. This value was approximately twofold higher than the yield in the complex medium. Physical properties of BC from the complex and molasses media were studied using Fourier-transform infrared (FT-IR) spectroscopy and X-ray diffractometer. By FT-IR, all the BC were found to be of cellulose type І, the same as typical native cellulose. The relative crystallinity of BC produced in the complex and molasses media were 83.02 and 67.27%, respectively. These results suggest that molasses and CSL can be useful low-cost substrates for BC production by Acetobacter sp. V6 without supplementation with expensive nitrogen complexes such as yeast extract and polypeptone, leading to the reduction in the production costs.
Keywords: Cellulose; Cost-effective medium; Acetobacter strain; Molasses; Corn steep liquor

Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger Z-25 in Pichia pastoris by Yao Guo; Fengxia Lu; Haizhen Zhao; Yanchong Tang; Zhaoxin Lu (498-509).
A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting revealed that rGOD exhibited a K m value of 16.95 mM and a K cat value of 484.26 s−1.
Keywords: Glucose oxidase; Aspergillus niger ; Overexpression; Pichia pastoris ; Properties

Scale up and Application of Biosurfactant from Bacillus subtilis in Enhanced Oil Recovery by Hossein Amani; Mohammad Reza Mehrnia; Mohammad Hossein Sarrafzadeh; Manouchehr Haghighi; Mohammad Reza Soudi (510-523).
There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y p/x), biosurfactant on sucrose (Y p/s), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g−1, 0.18 g g−1, and 0.03 g l−1 h−1, respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y x/s, Y p/x, Y p/s, and Y of 0.42 g g−1, 0.595 g g−1, 0.25 g g−1, and 0.057 g l−1 h−1, respectively. The biosurfactant maximum production, 2.5 g l−1, was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient (K L a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s−1, respectively. Comparison of K L a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with K L a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.
Keywords: Bacillus subtilis ; Bioreactor; Biosurfactant; Enhanced oil recovery; Foam; Scale up

Overall measurement of methanogenic activity of sludge and or slurry is thought as a key for understanding the basic physiology of anaerobic consortia involved in anaerobic digestion process of an alternative biomass. In this study, the methanogenic activity of biogas plant slurry was used to evaluate the anaerobic digestion of ossein factory wastes such as sinews and primary clarified bone waste (PCBW) and cyanobacterial biomass in standard assay conditions. A maximum methanogenic activity was reported here when ossein factory wastes mixed with cyanobacterial biomass in specific proportions in which sinews and PCBW alone also favored to a significant methane yield. Cyanobacterial biomass alone did not give a desirable methanogenic activity. Approximately 48% of total solids were destroyed from these wastes after 30 days. This study gives information on the use of these wastes with suitable proportions for taking an effort in a large-scale anaerobic digestion in an effective way of ossein factory.
Keywords: Ossein factory waste; Cyanobacterial biomass; Methanogenic activity; Biogas; Methane; Anaerobic digestion

Profiling Differential Expression of Cellulases and Metabolite Footprints in Aspergillus terreus by Asiya Nazir; Rohit Soni; H. S. Saini; Amarjeet Kaur; B. S. Chadha (538-547).
This study reports differential expression of endoglucanase (EG) and β-glucosidase (βG) isoforms of Aspergillus terreus. Expression of multiple isoforms was observed, in presence of different carbon sources and culture conditions, by activity staining of poly acrylamide gel electrophoresis gels. Maximal expression of four EG isoforms was observed in presence of rice straw (28 U/g DW substrate) and corn cobs (1.147 U/ml) under solid substrate and shake flask culture, respectively. Furthermore, the sequential induction of EG isoforms was found to be associated with the presence of distinct metabolites (monosaccharides/oligosaccharides) i.e., xylose (X), G1, G3 and G4 as well as putative positional isomers (G1/G2, G2/G3) in the culture extracts sampled at different time intervals, indicating specific role of these metabolites in the sequential expression of multiple EGs. Addition of fructose and cellobiose to corn cobs containing medium during shake flask culture resulted in up-regulation of EG activity, whereas addition of mannitol, ethanol and glycerol selectively repressed the expression of three EG isoforms (Ia, Ic and Id). The observed regulation profile of βG isoforms was distinct when compared to EG isoforms, and addition of glucose, fructose, sucrose, cellobiose, mannitol and glycerol resulted in down-regulation of one or more of the four βG isoforms.
Keywords: Differential expression; Endoglucanase (EG); β-Glucosidase (βG); Multiple isoforms; Regulation; Metabolite footprint profile

Thermodynamics and Kinetics of Heat Inactivation of a Novel Keratinase from Chryseobacterium sp. Strain kr6 by Silvana Terra Silveira; Franciani Casarin; Sabrine Gemelli; Adriano Brandelli (548-560).
A novel keratinase from Chryseobacterium sp. strain kr6 was purified to homogeneity by (NH4)2SO4 precipitation, gel permeation on Sephadex G-100, and Q-Sepharose Fast Flow anion-exchange chromatography. The molecular weight of the purified enzyme was around 20 kDa. Kinetic and thermodynamic parameters for thermal inactivation were determined. The influence of Ca2+ and Mg2+ ions and purification degree on the enzyme stability was evaluated in the range of 50 to 60 °C. The results showed that first-order kinetics explained well the thermal denaturation of the keratinase in this temperature interval. The presence of Ca2+ increases significantly the enzyme stability. Compared with the controls, the half-life of the purified enzyme after two purification steps in the presence of Ca2+ increased 7.3, 20.2, and 9.8 fold at 50, 55, and 60 °C, respectively. Thermodynamics parameters for thermal inactivation were also determined.
Keywords: Biocatalysis; Enzyme; Kinetics; Microbial; Protease; Thermal inactivation

Sugarcane bagasse and chicken manure were anaerobically fermented to carboxylic acids using a mixed culture of marine microorganisms at 55 °C. Using the MixAlco process— an example of consolidated bioprocessing— the resulting carboxylate salts can be converted to mixed alcohol fuels or gasoline. To enhance digestibility, sugarcane bagasse was lime pretreated with 0.1 g Ca(OH)2/g dry biomass at 100 °C for 2 h. Four-stage countercurrent fermentation of 80% sugarcane bagasse/20% chicken manure was performed at various volatile solids (VS) loading rates and liquid residence times. Calcium carbonate was used as a buffer during fermentation. The highest acid productivity of 0.79 g/(L day) occurred at a total acid concentration of 21.5 g/L. The highest conversion (0.59 g VS digested/g VS fed) and yield (0.18 g total acids/g VS fed) occurred at a total acid concentration of 15.5 g/L. The continuum particle distribution model (CPDM) predicted the experimental total acid concentrations and conversions at an average error of 10.14% and 12.68%, respectively. CPDM optimizations show that high conversion (>80%) and total acid concentration of 21.3 g/L are possible with 300 g substrate/(L liquid), 30 days liquid residence time, and 3 g/(L day) solid loading rate. Thermophilic fermentation has a higher acetate content (∼63 wt%) than mesophilic fermentation (∼39 wt%).
Keywords: Sugarcane bagasse; Consolidated bioprocessing; CBP; Carboxylic acids; Bioconversion; MixAlco; CPDM; Thermophilic fermentation; Acetic acid

Bioactive Secondary Metabolites from a New Terrestrial Streptomyces sp. TN262 by Lobna Elleuch; Mohamed Shaaban; Slim Smaoui; Lotfi Mellouli; Ines Karray-Rebai; Lilia Fourati-Ben Fguira; Khaled A. Shaaban; Hartmut Laatsch (579-593).
During our search for Streptomyces spp. as new producers of bioactive secondary metabolites, the ethyl acetate extract of the new terrestrial Streptomyces isolate TN262 delivered eight antimicrobially active compounds. They were identified as 1-acetyl-β-carboline (1), tryptophol (2), cineromycin B (3), 2,3-dihydrocineromycin B (4), cyclo-(tyrosylprolyl) (5), 3-(hydroxyacetyl)-indole (6), brevianamide F (7), and cis-cyclo-(l-prolyl-l-leucyl) (8). Three further metabolites were detected in the unpolar fractions using GC–MS and tentatively assigned as benzophenone (9), N-butyl-benzenesulfonamide (10), and hexanedioic acid-bis-(2-ethylhexyl) ester (11). This last compound is known as plasticizer derivatives, but it has never been described from natural sources. In this article, we describe the identification of the new Streptomyces sp. isolate TN262 using its cultural characteristics, the nucleotide sequence of the corresponding 16S rRNA gene and the phylogenetic analysis, followed by optimization, large-scale fermentation, isolation of the bioactive constituents, and determination of their structures. The biological activity of compounds (2), (3), (4), and those of the unpolar fractions was addressed as well.
Keywords: New Streptomyces sp.TN262; Identification; Antimicrobial activities; Macrolides; Natural hexanedioic acid-bis-(2-ethylhexyl) ester

Fermentation Efficiency of Cells Immobilized on Delignified Brewers' Spent Grains after Low- and High-Temperature Thin Layer Thermal Drying by Konstantina Tsaousi; Athanasios A. Koutinas; Argyro Bekatorou; Paul Loukatos (594-606).
Low-cost dried yeasts immobilized on delignified brewers' spent grains for use in wine making and brewing were produced by simple thermal drying techniques. To optimize the thermal drying process, vacuum and air stream conditions were examined. Drying of thin layers of the biocatalysts was performed at low (30–38 °C) and high temperatures (40–70 °C). The fermentation efficiency of the thermally dried biocatalysts was acceptable, with immobilized cells showing a significantly higher thermotolerance compared with free cells. Immobilized cells dried at high temperatures presented slightly improved glucose fermentation efficiency compared with the low-temperature dried biocatalysts. Gas chromatography–mass spectrometry analysis of aroma volatiles of the fermented products revealed an increase of esters, lower higher alcohol formation, and significantly lower concentration of carbonylic compounds.
Keywords: Thermal drying; Brewers' spent grains; Yeast; Immobilization; Volatiles

Rhizopus oryzae immobilized in calcium alginate was applied in lactic acid fermentation with unhydrolyzed raw sweet potato powders as the sole carbon source. The effects of sodium alginate concentration, calcium chloride concentration, and the immobilized bead diameter on lactic acid production were investigated. Increase in sodium alginate concentration during the gelation process would harden the immobilized capsule, which led to a decrease in lactic acid production. The increase in calcium chloride would increase the thickness of the immobilized capsule, which would increase the mass transfer resistance. Nevertheless, while the calcium chloride was lower than 15%, it would not have obvious effects on lactic acid production. A larger bead could have more space for cell growth, which led to the maximum lactic acid production observed at the 5-mm bead diameter. Moreover, results of repeated-batch operation suggested that immobilized cells could have higher stability in lactic acid production than free cells. The total cumulative lactic acid in immobilized-cell operation could increase by 55% as compared with free-cell operation after 216 h (seven repeated-batches), and no loss of amylolytic activity was observed. The results indicated that immobilized R. oryzae by Ca-alginate could be suitable for lactic acid production from unhydrolyzed raw potato powders.
Keywords: Rhizopus oryzae ; Repeated-batch; Immobilization conditions; Bead size; Calcium chloride