Applied Biochemistry and Biotechnology (v.171, #3)

Xylose mother liquor (XML) is a by-product of xylose production through acid hydrolysis from corncobs, which can be used potentially for alternative fermentation feedstock. Sixteen Clostridia including 13 wild-type, 1 industrial strain, and 2 genetically engineered strains were screened in XML, among which the industrial strain Clostridium acetobutylicum EA 2018 showed the highest titer of solvents (12.7 g/L) among non-genetic populations, whereas only 40 % of the xylose was consumed. An engineered strain (2018glcG-TBA) obtained by combination of glcG disruption and expression of the d-xylose proton-symporter, d-xylose isomerase, and xylulokinase was able to completely utilize glucose and l-arabinose, and 88 % xylose in XML. The 2018glcG-TBA produced total solvents up to 21 g/L with a 50 % enhancement of total solvent yield (0.33 g/g sugar) compared to that of EA 2018 (0.21 g/g sugar) in XML. This XML-based acetone–butanol–ethanol fermentation using recombinant 2018glcG-TBA was estimated to be economically promising for future production of solvents.
Keywords: Butanol; Clostridium acetobutylicum ; Xylose pathway engineering; Xylose mother liquor

Application of Yeast Candida utilis to Ferment Eisenia bicyclis for Enhanced Antibacterial Effect by Sung-Hwan Eom; Dae-Sung Lee; Young Mi Kang; Kwang-Tae Son; You-Jin Jeon; Young-Mog Kim (569-582).
In this study, fermentation broth of Eisenia bicyclis with Candia utilis YM-1 exhibited enhanced antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and food-borne pathogenic bacteria. To perform a more detailed investigation on the antibacterial activity, the fermented broth of E. bicyclis was extracted with methanol and further fractionated with organic solvents. After 1-day fermentation, the ethyl acetate (EtOAc)-soluble extract exhibited the highest anti-MRSA activity with minimum inhibitory concentration values ranging from 128 to 512 μg/mL, suggesting that the fermentation of E. bicyclis with C. utilis YM-1 may enhance antibacterial activity against MRSA. This effect was correlated to the result obtained by an increase in total phenolic contents in EtOAc-soluble extract. In addition, high-performance liquid chromatography analysis revealed that eckol, dieckol, dioxinodehydroeckol, and phlorofucofuroeckol-A contents in the EtOAc-soluble extract increased significantly. Thus, these results show that anti-MRSA activity of E. bicyclis fermented with C. utilis most likely originated from phlorotannins and allow the possible application of a variety of seaweed functional foods.
Keywords: Antimicrobial activity; Eisenia bicyclis ; Fermentation; MRSA; MIC; HPLC

FLAG, a short hydrophilic peptide consisting of eight amino acids (DYKDDDDK), has been widely used as a fusion tag for the purification and detection of a wide variety of recombinant proteins. One of the monoclonal antibodies against this peptide, anti-FLAG M2, recognises a FLAG peptide sequence at the N terminus, Met-N terminus, C terminus, or internal site of a fusion protein and has been extremely useful for the detection, identification, and purification of recombinant proteins. Nevertheless, detailed binding specificity of anti-FLAG M2 has yet to be determined. In the current study, a phage display combinatorial peptide library was used to determine that the motif DYKxxD encompasses the critical amino acid residues responsible for the binding of FLAG peptide to this antibody. This study demonstrates the utility of phage display technology and helps to elucidate the mode of action of this detection system.
Keywords: FLAG; Monoclonal antibody; Phage display; Combinatorial peptide; Epitope tag; Anti-FLAG M2; Mapping

Enzymatic reactions are very basic processes in biological systems, and parameters related to enzymatic reactions always provide good indicators for understanding of mechanisms underlined in enzymatic reactions, for better controlling of enzymatic reactions, and for comparison of different enzymes. In this mini-review: first, parameters in enzymatic reactions were briefly reviewed from three different standpoints; second, predictions of parameters in enzymatic reactions without information on enzyme structure were shortly reviewed from viewpoints of geometric approach, graphic approach and compartmental approach; third, predictions of parameters in enzymatic reaction with information on enzyme structure were reviewed from the points of view of modeling, with 19 currently available databases, and 17 software packages and web servers; fourth, the current state of prediction on parameters in enzymatic reaction in biofuel industry with respect to cellulolytic enzymes were reviewed; fifth, the pros and cons for future development were discussed; and finally, a worked example was given in the Appendix to describe the whole procedures of prediction of enzymatic parameters in reactions.
Keywords: Biofuel; Enzyme; Prediction; Review

Pigment Production by Filamentous Fungi on Agro-Industrial Byproducts: an Eco-Friendly Alternative by Fernanda Cortez Lopes; Deise Michele Tichota; Jamile Queiroz Pereira; Jéferson Segalin; Alessandro de Oliveira Rios; Adriano Brandelli (616-625).
The search for new sources of natural pigments has increased, mainly because of the toxic effects caused by synthetic dyes used in food, pharmaceutical, textile, and cosmetic industries. Fungi provide a readily available alternative source of natural pigments. In this context, the fungi Penicillium chrysogenum IFL1 and IFL2, Fusarium graminearum IFL3, Monascus purpureus NRRL 1992, and Penicillium vasconiae IFL4 were selected as pigments producers. The fungal identification was performed using ITS and part of the β-tubulin gene sequencing. Almost all fungi were able to grow and produce water-soluble pigments on agro-industrial residues, with the exception of P. vasconiae that produced pigments only on potato dextrose broth. The production of yellow pigments was predominant and the two strains of P. chrysogenum were the largest producers. In addition, the production of pigments and mycotoxins were evaluated in potato dextrose agar using TOF-MS and TOF-MS/MS. Metabolites as roquefortine C, chrysogine were found in both extracts of P. chrysogenum, as well fusarenone X, diacetoxyscirpenol, and neosolaniol in F. graminearum extract. In the M. purpureus extract, the pigments monascorubrin, rubropunctatin, and the mycotoxin citrinin were found. The crude filtrates have potential to be used in the textile industry; nevertheless, additional pigment purification is required for food and pharmaceutical applications.
Keywords: Agro-industrial wastes; Pigments; Eco-friendly; TOF-MS; Fungi

Improving the Mixing Performances of Rice Straw Anaerobic Digestion for Higher Biogas Production by Computational Fluid Dynamics (CFD) Simulation by Fei Shen; Libin Tian; Hairong Yuan; Yunzhi Pang; Shulin Chen; Dexun Zou; Baoning Zhu; Yanping Liu; Xiujin Li (626-642).
As a lignocellulose-based substrate for anaerobic digestion, rice straw is characterized by low density, high water absorbability, and poor fluidity. Its mixing performances in digestion are completely different from traditional substrates such as animal manures. Computational fluid dynamics (CFD) simulation was employed to investigate mixing performances and determine suitable stirring parameters for efficient biogas production from rice straw. The results from CFD simulation were applied in the anaerobic digestion tests to further investigate their reliability. The results indicated that the mixing performances could be improved by triple impellers with pitched blade, and complete mixing was easily achieved at the stirring rate of 80 rpm, as compared to 20–60 rpm. However, mixing could not be significantly improved when the stirring rate was further increased from 80 to 160 rpm. The simulation results agreed well with the experimental results. The determined mixing parameters could achieve the highest biogas yield of 370 mL (g TS)−1 (729 mL (g TSdigested)−1) and 431 mL (g TS)−1 (632 mL (g TSdigested)−1) with the shortest technical digestion time (T 80) of 46 days. The results obtained in this work could provide useful guides for the design and operation of biogas plants using rice straw as substrates.
Keywords: CFD simulation; Rice straw; Anaerobic digestion; Mixing performances

Synthesis, Characterization, and Antimicrobial Activity of Poly(acrylonitrile-co-methyl methacrylate) with Silver Nanoparticles by M. R. El-Aassar; Elsayed E. Hafez; Moustafa M. G. Fouda; Salem S. Al-Deyab (643-654).
Nanotechnology is expected to open some new aspects to fight and prevent diseases using atomic-scale tailoring of materials. The main aim of this study is to biosynthesize silver nanoparticles (AgNPs) using Trichoderma viride (HQ438699); the metabolite of this fungus will help either in reduction of the silver nitrate-adding active materials which will be loaded on the surface of the produced AgNPs. Poly(acrylonitrile-co-methyl methacrylate) copolymer (poly (AN-co-MMA)) was grafted with the prepared AgNPs. The poly(AN-co-MMA)/AgNPs were examined against ten different pathogenic bacterial strains, and the result was compared with another four different generic antibiotics. The produced poly(AN-co-MMA)/AgNPs showed high antibacterial activity compared with the four standard antibiotics. Moreover, the grafting of these AgNPs into the copolymer has potential application in the biomedical field.
Keywords: Nanomaterials; Polymer; Antimicrobial; Silver nanoparticles

Cocoa butter equivalent (CBE) was prepared by enzymatic acidolysis reaction of substrate consisting of refined palm mid fraction oil and palmitic–stearic fatty acid mixture. The reactions were performed in a batch reactor at a temperature of 60 °C in an orbital shaker operated at 160 RPM. Different mass ratios of substrates were explored, and the composition of the five major triacylglycerols (TAGs) of the structured lipids was identified and quantified using cocoa butter certified reference material IRMM-801. The reaction resulted in production of cocoa butter equivalent with the TAGs' composition (1,3-dipalmitoyl-2-oleoyl-glycerol 30.7 %, 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol 40.1%, 1-palmitoy-2,3- dioleoyl glycerol 9.0 %, 1,3-distearoyl-2-oleoyl-glycerol 14.5 %, and 1-stearoyl-2,3-dioleoyl glycerol 5.7 %) and with onset melting temperature of 31.6 °C and peak temperature of 40.4 °C which are close to those of cocoa butter. The proposed kinetics model for the acidolysis reaction presented the experimental data very well. The results of this research showed that palm mid fraction oil TAGs could be restructured to produce value added product such as CBE.
Keywords: Cocoa butter equivalent; Lipase; Triacylglycerol; Acidolysis; Interesterification; Palm oil; Structured lipids

Biochemical, Immunological and Kinetic Characterisation of Thiol Protease Inhibitor (Cystatin) from Liver by Aaliya Shah; Medha Priyadarshini; Mohd Shahnawaz Khan; Mohammad Aatif; Fakhra Amin; Bilqees Bano (667-675).
Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6–8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.
Keywords: Liver cystatin (LC); Thiol proteinase inhibitor; Kinetics of inhibition; Ultraviolet and fluorescence spectroscopy

Ficus carica Latex-Mediated Synthesis of Silver Nanoparticles and Its Application as a Chemophotoprotective Agent by Hemant P. Borase; Chandrashekhar D. Patil; Rahul K. Suryawanshi; Satish V. Patil (676-688).
The present work provides scientific support on the use of latex of Ficus carica to synthesize stable silver nanoparticles (AgNPs). AgNPs synthesized immediately after the addition of latex to silver nitrate solution at room temperature. Synthesized nanoparticles were of spherical shape with average size of 163.7 nm. Fourier transform infrared spectroscopy analysis revealed capping of proteins and phenolic compound on AgNPs, while X-ray diffraction analysis confirmed the fcc nature of AgNPs. Particles formed were stable for a long time (6 months). It was found that incorporation of AgNPs with 2 and 4 % concentration exhibits synergistic increase in sun protection factor of commercial sunscreen and natural extracts ranging from 01 to 12,175 % than control. Further characterization of latex and AgNPs revealed total phenolic content of 98.75 and 94.88 μg/ml. The ferric ion reduction potentials of latex and AgNPs were 79.69 and 18.79 %. Reduction potential of ascorbic acid was synergistically increased after cumulative preparation of ascorbic acid with latex and AgNPs and found to be 106.76 and 101.50 % for ascorbic acid + latex and ascorbic acid + AgNPs, respectively.
Keywords: Ficus carica ; Nanoparticles; Sun protection factor; UV protection; Antioxidant

Oil Crop Biomass Residue-Based Media for Enhanced Algal Lipid Production by Zhen Wang; Xiaochen Ma; Wenguang Zhou; Min Min; Yanling Cheng; Paul Chen; Jian Shi; Qin Wang; Yuhuan Liu; Roger Ruan (689-703).
The aim of this study was to evaluate the use of hydrolysates from acid hydrolysis of four different oil crop biomass residues (OCBR) as low cost culture media for algae growth. The one-factor-at-a-time method was used to design a series of experiments to optimize the acid hydrolysis conditions through examining the total nitrogen, total phosphorus, chemical oxygen demand, and ammonia nitrogen in the hydrolysates. The optimal conditions were found to be using 3 % sulfuric acid and hydrolyzing residues at 90 °C for 20 h. The hydrolysates (OCBR media) produced under the optimal conditions were used to cultivate the two algae strains, namely UM258 and UM268. The results from 5 days of cultivation showed that the OCBR media supported faster algae growth with maximal algal biomass yield of 2.7 and 3 g/L, respectively. Moreover, the total lipids for UM258 and UM268 were 54 and 35 %, respectively, after 5 days of cultivation, which suggested that the OCBR media allowed the algae strains to accumulate higher lipids probably due to high C/N ratio. Furthermore, over 3 % of omega-3 fatty acid (EPA) was produced for the two algae strains. In conclusion, OCBR media are excellent alternative for algae growth and have a great potential for large-scale production of algae-based ingredients for biodiesel as well as high-value food and pharmaceutical products.
Keywords: Oil crop biomass residue (OCBR); Acid hydrolysis; Facultative heterotrophic microalgae; Lipid; Omega-3 fatty acid

Maximizing Hydrogen Production and Substrate Consumption by Escherichia coli WDHL in Cheese Whey Fermentation by Luis Manuel Rosales-Colunga; Zazil Donaxí Alvarado-Cuevas; Elías Razo-Flores; Antonio De León Rodríguez (704-715).
Fermentative hydrogen production is strongly affected by pH. In order to maximize hydrogen production and substrate consumption in Escherichia coli ΔhycA, ΔlacI (WDHL) cheese whey fermentation, the influence of pH control at values of 5.5, 6, and 6.5 was studied in batch stirred-tank bioreactors. From the conditions evaluated, pH 6.5 was the best condition, at which the highest cumulative hydrogen production and yield (1.78 mol H2/mol lactose) were obtained. Moreover, at this pH, all carbohydrates from the cheese whey were consumed, and a mix of ethanol and organic acids, mainly lactate, were produced from glucose, whereas galactose yielded acetate, ethanol, and succinate. Operating the reactor at pH 5.5 resulted in the highest maximum specific production rate, but smaller hydrogen yield because only glucose was metabolized and galactose was accumulated. At pH 6, not all cheese whey carbohydrates were consumed, and it was not favorable for hydrogen production. Lactose consumption and growth kinetics were not affected by the pH. The results show the importance of controlling pH to maximize hydrogen production and substrate consumption using cheese whey as substrate.
Keywords: E. coli ; Fermentative metabolism; Lactose; Biohydrogen; Biofuels

In this study, covalent immobilization of the horseradish peroxidase (HRP) onto various polysulfone supports was investigated. For this purpose, different polysulfones were methacrylated with methacryloyl chloride, and then, nonwoven fabric samples were coated by using solutions of these methacrylated polysulfones. Finally, support materials were immersed into aquatic solution of HRP enzyme for covalent immobilization. Structural analysis of enzyme immobilization onto various polysulfones was confirmed with Fourier transform infrared spectroscopy, atomic force microscopy, and proton nuclear magnetic resonance spectroscopy. Decolorization of textile diazo (Acid Black 1) and anthraquinone (Reactive Blue 19) dyes was investigated by UV–visible spectrophotometer. Covalently immobilized enzyme has been used seven times in freshly prepared dye solutions through 63 days. Dye decolorization performance of the immobilized systems was observed that still remained high (70 %) after reusing three times. Enzyme activities of immobilized systems were determined and compared to free enzyme activity at different conditions (pH, temperature, thermal stability, storage stability). Enzyme activities of immobilized systems and free enzyme were also investigated at the different temperatures and effects of temperature and thermal resistance for different incubation time at 50 °C. In addition, storage activity of free and immobilized enzymes was determined at 4 °C at different incubation days.
Keywords: Decolorization; Horseradish peroxidase; Immobilized enzyme; Polymer support; Polysulfone; Textile dyes

Cell Morphology Variations of Klebsiella pneumoniae Induced by Acetate Stress Using Biomimetic Vesicle Assay by Shengguo Lu; Yuwang Han; Xujia Duan; Fang Luo; Lingyan Zhu; Shuang Li; He Huang (731-743).
Supplementation with acetate under low levels was used as a novel approach to control the morphological development of Klebsiella pneumoniae aimed to improve 1,3-propanediol (1,3-PD) production. A full range of morphological types formed from rod shape to oval shape even round shape in response to different concentrations of acetate. The cell growth and 1,3-PD productions in the shake flasks with 0.5 g/L acetate addition were improved by 9.4 and 28.37 %, respectively, as compared to the control, while the cell became shorter and began to lose its original shape. The cell membrane penetration by acetate was investigated by the biomimetic vesicles, while higher concentration of acetate led to more moderate colorimetric transitions. Moreover, the percentage composition of unsaturated fatty acid (UFA) was increased as well as the increased concentrations of acetate, whereas higher UFA percentage, higher fluidity of bacterial cell membrane.
Keywords: Acetate stress; Biomimetic vesicle assay; 1,3-Propanediol; Cell morphology; Klebsiella pneumoniae ; Membrane phospholipids analysis

Affinity Matrices of Cratylia mollis Seed Lectins for Isolation of Glycoproteins from Complex Protein Mixtures by Thiago Henrique Napoleão; Teodomiro Gomes dos Santos-Filho; Emmanuel Viana Pontual; Rodrigo da Silva Ferreira; Luana Cassandra Breitenbach Barroso Coelho; Patrícia Maria Guedes Paiva (744-755).
This work reports the use of matrices containing Cratylia mollis lectins (Cramoll 1,2,3-Sepharose and Cramoll 3-Sepharose) for isolation of glycoproteins from fetal bovine serum, human colostrum, hen egg white, and human blood plasma. Cramoll 1,2,3-Sepharose was able to bind a glycoprotein from fetal bovine serum which showed the same fetuin electrophoretic profile. The data indicate that this protein adsorbed to the matrix by interaction with Cramoll 3. Cramoll 1,2,3-Sepharose was not efficient to retain glycoproteins from human colostrum or commercial ovalbumin. Cramoll 3-Sepharose bound ovalbumin, and the support retained protein from hen egg white. Protein peaks eluted from the column with 1.0 M NaCl or 0.3 M galactose showed apparent molecular mass of ovalbumin. Two main proteins from blood plasma with apparent molecular mass 67 (similar to albumin) and 50 kDa (similar to fetuin) adsorbed on Cramoll 3-Sepharose and were eluted with 1.0 M NaCl as a single peak. Elution of adsorbed plasma proteins with 0.3 M galactose was less selective than with 1.0 M NaCl as revealed by SDS-PAGE. In conclusion, the Cramoll 1,2,3-Sepharose and Cramoll 3-Sepharose matrices were useful to separate glycoproteins from complex protein mixtures, and the adsorption phenomena was a carbohydrate-dependent event.
Keywords: Cratylia mollis ; Lectin affinity chromatography; Ovalbumin; Fetuin; Albumin

Asymmetric reduction of alkyl-3-oxobutanoates mediated by Candida parapsilosis ATCC 7330 resulted in optically pure alkyl-3-hydroxybutanoates in good yields (up to 72 %) and excellent enantiomeric excess (up to >99 %). A detailed and systematic optimisation study was necessary and was carried out to avoid the undesired transesterification reaction during the course of asymmetric reduction. Under optimised conditions, the (S)-alkyl hydroxyesters were produced predominantly except for the methyl ester which formed the (R)-enantiomer. To the best of our knowledge, the biocatalytic asymmetric reduction of isoamyl-3-oxobutanoate to (S)-isoamyl-3-hydroxybutanoate is reported here for the first time.
Keywords: Asymmetric reduction; Alkyl-3-oxobutanoates; Transesterification; Candida parapsilosis ATCC 7330; Solvent studies

The paper deals with the exploitation of Ipomoea carnea as a feedstock for the production of bioethanol. Dilute acid pretreatment under optimum conditions (3 %H2SO4, 120 °C for 45 min) produced 17.68 g L−1 sugars along with 1.02 g L−1 phenolics and 1.13 g L−1 furans. A combination of overliming and activated charcoal adsorption facilitated the removal of 91.9 % furans and 94.7 % phenolics from acid hydrolysate. The pretreated biomass was further treated with a mixture of sodium sulphite and sodium chlorite and, a maximum lignin removal of 81.6 % was achieved. The enzymatic saccharification of delignified biomass resulted in 79.4 % saccharification with a corresponding sugar yield of 753.21 mg g−1. Equal volume of enzymatic hydrolysate and acid hydrolysate were mixed and used for fermentation with a hybrid yeast strain RPRT90. Fermentation of mixed detoxified hydrolysate at 30 °C for 28 h produced ethanol with a yield of 0.461 g g−1. A comparable ethanol yield (0.414 g g−1) was achieved using a mixture of enzymatic hydrolysate and undetoxified acid hydrolysate. Thus, I. carnea biomass has been demonstrated to be a potential feedstock for bioethanol production, and the use of hybrid yeast may pave the way to produce bioethanol from this biomass.
Keywords: Bioethanol; Ipomoea carnea ; Hybrid yeast; Hydrolysate; Fermentation

The mammalian matrix metalloproteinase-9 (MMP-9), which might play a role in ovulation, uterus remodeling, embryo development, and implantation in mammals, is one of the potential functional candidate genes for porcine reproductive traits. In this study, the entire genomic sequence of porcine MMP-9 (pMMP-9) gene was established; it contains 13 exons and 12 introns. Real-time PCR analysis revealed that pMMP-9 is highly expressed in the Minzhu uterus before puberty and decreases significantly after sexual maturity (p < 0.05). Two single-nucleotide polymorphisms (A3011G and T5079C) that can be detected by PCR restriction fragment length polymorphism (PCR-RFLP) were discovered and tested for statistical associations with litter size traits in a crossbred population (Line DIV) derived from Landrace, Large White, Chinese Tongcheng and/or Chinese Meishan pigs. For A3011G, the GG genotype was associated with a significantly higher (p < 0.05) number of live births than those recorded for AA sows and the additive effect was significant (p < 0.05). The T5079C marker is not associated with litter size in this population. Further studies are needed to confirm the results of this study.
Keywords: MMP-9 ; Expression; Polymorphism; Litter size; Pig

Acidic Proteases from Monterey Sardine (Sardinops sagax caerulea) Immobilized on Shrimp Waste Chitin and Chitosan Supports: Searching for a By-product Catalytic System by Jesus Aaron Salazar-Leyva; Jaime Lizardi-Mendoza; Juan Carlos Ramirez-Suarez; Elisa Miriam Valenzuela-Soto; Josafat Marina Ezquerra-Brauer; Francisco Javier Castillo-Yañez; Ramon Pacheco-Aguilar (795-805).
Solid wastes generated from the seafood industry represent an important environmental pollutant; therefore, utilization of those wastes for the development of processing biochemical tools could be an attractive and clean solution for the seafood industry. This study reports the immobilization of semi-purified acidic proteases from Monterey sardine stomachs onto chitin and chitosan materials extracted from shrimp head waste. Several supports (chitosan beads, chitosan flakes, and partially deacetylated flakes) were activated either with genipin or Na-tripolyphosphate and evaluated as a mean to immobilize acidic proteases. The protein load varied within the 67–91 % range on different supports. The immobilization systems based on chitosan beads achieved the highest protein loads but showed the lowest retained catalytic activities. The best catalytic behavior was obtained using partially deacetylated chitin flakes activated either with genipin or Na-tripolyphosphate. According to results, the immobilization matrix structure, as well as acetylation degree of chitin–chitosan used, has considerable influence on the catalytic behavior of immobilized proteases. Partially deacetylated chitin flakes represent a suitable option as support for enzyme immobilization because its preparation requires fewer steps than other supports. Two abundant seafood by-products were used to obtain a catalytic system with enough proteolytic activity to be considered for biotechnological applications in diverse fields.
Keywords: Chitin; Chitosan; Acidic proteases; Fisheries by-products; Enzyme immobilization

Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.
Keywords: Fatty acid; Immobilized yeast; Kluyveromyces marxianus ; Thermal stress