Current Biotechnology (v.4, #3)

Background: The big wave of the successes in glycobiology is also embracing the marine domain. The major inherent structural complexity of carbohydrate molecules greater than that of proteins and nucleic acids, adds challenges for the control of pharmaceutical preparations based on such complex biomolecules. More effort is needed to overcome current limitations for the use of marine polysaccharides in pharmaceutics.
Methods: From an overview of literature, a foresight is possible for the next decade, related to an expected increase of studies about marine oligosaccharides. These topics will be covered in this review covering the recent literature related to the subject.
Results: Scientific interest for marine oligosaccharides is spread worldwide: in western countries, and recently especially in Europe, the research funding Horizon 2020 programs, with the starting enthusiasm focused on marine domain, will make possible big research efforts to unlock biotechnological potential of the sea and oceans. In the eastern countries traditional interests for marine biomasses in food technology are in a renewing era with discovery of therapeutic potential of constituent carbohydrate molecules.
Conclusion: The situation analyzed is multifaceted including different elements such as sustainable supply of marine oligosaccharides by specific and high-yield enzymatic hydrolysis, new enabling methodologies for straightforward purification techniques, fine structural detail information including secondary structures, activity assay, mechanism(s) of action of these molecules, etc. Flourishing of molecular technologies suitable for interdisciplinary study of these molecules will be possible.


Biochemical and Structural Characterization of Alginate Lyases: An Update by Bing Wang, Shi-Qi Ji, Ming Lu, Fu-Li Li (223-239).
Background: Alginates, a complex copolymer of ?-L-guluronate (G) and its C5 epimer ?-Dmannuronate (M), can be disassembled by alginate lyases to monosaccharides or alginate oligosaccharides, and the latter exhibits many fascinating bioactivities. A lot of new alginate lyases have been identified in many resources, especially in marine organisms. Elucidating the natural paradigms of alginate conversion will enable us to utilize algal biomass efficiently and enhance our understanding of how microorganisms evolved with plants. Crystal structures of different polysaccharide lyase (PL) families have been solved by X-ray diffraction, which will improve our understanding in molecule-level catalytic mechanisms.
Methods: The researches related to alginate lyases after 2000 are reviewed, and data is sorted and analyzed. Protein sequences alignment and phylogenetic tree are employed to discuss different groups of alginate lyases. Furthermore, viewpoints about the studies of alginate lyases are proposed. Results: In this review, the latest resources of alginate lyases are summarized, progress of alginate lyases on structuralfunctional relationships are presented. The biochemical characterization, biological role, and the promising applications of alginate lyases also are discussed.
Conclusion: New alginate lyases have sprung up throughout the marine and terrestrial organisms. Structure-function analyses of alginate lyases will shed light on the understanding of catalytic mechanism of alginate lyases. However, the mechanisms of bioactivities of alginates are still unclear. Extensive investigation of alginate lyases is crucial for the development of alginate applications. Besides, a standard and meaningful naming system for alginate lyases may be needed.


Characterization of an eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia Yezoensis by Akira Inoue, Chieco Mashino, Toshiki Uji, Naotsune Saga, Koji Mikami, Takao Ojima (240-248).
Background: Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL- 7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis.
Methods: The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated.
Results: PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage.
Conclusion: This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.


Bioinformatics-Inspired Isolation of Akaemycin, a New Macrolide from Marine Streptomyces sp. by Tao Zhou, Seizo Sato, Hisayuki Komaki, Yasuhiro Igarashi (249-253).
Background: Investigation of products from marine Streptomyces sp. NPS554 led to the discovery of lorneic acids and akaeolide. Genome analysis of this strain predicted the presence of more unidentified secondary metabolites. In this study, we further examined the products of strain NPS554 to obtain novel compounds predicted by bioinfomatical screening.
Methods: Culture extract of strain NPS554 was fractionated by different chromatography methods. The chemical structure was elucidated by spectroscopic analysis. The absolute configuration was further determined by PKS gene analysis. Antimicrobial assay was conducted to evaluate the biological activity of the new compound.
Results & Conclusion: Akaemycin, a novel macrolide compound was isolated as a yellow amorphous solid. Its structure was elucidated by MS and NMR spectroscopic analyses. Its spectroscopically defined structure was consistent with the structure predicted from the annotation of orphan type I PKS gene clusters in the NPS554 genome. Stereochemistry of akaemycin was proposed on the basis of ROESY and bioinformatical analysis. Antimicrobial assay showed activity against Gram-positive bacteria and filamentous fungi.


Objective: This is a study on the condition optimization of production of l-arginine deiminase (ADI) by Vibrio alginolytics 1374. To increase the production quantity, it could be helpful in the production of ADI for the therapeutic use in the future.
Method: Vibrio alginolyticus 1374; GU726873 is a gram negative rod shaped marine bacteria isolated from chirala beach of Andhra Pradesh. It was screened for the enzyme production in minimal arginine medium by dye based method. Further the activity was confirmed calorimetrically by estimating the levels of l-citrulline. The potential of isolated novel strain for ADI production was analyzed under submerged fermentation with different process parameters and medium constituents. The enzyme thus obtained was purified by ammonium sulphate fractionation, ion exchange and gel permeation chromatography. The purity of enzyme was detected by polyacrylamide gel electrophoresis.
Result: The maximum yield of enzyme production was achieved in a seawater based medium at pH 8, 37°C, 2% inoculum concentration and 2% l-arginine concentration for 120 h. The medium when supplemented with carbon source, it improved the enzyme production from 172 to 192 IU/ml with 2% maltose. Addition of 2% soybean meal also improved the ADI production (183.56 IU/ml). The enzyme was purified to near homogeneity (1161.2 fold).The enzyme was found to have specific activity of about 280.6 IU/mg with molecular weight of about 48kDa.
Conclusion: This novel strain has an immense potential as an industrial organism for the large scale production of ADI for the therapeutic use in the future.


Background: Molluscan larval ontogeny is a highly conserved process comprising three principal developmental stages; trochophore, veliger and metamorphosis into the juvenile. A characteristic that is unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch in bivalves. These shells vary in morphology, mineralogy and microstructure. The discrete temporal transitions in shell biomineralization between these larval stages are utilized in this study to investigate transcriptional involvement in several distinct biomineralization events.
Methods: Scanning electron microscopy, X-ray diffraction and microarray differential gene expression analysis were used to document temporal transitions in morphology, mineralogy and microstructure of larval shells and the genes directing their biomineralization.
Results: P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis are described in terms of mineralogy and microstructure of each shelled stage as well as establishing a timeline for transitions in biomineralization. P. maxima larval samples most representative of these biomineralization distinctions and transitions were analyzed for differential gene expression with the microarray platform PmaxArray 1.0. A number of known shell matrix genes and novel transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. However, only a single transcript, PM066, was noted as being expressed before and after the transition to the adult shell design. No other known/putative adult shell matrix genes from P. maxima were detected in association with the larval shells prodissococh I and II, suggesting that their expression is either below detection limits or an almost entirely different set of genes is potentially responsible for larval and adult shell mineralization.
Conclusion: This interdisciplinary investigation has linked the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of bivalve development and shell biomineralization.


Applications of Natural and Artificial Phycobiliproteins in Solar Cells by Jianfei Ma, Huaxin Chen, Song Qin, Hanzhi Lin (275-281).
Background: Phycobiliproteins are located in phycobilisomes, which are huge pigmentprotein complexes that function as light harvesting antennas for photosynthesis in cyanobacteria, red algae and other uncommon species.
Methods: The research progress of structures of the pigmented-covalently-binding phycobiliproteins were reviewed, including PhycoCyanin (PC), AlloPhycoCyanin (APC), PhycoErythroCyanin/PhycoErythrin (PEC/PE) and linkers. In addition, the progress of their combinational biosynthesis was also visited.
Results: We summarized advances in the development of phycobiliproteins-Sensitized Solar Cells (Dye Sensitized Solar Cells, DSSC) with high photoelectric transfer efficiency and low cost. Meantime, the efficiencies were compared on phycobilisome-based and bacteriochlorophyll/ chlorophyll-Sensitized Solar Cells. It was also discussed that directional molecular design of phycobiliproteins, for artificial high-level structure.
Conclusion: To accomplish higher level assembling of in vitro synthesis of phycobiliproteins requires technology for multichromophore attachment to apo-phycobiliproteins, and an understanding of linkers' function in high-level phycobilisome assembly.


Desalination Plants: Technology to Supply Water to Mining Processes and Local Populations, Opportunities and Environmental Impact by Pamela Chavez-Crooker, Johanna Obreque-Contreras, Danilo Pérez-Flores, Andrea Contreras-Vera (282-295).
Background: Mining operations generate high demand of water resources in the areas where they operate; in Chile, the majority of major mining operations are concentrated in the north, close to the Atacama Desert, the driest desert in the world. Fresh water is no longer available in big quantities in the region; for this reason, new alternatives have been developed. The use of seawater and desalination plants has increased in the past years and significant projects are underway. Unfortunately, desalination plant discharge may have an environmental impact that needs to be studied and monitored. In this chapter, we attempt to review state of the art desalination technologies, their environmental impact, and the current situation in Chile, together with our recommendations to control and diminish any negative impacts desalination plants could potentially produce.
Conclusion: In order to protect and preserve the marine environment and make use of seawater as a source of industrial water, it is important to study, measure, and evaluate the potential impacts of the desalination plant on the marine environment, implemented by multidisciplinary teams, in order to mitigate ecological harm. The benefits and economic impacts of a new source of water must also be evaluated taking into account the projected water needs of industrial operations in the region. Under these conditions, desalination plants have the potential to be a viable and sustainable technology.

Background: Global aquaculture production has increased continuously over the last five decades, and particularly in China. Its aquaculture has become the fastest growing and most efficient agri-sector, with production accounting for more than 70% of the world's aquaculture output. In the new century, with serious challenges regarding population, resources and the environment, China has been working to develop high-quality, effective, healthy, and sustainable blue agriculture through the application of modern biotechnology. Sound knowledge related to the biology and ecology of aquatic organisms has laid a solid foundation and provided the innovation and technology for rapid development of the aquaculture industry. Marine biotechnology, which is enabling solutions for ocean productivity and sustainability, has been promoted since the last decades of the 20th Century in China.
Objective: In this article, priority areas of research, mainly genetic breeding, omics studies, novel production systems, biosecurity, bioprocesses and biorefinery, as well as the major progress of marine biotechnology R&D in China are reviewed.
Conclusion: Current innovative achievements in China are not enough and the level and frequency of academic advancements must be improved. International cooperation and assistance remain crucial for the success of marine biotechnology.

The Marine Aquaculture Biotechnology Status and its Sustainability in Taiwan by Jenn-Kan Lu, Hsin-Yiu Chou, Hong-Yi Gong, Pinwen P. Chiou, Chang-Wen Huang, Li- Li Chen, Jen-Leih Wu (311-318).
Background: Aquaculture faces many challenges over the next decade, notably, combating diseases andepizootics, broodstock improvement and domestication, development of appropriate feeds and feeding mechanisms, hatchery and grow-out technology, as well as water-quality management. These all present considerable scope for biotechnological and other technology interventions. Aquaculture biotechnology can be described as the scientific application of biological concepts that enhance the productivity and economic viability of its various industrial sectors (Liao and Chao, 1997) [71].
Objective: This paper is to introduce the conception of sustainable aquaculture, status, objective and how to enter the way of sustainability in Taiwan. The paper provides an overview of the current trends in marine aquaculture and highlights how the Taiwan aquaculturists employed various technological innovations that occurred in the management and production of aquatic resources. The purpose of the paper is to explain the role technological innovation played in meeting the growing worldwide consumer demand for aquatic species, and in dealing with scientific uncertainty regarding the potential negative impact of these advances on the aquatic environment, fish health and the socioeconomic circumstances.
Methods: We use the OECD definition of biotechnology: “The application of science and technology to living organisms, as well as parts, products and models thereof, to alter living or non-living materials for the production of knowledge, goods and services”. To revolutionize aquaculture, it is essential to apply modern biotechnology techniques. Taiwan aquaculturists conducting biotechnology research would adopt at least one of six technologies or processes, biotechnological techniques include (a) water environmental bioremediation technology, (b) -omics, bioinformatics and molecular breeding program to select superior aquatic seeds, (c) molecular nutrition and feed biotechnology to develop functional feed and additives, and(d) biosecurity management strategies for aquatic animals, (e) smart aquaculture facility ; including recirculation system and aquaponics system, and (f) application of ICT and IoT technology to establish national wide seafood traceability system.
Results: We find it useful to follow up on this overall definition, and elaborate on its practical applications within the marine culture biotechnology sector. One way of following up is to divide into six different researches and business sub-areas, as follows:
a) Microbial agents for environmental remediation, bio-control and probiotics; b) New genetic markers and breeding program to speed up the selection of new strain with good traits for industrial use; c) To develop green functional feed additives by nutrigenomics and molecular nutrition concepts, economical alternatives to fish meal as a protein source in aquaculture feeds; d) Providing new techniques for biosecurity management of aquatic species; e) Seafood traceability to ensuring bio-safety and food safety of aquaculture and fisheries; f) Smart aquaculture facility, including recirculation system and aquaponics system
Conclusion: The paper briefly reports the current progress in Taiwan and thrust areas in the use of probiotics for water environmental remediation, molecular markers for fish breeding and production of monosex and transgenesis, biotechnology in aquaculture nutrition and health management, gene banking, intelligent aquaculture facility and the seafood traceability system. The conclusions recapitulate some of the findings gained in this interdisciplinary analysis on the role of marine culture biotechnology in the management of aquatic resources, and emphasize the long-run perspective of technological innovation in fish and shellfish production.

Drug-Loaded Nanocarriers in Tumor Targeted Drug Delivery by Meerza Abdul Razak, Shankarappa Devi Prasad Boggupalli, Buddolla Viswanath (319-344).
Background: Cancer cells are more susceptible than normal cells to the effect of chemotherapeutic agents and most of the anticancer drugs can cause harm to the normal cells. Optimum dose and frequency are both important factors in the persistence of cancer cells during cancer chemotherapy. Now attempts have been focusing on efforts to kill cancer cells by more specific targeting while sparing the normal cells by drug loaded nanocarriers.
Methods: We review technical papers and other articles related to nanoparticles, which have been developed as effective target specific strategies for cancer treatment, acting as nanocarriers and also as active agents.
Results: This review will allow to gain a more general view of the various drug loaded nanocarriers which offers a predominantly unique set of chemical, physical and photonic properties for better drug delivery to the tumor tissues based on morphological and functional differences between normal and tumor tissues.
Conclusion: Nanoparticulate delivery systems in cancer therapies provide better penetration of therapeutic and diagnostic substances with the cancerous tissue in comparison to conventional cancer therapies. Thus nanocarriers can be used for strategic development of novel drug delivery systems and reformulating existing drugs to enhance effectiveness.

Regioselective Oxygenation of Polyunsaturated Fatty Acids by the Thermostable P450 from Thermus thermophilus HB27 by Shibdas Banerjee, Dwaipayan Dutta Gupta, Shyamalava Mazumdar (345-356).
Background: CYP175A1 is a thermophilic P450 with high potential to invoke as an industrially viable biocatalyst though very little is known about the natural substrate of this enzyme. The crystal structure of CYP175A1 is similar to its mesophilic analogue CYP102A1, which is a fatty acid metabolizing enzyme (J Biol Chem 1990; 265: 4233-9). We have earlier shown that CYP175A1 catalyzes regioselective mono-oxygenation of different monounsaturated fatty acids (Biochemistry 2012; 51: 7880-90). The present work highlights screening of several important polyunsaturated fatty acids for their potentiality as substrate of this orphan P450 enzyme.
Methods: Both molecular modeling/docking as well as spectrophotometric screening of the substrates along with detailed activity assay and mass spectrometric identification of the products have been carried out to determine the reactivity and selectivity of oxygenation of polyunsaturated fatty acids by CYP175A1.
Results: Our investigations showed that polyunsaturated fatty acids (arachidonic acid, linoleic acid, ?-linolenic acid and - linolenic acid) can be oxygenated by the enzymatic action of CYP175A1 although the enzyme did not show any detectable activity on the corresponding saturated analogues. Results further showed that unlike the monounsaturated fatty acids, the polyunsaturated fatty acids undergo mono- as well as di-oxygenation reactions. Analyses of products also suggested that the monooxygenation of these fatty acids is highly regioselective and is regulated by the number and position of the double bonds in the fatty acids.
Conclusion: CYP175A1 may have important roles in lipid metabolism and thus can be used for regioselective oxygenation of different fatty acids. The present study also improves our current understanding on the nature of the enzyme pocket and of the possible natural substrate of this orphan enzyme.

Introduction: Understanding the factors responsible for enzyme thermostability and discriminating them from mesophilic proteins is one of the most important concerns in engineering new proteins. The primary structure of a protein is an important factor in determining L-asparaginase thermostability and it can be improved by adjusting external environmental factors. L-asparaginase II possesses antitumor activity and expressed in the periplasmic space of the bacterial membranes as it catalyzes the conversion of L-asparagine to L-aspartic acid
Methods: In present study, in silico analysis of amino acid sequences of ten mesophilic and ten thermophilic bacterial L-asparaginases has been done. The sequences were subjected to multiple sequence alignment (MSA), discovering individual amino acid composition, and phylogenetic tree construction.
Results: The multiple sequence alignment analysis of amino acid sequences has shown clear differences between mesophilic and thermophillic asparaginases in terms of position specific presence of conserved amino acids. The physiochemical properties of these two groups of L-asparaginase enzyme also differ in number of amino acids, molecular weight, positively charged ions, i.e. Arg + Lys, aliphatic index, Grand Average of Hydropathacity (GRAVY) and the compositions of different amino acid residues. The comparative study of amino acid sequence and physicochemical properties indicate the affiliation of protein from L-asparaginase enzyme of nitrilase superfamily.
Conclusion: The present finding will be helpful in identification and elucidation of the extent of thermostability among Lasparaginases from the large number of sequenced microbial genomes for current as well as future biotechnological applications.

Background: Poly-?-hydroxybutyrate (PHB) from cyanobacteria is considered to be a potentially viable source for degradable plastic. Yet, to date, only a modest level of research has been reported in this area. PHB has variety of properties such as natural origin, biocompatibility, biodegradability, piezoelectricity, streospecificity, thermoplasticity and optical activity, which make it suitable for various applications in health industry. Methods: This article elucidates comprehensive information and knowledge with regard to several stress conditions (e.g., salt stress, heat and chilling stresses, heavy metal supplementation, limitation of gas exchange and nutrient deficiency) to enhance PHB production from a diazotrophic cyanobacterium, Aulosira fertilissima CCC 444. Results: The results showed that cultures reached a maximum biomass concentration of 631 mg l-1, regardless of initial inoculum levels. However, with an initial inoculum of 80 mg dry cell weight (dcw) l-1, maximum PHB accumulation was achieved under regular light-dark cycles. A. fertilissima supplemented with heavy metals showed enhancement in PHB accumulation up to 18.7% of dcw in 1.0 mg l-1 Ni-supplemented medium for 7 days, followed by 17.6% (dcw) in 1.5 mg l-1 Cu-supplemented medium. NaCl stress, heat and chilling stresses and deficiencies of potassium, calcium, and magnesium had negative effects on PHB accumulation. Interestingly, supplementation of 0.5% acetate at the initiation of gas exchange limitation boosted the accumulation of PHB to 48.7% (dcw) on 14 days of incubation, a value 7-fold higher compared to the control.
Conclusion: Gas exchange limitation with carbon supplementation found to be an appropriate condition for enhanced PHB accumulation in cyanobacteria. Thus, using cyanobacteria for PHB production is one of the most promising ecofriendly ways as these are oxygen evolving photoautotrophic organisms; require minimal inorganic nutrients for growth and due to aquatic in nature, cyanobacteria do not compete for arable land for their cultivation. Currently, intensive research is essential in this area for ingenious utilization of cyanobacterial biomass to produce environmentally affordable bioplastic.

Background: ?-galactosidase is an enzyme that hydrolyzes lactose to glucose and galactose. Lactose is the major sugar present in milk and its hydrolysis makes milk fit for consumption by lactose intolerant people. Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry. Thus, the continuous efforts are going on to enhance ?-galactosidase production and operational stability of immobilized preparations. The present paper deals with isolation of bacteria using soil sample enriched with whey, identification of bacteria, ?-galactosidase production and whole cell immobilization to evaluate the potential of isolated bacteria for industrial application.
Methods: The identification of isolated bacteria was carried out using 16S r-RNA based technology. The fermentation medium was optimized using response surface design and permeabilized whole cells immobilization was carried out using gelatine-alginate gel.
Results: The isolated bacterial strain was identified as Pantoea septic, which produced 2.0±0.06 fold higher ?- galactosidase activity than the known bacterial strain when tested under similar experimental conditions. The fermentation medium optimized using response surface design resulted into 26.45±0.52% higher ?-galactosidase activity than the nonoptimized media. Toluene treated isolated bacterial whole cells showed 4.86±1.4 fold higher ?-galactosidase activity than the untreated cells with negligible enzyme leakage. The maximum activity for immobilized enzyme was obtained at pH 5.5 and temperature 60 °C. After ten consecutive cycles, immobilized whole cells displayed 80.4±2.3% enzyme activity of its initial activity.
Conclusion: The isolated bacterial strain, which produced 2.0±0.06 fold higher ?-galactosidase activity than the known bacterial strain when tested under similar experimental conditions prove its novelty. Secondly, the isolated bacterial strain which was identified as Pantoea septic has not been reported as ?-galactosidase producing strain so far. There is a possibility that the further genetic modification of isolated bacteria may give highly active and stable ?-galactosidase producing strain in comparison with the microbes being used currently at industrial level operations. In addition, the immobilized preparation has retained 80.4±2.3% enzyme activity after ten consecutive cycles. All these results strongly suggest the high potential of isolated bacteria (Pantoea septica) for industrial applications.

Purification and Characterization of ?-Glucosidase Produced by Aspergillus terreus Under Solid State Fermentation by Noura El-Ahmady El-Naggar, S.A. Haroun, Ahmed Abd ElRazak, Eman A. Owis, A.A. Sherief (380-386).
Background: ?-glucosidase is one of the important components of cellulase enzyme complex which acts synergistically with endoglucanase and exoglucanase to hydrolyze cellulosic substances into glucose. ?-glucosidase is widely being used in biofuels, food, textile, cosmetics, leather, detergent, pulp, pharmaceutical and paper industries. It prevents the discoloration of fruit juices and helps in the enzymatic release of aromatic compounds from flavorless glucosidic precursors present in fruits and fermenting products. It has also several other potential applications including the increase of bioaccessibility of isoflavones in human intestine.
Methods: Purification of ?-glucosidase from Aspergillus terreus strain EMOO 6-4 was carried out in a two-step procedure; ammonium sulphate was used for the enzyme precipitation, then further separation occurs by gel filtration method using Sephadex G100. Characterization of the enzyme was carried out to determine the optimum incubation period, pH level, temperature of the enzyme activity and the effect of different concentration of the specific substrate on the enzyme activity.
Results: The optimum reaction incubation period for maximum ?-glucosidase activity produced by Aspergillus terreus strain EMOO 6-4 was 60 min. The optimum reaction pH and temperature were 5.5 and 35°C, respectively. The enzyme showed the highest activity at substrate concentration 0.0136 M. The enzyme is thermo stable up to 40 min at 50°C with calculated half-life of approximately 173.49 hours. The molecular weight of the purified ?-glucosidase determined by SDS-PAGE was found to be 120 kDa.
Conclusion: Aspergillus terreus strain EMOO 6-4 as wild isolate was found to be a good producer of ?-glucosidase enzyme. The enzyme thermostability and high activity in an acidic pH (5.5) could find a profound use of the enzyme in saccharification of lignocellulosic wastes; that can help in final biodegradation of agricultural wastes to produce glucose as high energy source that can be used in different industries.