Search results for: enzyme production from fungi

Authors: Nuha Mansour Alhazmi, Magda Mohamed Aly, Fardus M. Bokhari, Ahmed Bahieldin, Sherif Edris

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Out of 30 fungal isolates, 2 new isolates were proven to degrade poly-β-hydroxybutyrate (PHB). Enzyme assay for these isolates indicated the optimal environmental conditions required for depolymerase enzyme to induce the highest level of biopolymer degradation. The two isolates were basically characterized at the morphological level as Trichoderma asperellum (isolate S1), and Aspergillus fumigates (isolate S2) using standard approaches. The aim of the present study was to characterize these two isolates at the molecular level based on the highly diverged rRNA gene(s). Within this gene, two domains of the ribosome large subunit (LSU) namely internal transcribed spacer (ITS) and 26S were utilized in the analysis. The first domain comprises the ITS1/5.8S/ITS2 regions ( > 500 bp), while the second domain comprises the D1/D2/D3 regions ( > 1200 bp). Sanger sequencing was conducted at Macrogen (Inc.) for the two isolates using primers ITS1/ITS4 for the first domain, while primers LROR/LR7 for the second domain. Sizes of the first domain ranged between 594-602 bp for S1 isolate and 581-594 bp for S2 isolate, while those of the second domain ranged between 1228-1238 bp for S1 isolate and 1156-1291 for S2 isolate. BLAST analysis indicated 99% identities of the first domain of S1 isolate with T. asperellum isolates XP22 (ID: KX664456.1), CTCCSJ-G-HB40564 (ID: KY750349.1), CTCCSJ-F-ZY40590 (ID: KY750362.1) and TV (ID: KU341015.1). BLAST of the first domain of S2 isolate indicated 100% identities with A. fumigatus isolate YNCA0338 (ID: KP068684.1) and strain MEF-Cr-6 (ID: KU597198.1), while 99% identities with A. fumigatus isolate CCA101 (ID: KT877346.1) and strain CD1621 (ID: JX092088.1). Large numbers of other T. asperellum and A. fumigatus isolates and strains showed high level of identities with S1 and S2 isolates, respectively, based on the diversity of the first domain. BLAST of the second domain of S1 isolate indicated 99 and 100% identities with only two strains of T. asperellum namely TR 3 (ID: HM466685.1) and G (ID: KF723005.1), respectively. However, other T. species (ex., atroviride, hamatum, deliquescens, harzianum, etc.) also showed high level of identities. BLAST of the second domain of S2 isolate indicated 100% identities with A. fumigatus isolate YNCA0338 (ID: KP068684.1) and strain MEF-Cr-6 (ID: KU597198.1), while 99% identities with A. fumigatus isolate CCA101 (ID: KT877346.1) and strain CD1621 (ID: JX092088.1). Large numbers of other A. fumigatus isolates and strains showed high level of identities with S2 isolate. Overall, the results of molecular characterization based on rRNA diversity for the two isolates of T. asperellum and A. fumigatus matched those obtained by morphological characterization. In addition, ITS domain proved to be more sensitive than 26S domain in diversity profiling of fungi at the species level.

Keywords: Aspergillus fumigates, Trichoderma asperellum, PHB, degradation, BLAST, ITS, 26S, rRNA

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Authors: Deepanwita Deka, Dhruva Kumar Jha

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Endophytes are the microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects. Endophytes are rich source of therapeutic substances like antimicrobial, anticancerous, herbicidal, insecticidal, immunomodulatory compounds. Brucea mollis, commonly known as Quinine in Assam, belonging to the family Simaroubaceae, is a shrub or small tree, recorded as endangered species in North East India by CAMP survey in 2003. It is traditionally being used as antimalarial and antimicrobial agent and has antiplasmodial, cytotoxic, anticancer, diuretic, cardiovascular effect etc. Being endangered and medicinal; this plant may host certain noble endophytes which need to be studied in depth. The aim of the present study was isolation and identification of potent endophytic fungi from Brucea mollis, an endangered medicinal plant, to protect it from extinction due to over use for medicinal purposes. Aseptically collected leaves, barks and roots samples of healthy plants were washed and cut into a total of 648 segments of about 2 cm long and 0.5 cm broad with sterile knife, comprising 216 segments each from leaves, barks and roots. These segments were surface sterilized using ethanol, mercuric chloride (HgCl2) and aqueous solution of sodium hypochlorite (NaClO). Different media viz., Czapeck-Dox-Agar (CDA, Himedia), Potato-Dextrose-Agar (PDA, Himedia), Malt Extract Agar (MEA, Himedia), Sabourad Dextrose Agar (SDA, Himedia), V8 juice agar, nutrient agar and water agar media and media amended with plant extracts were used separately for the isolation of the endophytic fungi. A total of 11 fungal species were recovered from leaf, bark and root tissues of B. mollis. The isolates were screened for antimicrobial, antioxidant and enzymatic activities using certain protocols. Cochliobolus geniculatus was identified as the most dominant species. The mycelia sterilia (creamy white) showing highest inhibitory activity against Candida albicans (MTCC 183) was induced to sporulate using modified PDA media. The isolate was identified as Geosmithia pallida. The internal transcribed spacer of rDNA was sequenced for confirmation of the taxonomic identity of the sterile mycelia (creamy white). The internal transcribed spacer r-DNA sequence was submitted to the NCBI (KU693285) for the first time from India. G. pallida and Penicillium showed highest antioxidant activity among all the isolates. The antioxidant activity of G. pallida and Penicillium didn’t show statistically significant difference (P˃0.05). G. pallida, Cochliobolus geniculatus and P. purpurogenum respectively showed highest cellulase, amylase and protease activities. Thus, endopytic fungal isolates may be used as potential natural resource of pharmaceutical importance. The endophytic fungi, Geosmithia pallida, may be used for synthesis of pharmaceutically important natural products and consequently can replace plants hitherto used for the same purpose. This study suggests that endophytes should be investigated more aggressively to better understand the endophyte biology of B. mollis.

Keywords: Antimicrobial activity, antioxidant activity, Brucea mollis, endophytic fungi, enzyme activity, Geosmithia pallida

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Authors: Tadesse Kebede, Orkun Baris Kovanci

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Tomatoes leaf minor (Tutaabasoluta) is one of the most economically important insect pest in tomatoes production. The use of biological control such as entomopathogen fungi isolates would be a long-term and cost-effective solution to control insects pest. Therefore, identifying the most virulent and pathogenic entomopathogen fungi is one of the basic requirements for effective management options to combat Tomatoes leaf minor (Tutaabasoluta). Furthermore, the pathogenicity and virulence difference among entomopathogenfungus strains is not widely well investıgated. The current study was therefore initiated to test the pathogenicity of some entomopathogenic fungus isolates against Tutaabsoluta. The experiment was conducted at Bursa Uludag University, Agiculutre faculty, horticulture department glasshouse in 2020/2021. Tutabasoluta adult were collected, and masslarvae were reared in a growth chamber. Then, ten third instar larvae were inoculated with four entomopathogen fungi isolates (Beuaveriabassania Ak-10, Beuaveriabassania Ak-14, Metarhziumanisoplai Ak-11, and Metarhziumanisoplai Ak-12) with different inoculum suspension (0, 1x10⁶, 1x10⁷,,4 × 10⁸, 4× 10⁹ and 1×10¹⁰ conidia /ml) in a factorial experiment arranged in randomized complete block design with three replication. Mortality data assessment was done on the 3rd, 5thand 7th days after treatment and analyzed. The analysis of variance for mortality rate revealed significant variations (p<0.05) among entomoptahogen fungi isolates and conidia concentrations. The results revealed thatMetarhziumanisoplai Ak-12was found to show the lowest mortality percentage80.77%, highest LC50 2.3x108, and the longest incubation period, LT50, 4.9 and LT90, 9.9daysand considered to be less pathogenic fungi. On the other hand, Beuaveriabassania Ak-10 isolate showed the highest mortality percentage, 91%, and the lowest LT50, 4, and LT90, 7.6 values at 1×10¹⁰ conidia /ml, followed by Beuaveriabassania Ak-14 and being considered as the most aggressive bio-agent. Metarhziumanisoplai Ak-11 was determined as moderately virulent, having a mortality rate 27-81%. Results also revealed that among conidia concentrations, 1x10⁹ and 1x10¹⁰ suspensions is the most effective, while 1x10⁶ conidia/ml concentration is the least effective. Hence, results indicated that EPF tested were effective against T. absoluta larvae. As the current work revealed the potential variation among entomopathogen fungi isolates and concentration against third instar larvae.

Keywords: tuta absoluta, tomato, metarhizium anisopliae, beauveria bassiana, biological control

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Authors: Afsheen Aman, Zainab Bibi, Shah Ali Ul Qader

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Introduction: Xylan is a heteropolysaccharide composed of xylose monomers linked together through 1,4 linkages within a complex xylan network. Owing to wide applications of xylan hydrolytic products (xylose, xylobiose and xylooligosaccharide) the researchers are focusing towards the development of various strategies for efficient xylan degradation. One of the most important strategies focused is the use of heat tolerant biocatalysts which acts as strong and specific cleaving agents. Therefore, the exploration of microbial pool from extremely diversified ecosystem is considerably vital. Microbial populations from extreme habitats are keenly explored for the isolation of thermophilic entities. These thermozymes usually demonstrate fast hydrolytic rate, can produce high yields of product and are less prone to microbial contamination. Another possibility of degrading xylan continuously is the use of immobilization technique. The current work is an effort to merge both the positive aspects of thermozyme and immobilization technique. Methodology: Geobacillus stearothermophilus was isolated from soil sample collected near the blast furnace site. This thermophile is capable of producing thermostable endo-β-1,4-xylanase which cleaves xylan effectively. In the current study, this thermozyme was immobilized within a synthetic and a non-synthetic matrice for continuous production of metabolites using entrapment technique. The kinetic parameters of the free and immobilized enzyme were studied. For this purpose calcium alginate and polyacrylamide beads were prepared. Results: For the synthesis of immobilized beads, sodium alginate (40.0 gL-1) and calcium chloride (0.4 M) was used amalgamated. The temperature (50°C) and pH (7.0) optima of immobilized enzyme remained same for xylan hydrolysis however, the enzyme-substrate catalytic reaction time raised from 5.0 to 30.0 minutes as compared to free counterpart. Diffusion limit of high molecular weight xylan (corncob) caused a decline in Vmax of immobilized enzyme from 4773 to 203.7 U min-1 whereas, Km value increased from 0.5074 to 0.5722 mg ml-1 with reference to free enzyme. Immobilized endo-β-1,4-xylanase showed its stability at high temperatures as compared to free enzyme. It retained 18% and 9% residual activity at 70°C and 80°C, respectively whereas; free enzyme completely lost its activity at both temperatures. The Immobilized thermozyme displayed sufficient recycling efficiency and can be reused up to five reaction cycles, indicating that this enzyme can be a plausible candidate in paper processing industry. Conclusion: This thermozyme showed better immobilization yield and operational stability with the purpose of hydrolyzing the high molecular weight xylan. However, the enzyme immobilization properties can be improved further by immobilizing it on different supports for industrial purpose.

Keywords: immobilization, reusability, thermozymes, xylanase

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Authors: Hakan Arslan, Deniz Ekinci, Alper Gungor, Gurkan Bilir, Omer Tas, Mehmet Altun

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Drought is one of the major abiotic stresses affecting the agricultural production worldwide. In this study, Leaf samples were taken from the carrot plants grown under drought stress conditions during the harvesting period. The plants were irrigated in three irrigation interval (4, 6 and 8 days) and Irrigation water regime was set up in pots. The changes in activities of antioxidant enzymes such as glutathione reductase (GR), glutathione s-transferase (GST), superoxide dismutase (SOD)) in leaves of black carrot were investigated. The activities of antioxidant enzymes (GR, GST, SOD) were varied significantly with irrigation intervals. The highest value of GR, GST and SOD were determined in the irrigation interval of 6 days. All antioxidant activity values were decreased in 8 days of irrigation interval. As a result of the study, it has been suggested that optimum irrigation intervals for plants can be used in antioxidant enzymes.

Keywords: antioxidant enzyme, carrot, drought, irrigation interval

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Authors: J. B. Minari, O. B. Oloyede, A. A. Odutuga

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Myeloperoxidase is the most abundant enzyme found in the polymorphonuclear neutrophil and is known to play a central role in the host defense system of the leukocyte. The enzyme has been reported to interact with some drugs to generate free radical which inhibits its activity. This study investigated the effects of artesunate on the activity of the enzyme and the subsequent effect on the host immune system. In investigating the effects of the drugs on myeloperoxidase, the influence of concentration, pH, partition ratio estimation and kinetics of inhibition were studied. This study showed that artesunate is concentration-dependent inhibitor of myeloperoxidase with an IC50 of 0.078mM. Partition ratio estimation showed that 60 enzymatic turnover cycles are required for complete inhibition of myeloperoxidase in the presence of artesunate. The influence of pH on the effect of artesunate on the enzyme showed least activity of myeloperoxidase at physiological pH. The kinetic inhibition studies showed that artesunate caused a competitive inhibition with an increase in the Km value from 0.12mM to 0.26mM and no effect on the Vmax value. The Ki value was estimated to be 2.5mM. The results obtained from this study show that artesunate is a potent inhibitor of myeloperoxidase and it is capable of inactivating the enzyme. It is considered that the inhibition of myeloperoxidase in the presence of artesunate as revealed in this study may partly explain the impairment of polymorphonuclear neutrophil and consequent reduction of the strength of the host defense system against secondary infections.

Keywords: myeloperoxidase, artesunate, inhibition, nuetrophill

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Authors: Muhammad Adeel Arshad, Shaukat Ali Bhatti, Faiz-ul Hassan

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Manipulating microbial ecosystem in the rumen is considered as an important strategy to optimize production efficiency in ruminants. In the past, antibiotics and synthetic chemical compounds have been used for the manipulation of rumen fermentation. However, since the non-therapeutic use of antibiotics has been banned, efforts are being focused to search out safe alternative products. In tropics, crop residues and forage grazing are major dietary sources for ruminants. Poor digestibility and utilization of these feedstuffs by animals is a limiting factor to exploit the full potential of ruminants in this area. Hence, there is a need to enhance the utilization of these available feeding resources. One of the potential strategies in this regard is the use of direct-fed microbes. Bacteria and fungi are mostly used as direct-fed microbes to improve animal health and productivity. Commonly used bacterial species include lactic acid-producing and utilizing bacteria (Lactobacillus, Streptococcus, Enterococcus, Bifidobacterium, and Bacillus) and fungal species of yeast are Saccharomyces and Aspergillus. Direct-fed microbes modulate microbial balance in the gastrointestinal tract through the competitive exclusion of pathogenic species and favoring beneficial microbes. Improvement in weight gain and feed efficiency has been observed as a result of feeding direct-fed bacteria. The use of fungi as a direct-fed microbe may prevent excessive production of lactate and harmful oxygen in the rumen leading to better feed digestibility. However, the mechanistic mode of action for bacterial or fungal direct-fed microbes has not been established yet. Various reports have confirmed an increase in dry matter intake, milk yield, and milk contents in response to the administration of direct-fed microbes. However, the application of a direct-fed microbe has shown variable responses mainly attributed to dosages and strains of microbes. Nonetheless, it is concluded that the inclusion of direct-fed microbes may mediate the rumen ecosystem to manage lactic acid production and utilization in both clinical and sub-acute rumen acidosis.

Keywords: microbes, roughages, rumen, feed efficiency, production, fermentation

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Authors: Yung-Chen Hsu, Juan Hernandez, W. T. Evert Ting, Dawit Gizachew

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Mycotoxin contamination of foods and feeds poses a high risk for human and animal health. Ochratoxin A (OTA) is a ubiquitous mycotoxin produced by Aspergillus and Penicillium fungi. It exhibits nephrotoxicity, teratogenicity, mutagenicity, and immunotoxicity in both humans and animals. OTA has been detected in foods such as cereals, coffee, grapes, cocoa, wine, and spices. Consumption of food contaminated with OTA has been linked to kidney and liver diseases. Niger (Guizotia abyssinica) is an oil seed that is used for extracting cooking oil in countries like Ethiopia and India. The seed cake (a byproduct from oil extraction) is also used as dairy cattle feed in Ethiopia. It is also exported to North America and Europe to be used mainly as bird feed. To our knowledge, there have been no studies on the growth and production of OTA on niger seeds. In this study, the environment conditions that support OTA production including effects of water activity, temperature, and incubation time on growth and OTA production by A. fresenii and A. sulphureus were investigated.

Keywords: mycotoxin, ochratoxin A, aspergillus, niger seed

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Authors: Ali Bahri Lubis

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The study of dioxygenase enzymatic mechanism on tryptophan oxidation has been a wide interest since the reaction is rate-limiting step of kynurenine pathway. In this research, observation of tryptophan oxidation through h-IDO enzyme along with synthesis of enzyme products was conducted in order to comprehend how the enzyme works on distinct substrates. UV-vis spectrophotometry, LC-MS, H-NMR and HSQC measurement were carried out to characterise enzyme product. It is found that while tryptophan was oxidised to form Nformylkynurenine (NFK) as a major product and hydroxypyrroloindole amine carboxylic acid (HPIC) in cis and trans confirmed in HSQC, N-methyl tryptophan substrate was converted to NFK and trans HPIC only. Other intriguing results showed that 5-hydroxy- tryptophan and Stryptophan was degraded to become NFK and epoxide cyclic respectively. The formation of NFK was considered through dioxygenation pathway, however HPIC was formed via monooxygenation. The epoxide cyclic—considered as intermediate compound in the mechanism— from S-tryptophan was not able to cleave the epoxide ring since bond energy of epoxide was probably much stronger. This validates the enzymatic mechanism where the intermediate compound in the enzymatic mechanism is epoxide cyclic.

Keywords: tryptophan oxidation, heme-dioxygenases, N-formylkynurenine, hydroxypyrrroloindoleamine, monooxidation

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Authors: Adamu Muhammed Tukur

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Pulp and paper industry is one of the fastest growing industries due to an increased demand in paper products. For it to satisfy this ever increasing demand, it adopts new technological innovations some of which are proved to affect our environment negatively. Global consumption of paper has increased by 400% in the last four decades and this suggests that more research is required to assess the impact of industrial effluents to our environment and public health. Paper products are generally biodegradable, however, the processes involved in its production which involve the use of mainly bleaching agents and other non-biodegradable substances pose serious problem to the environment. There are more than 250 chemicals released in paper mill waste and some are xenobiotics. Different methods such as physical and chemical methods can be adopted for the remediation of the effluents but are proved to be costly and not safe to the environment. On the other hand, biological method is shown to be less costly and environmentally friendly. Microorganisms and their enzymes have shown a promising future for bioremediation of effluents related to paper mill. Many studies prove that one of the major pollutants in the paper mill effluent is phenol especially its chlorinated derivatives. Pentachlorophenol is extremely hazardous to living cells and therefore need to be removed from the environment. Microorganisms including bacteria and fungi have the potential to degrade phenolic compounds e.g. Bacillus stearothermiphilus, Pseudomonas putida, Coricus versicolor, Sphingomonas chlorophenolica, Fusarium sp, Bacillus subtilis and P. aeroginosa. Enzymes used for the degradation include phenol hydrooxylase, polyphenoloxylase, laccase, peroxidase among others. Lignin is another important pollutant and is resistant to microbial degradation but it has been proved that certain bacteria and fungi like can degrade it. Among the fungi white-rot fungi like Fomes lividus and Trametes vesicolor are the most important bioremediators. This review focused on use of microorganism to reduce or eradicate pollutants released from the paper industry. It can serve as a review for further research to be conducted especially in the field of Biotechnology.

Keywords: bioremediation, pulp and paper, pentachlorophenol, environment

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Authors: Elizabeth Starzycka-Korbas, Michal Starzycki, Wojciech Rybinski, Mirosława Dabert

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For a few years, the populations of pathogenic fungi occurring in winter oilseed rape in Malyszyn were analyzed. Brassica napus L. in Poland and in the world is a source of energy for both the men (oil), and animals, as post-extraction middling, as well as a motor fuel (oil, biofuel) therefore studies of this type are very important. The species composition of pathogenic fungi can be an indicator of seed yield. The occurrence of oilseed rape pathogens during several years were analyzed using the sequencing method DNA ITS. The results were compared in the gene bank using the program NCBI / BLAST. In field conditions before harvest of oilseed rape presence of pathogens infesting B. napus has been assessed. For example, in 2015, 150 samples have been isolated and applied to PDA medium for the identification of belonging species. From all population has been selected mycelium of 83 isolates which were sequenced. Others (67 isolates) were pathogenic fungi of the genus Alternaria which are easily to recognize. The population of pathogenic species on oilseed rape have been identified after analyzing the DNA ITS and include: Leptosphaeria sp. 38 (L. maculans 25, L. biglobosa 13), Alternaria sp. 29, Fusarium sp. 3, Sclerotinia sclerotiorum 7, heterogeneous 6, total of 83 isolates. The genus Alternaria sp. fungi wear the largest share of B. napus pathogens in particular years. Another dangerous species for oilseed rape was Leptosphaeria sp. Populations of pathogens in each year were different. The number of pathogens occurring in the field and their composition is very important for breeders and farmers because of the possible selection of the most resistant genotypes for sowing in the next growing season.

Keywords: B. napus, DNA ITS Sequencing, pathogenic fungi, population

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Authors: S. Ben-Shabat, T. Turgeman, O. Leubinski, N. Roth-Bejerano, V. Kagan-Zur, Y. Sitrit

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The ectomycorrhizal (ECM) desert truffle Terfezia boudieri produces edible fruit bodies and forms symbiosis with its host plant Helianthemum sessiliflorum (Cistaceae) in the Negev desert of Israel. The symbiosis is vital for both partners' survival under desert conditions. Under desert habitat conditions, ECMs must form symbiosis before entering the dry season. To secure a successful encounter, in the course of evolution, both partners have responded by evolving special signals exchange that facilitates recognition. Members of the Cistaceae family serve as host plants for many important truffles. Conceivably, during evolution a common molecule present in Cistaceae plants was recruited to facilitate successful encounter with ectomycorrhizas. Arbuscular vesicular fungi (AM) are promiscuous in host preferences, in contrast, ECM fungi show specificity to host plants. Accordingly, we hypothesize that H. sessiliflorum secretes a chemotrophic-signaling, which is common to plants hosting ECM fungi belonging to the Pezizales. However, thus far no signaling molecules have been identified in ECM fungi. We developed a bioassay for chemotrophic activity. Fractionation of root exudates revealed a substance with chemotrophic activity and molecular mass of 534. Following the above concept, screening the transcriptome of Terfezia, grown under chemoattraction, discovered genes showing high homology to G proteins-coupled receptors of plant pathogens involved in positive chemotaxis and chemotaxis suppression. This study aimed to identify the active molecule using analytical methods (LC-MS, NMR etc.). This should contribute to our understanding of how ECM fungi communicate with their hosts in the rhizosphere. In line with the ability of Terfezia to form also endomycorrhizal symbiosis like AM fungi, analysis of the mechanisms may likewise be applicable to AM fungi. Developing methods to manipulate fungal growth by the chemoattractant can open new ways to improve inoculation of plants.

Keywords: chemotrophic signal, Helianthemum sessiliflorum, Terfezia boudieri, ECM

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Authors: Mustafa Ümi̇t Ünal, Nafi̇z Çeli̇ktaş, Aysun Şener, Sara Betül Dolgun, Duygu Keser

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The aim of this study was to compare acid and base pretreatment of switchgrass for bioethanol production. Switchgrass was pretreated with sulfuric acid and sodium hydroxide at 0.5, 1.0 and 1.5% (v/v) at 120, 140, 180 °C for 10, 60 and 90. Optimization of enzymatic hydrolysis of the pretreated switchgrass samples were carried out using three different enzyme mixtures (22.5 mg cellulase and 75 mg cellobiase /g biomass; 45 mg cellulase and 150 mg cellobiase /g biomass; 90 mg cellulase and 300 mg cellobiase /g biomass). Samples were removed at 24-h interval for fermentable sugar analyses with HPLC. The results showed that use of 90 mg cellulase and 300 mg cellobiase/g biomass resulted in the highest fermentable sugar formation. Furthermore, the highest fermentable sugar yield was obtained by pretreatment at 120 °C for 10 min using 1.0 % sodium hydroxide.

Keywords: switchgrass, acid pretreatment, enzymatic hydrolysis, base pretreatment, ethanol production

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Authors: Pratiksha Dubey, Praveen Singh, Shantanu Sen Gupta, Anand K. Bachhawat

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5-oxoproline (pyroglutamic acid) is a cyclic lactam of glutamic acid. In the cell, it can be produced by several different pathways and is metabolized into glutamate with the help of the 5-oxoprolinase enzyme (OPLAH or OXP1). The inhibition of 5-oxoprolinase enzyme in mammals was found to result in heart failure and is thought to be a consequence of oxidative stress [1]. To analyze the consequences of 5-oxoproline accumulation more clearly, we are generating models for 5-oxoproline accumulation in yeast. The 5-oxoproline accumulation model in yeast is being developed by two different strategies. The first one is by overexpression of the mouse  -glutamylcyclotransferase enzyme. It degrades -glu-met dipeptide into 5-oxoproline and methionine taken by the cell from the medium. The second strategy is by providing high concentration of 5-oxoproline externally to the yeast cells. The intracellular 5-oxoproline levels in both models are being evaluated. In addition, the metabolic and cellular consequences are being investigated.

Keywords: 5-oxoproline, pyroglutamic acid, yeast, genetics

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Authors: Igor V. Grigoriev

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Understanding of biological processes encoded in fungi is instrumental in addressing future food, feed, and energy demands of the growing human population. Genomics is a powerful and quickly evolving tool to understand these processes. The Fungal Genomics Program of the US Department of Energy Joint Genome Institute (JGI) partners with researchers around the world to explore fungi in several large scale genomics projects, changing the fungal genomics landscape. The key trends of these changes include: (i) rapidly increasing scale of sequencing and analysis, (ii) developing approaches to go beyond culturable fungi and explore fungal ‘dark matter,’ or unculturables, and (iii) functional genomics and multi-omics data integration. Power of comparative genomics has been recently demonstrated in several JGI projects targeting mycorrhizae, plant pathogens, wood decay fungi, and sugar fermenting yeasts. The largest JGI project ‘1000 Fungal Genomes’ aims at exploring the diversity across the Fungal Tree of Life in order to better understand fungal evolution and to build a catalogue of genes, enzymes, and pathways for biotechnological applications. At this point, at least 65% of over 700 known families have one or more reference genomes sequenced, enabling metagenomics studies of microbial communities and their interactions with plants. For many of the remaining families no representative species are available from culture collections. To sequence genomes of unculturable fungi two approaches have been developed: (a) sequencing DNA from fruiting bodies of ‘macro’ and (b) single cell genomics using fungal spores. The latter has been tested using zoospores from the early diverging fungi and resulted in several near-complete genomes from underexplored branches of the Fungal Tree, including the first genomes of Zoopagomycotina. Genome sequence serves as a reference for transcriptomics studies, the first step towards functional genomics. In the JGI fungal mini-ENCODE project transcriptomes of the model fungus Neurospora crassa grown on a spectrum of carbon sources have been collected to build regulatory gene networks. Epigenomics is another tool to understand gene regulation and recently introduced single molecule sequencing platforms not only provide better genome assemblies but can also detect DNA modifications. For example, 6mC methylome was surveyed across many diverse fungi and the highest among Eukaryota levels of 6mC methylation has been reported. Finally, data production at such scale requires data integration to enable efficient data analysis. Over 700 fungal genomes and other -omes have been integrated in JGI MycoCosm portal and equipped with comparative genomics tools to enable researchers addressing a broad spectrum of biological questions and applications for bioenergy and biotechnology.

Keywords: fungal genomics, single cell genomics, DNA methylation, comparative genomics

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Authors: Prasanthi Malapati, R. Reshma, Vijay Soni, Perumal Yogeeswari, Dharmarajan Sriram

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In the present study, we attempted to develop Mycobacterium tuberculosis (Mtb) inhibitors by exploring the pharmaceutically underexploited enzyme targets which are majorly involved in cell wall biosynthesis of mycobacteria. For this purpose, glutamate racemase (coded by MurI gene) was selected. This enzyme racemize L-glutamate to D-glutamate required for the construction of peptidoglycan in the bacterial cell wall synthesis process. Furthermore this enzyme is neither expressed nor its product, D-glutamate is normally found in mammals, and hence designing inhibitors against this enzyme will not affect the host system as well act as potential antitubercular drugs. A library of BITS in house compounds were screened against Mtb MurI enzyme. Based on docking score, interactions and synthetic feasibility one hit lead was identified. Further optimization of lead was attempted and its derivatives were synthesized. Forty eight derivatives of 2-phenylbenzo[d]oxazole and 2-phenylbenzo[d]thiazole were synthesized and evaluated for Mtb MurI inhibition study, in vitro activities against Mtb, cytotoxicity against RAW 264.7 cell line. Chemical derivatization of the lead resulted in compounds NR-1213 AND NR-1124 as the potent M. tuberculosis glutamate racemase inhibitors with IC50 of 4-5µM which are remarkable and were found to be non-cytotoxic. Molecular dynamics, dormant models and cardiotoxicity studies of the most active molecules are in process.

Keywords: cell wall biosynthesis, dormancy, glutamate racemase, tuberculosis

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Authors: Kumaran Narayanan, Pei-Sheng Liew

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This paper adapts the bacteriophage N15 protelomerase enzyme to assemble linear chromosomes as vectors for gene expression in human cells. Phage N15 has the unique ability to replicate as a linear plasmid with telomeres in E. coli during its prophage stage of life-cycle. The virus-encoded protelomerase enzyme cuts its circular genome and caps its ends to form hairpin telomeres, resulting in a linear human-chromosome-like structure in E. coli. In mammalian cells, however, no enzyme with TelN-like activities has been found. In this work, we show for the first-time transfer of the protelomerase from phage into human and mouse cells and demonstrate recapitulation of its activity in these hosts. The function of this enzyme is assayed by demonstrating cleavage of its target DNA, followed by detecting telomere formation based on its resistance to recBCD enzyme digestion. We show protelomerase expression persists for at least 60 days, which indicates limited silencing of its expression. Next, we show that an intact human β-globin gene delivered on this linear chromosome accurately retains its expression in the human cellular environment for at least 60 hours, demonstrating its stability and potential as a vector. These results demonstrate that the N15 protelomerse is able to function in mammalian cells to cut and heal DNA to create telomeres, which provides a new tool for creating novel structures by DNA resolution in these hosts.

Keywords: chromosome, beta-globin, DNA, gene expression, linear vector

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Authors: Alla V. Goncharova, Tatyana A. Karpenyuk, Yana S. Tsurkan, Rosa U. Beisembaeva, Togzhan D. Mukasheva, Ludmila V. Ignatova, Ramza Z. Berzhanova

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In this work the effect of culturing conditions of filamentous fungi Penicillium raistrickii, Penicillium anatolicum, Fusarium sp. on biomass yield, the content of total lipids and fatty acids was studied. It has been established that in time the process of lipids accumulation correlated with biomass growth of cultures, reaching maximum values in stationary growth phase. Biomass yield and accumulation of general lipids was increased by adding zinc to the culture medium. The more intensive accumulation of biomass and general lipids was observed at temperature 18°C. Lowering the temperature of culturing has changed the ratio of saturated: Unsaturated fatty acids in the direction of increasing the latter.

Keywords: biomass, culturing conditions, fungi, fatty acids (FA), growth dynamics, lipids

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Authors: Zainal A. Muchlisin, Muhammad Iqbal, Abdullah A. Muhammadar

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The objective of the present study was to determine the optimum dose of papain enzyme in the diet for growing, survival rate and feed efficacy of climbing perch (Anabas testudineus). The study was conducted at the Laboratory of Aquatic of Faculty of Veterinary, Syiah Kuala University from January to March 2016. The completely randomized design was used in this study. Six dosages level of papain enzyme were tested with 4 replications i.e. 0 g kg-1 of feed, 20.0 g kg-1 feed, 22.5 g kg-1 of feed, 25.0 g kg-1 of feed, 27.5 g kg-1 of feed, and 30.0 g kg-1 of feed. The experimental fish fed twice a day at feeding level of 5% for 60 days. The results showed that weight gain ranged from 2.41g to 7.37g, total length gain ranged from 0.67cm to 3.17cm, specific growth rate ranged from 1.46 % day to 3.41% day, daily growth rate ranged from 0.04 g day to 0.13 g day, feed conversion ratio ranged from 1.94 to 3.59, feed efficiency ranged from 27.99% to 51.37%, protein retention ranged from 3.38% to 28.28%, protein digestibility ranged from 50.63% to 90.38%, and survival rate ranged from 88.89% to 100%. The highest rate for all parameters was found in the dosage of 3.00% papain enzyme kg feed. The ANOVA test showed that enzyme papain gave a significant effect on the weight gain, total length gain, daily growth rate, specific growth rate, feed conversion ratio, feed efficiency, protein retention, protein digestibility, and survival rate of the climbing perch (Anabas testudieus). The best enzyme papain dosage was 3.0%.

Keywords: betok, feed conversion ratio, freshwater fish, nutrition, feeding

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Authors: Sauradipta Ganguly, Akhato Sumi, Sanjeet Kumar Hom, Ajan T. Lotha

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Populus deltoides is a hardwood used predominantly for the manufacturing of plywood, matchsticks, and paper in India and hence has a higher economical significance. Wood-decaying fungi cause serious damage to Populus deltoides products, as the wood itself is perishable and vulnerable to decaying agents, decreasing their aesthetical value which in return results in significant monetary loss for the wood industries concerned. The aim of the study was to determine the antifungal activity of Cedrus deodara oil against three primary wood-decaying fungi namely white-rot fungi (Trametes versicolor), brown-rot fungi (Oligoporus placentus) and soft-rot fungi (Xylaria acuminata) on Populus deltoides samples under optimum laboratory conditions. The susceptibility of Populus deltoides samples on the fungal attack and the ability of deodar oil to control colonization of the wood rotting fungi on the samples were assessed. Three concentrations of deodar oil were considered for the study as treating solutions, i.e., 4%, 5%, and 6%. The Populus deltoides samples were treated with treating solutions, and the ability of the same to prevent a fungal attack on the samples were assessed using accelerated test in the laboratory at Biochemical Oxygen Demand incubator at temperature (25 ± 2°C) and relative humidity 70 ± 4%. Efficacy test and statistical analysis of deodar oil against Trametes versicolor, Oligoporus placentus, and Xylariaacuminataon P. deltoides samples exhibited light, minor and negligible mycelia growth at 4 %, 5% and 6% concentrations of deodar oil, respectively. Whereas, moderate to heavy attack was observed on the surface of the control samples. Statistical analysis further established that the treatments were statistically significant and had significantly inhibited fungal growth of all the three fungus spp by almost 3 to 5 times.

Keywords: populus deltoides, Trametes versicolor, Oligoporus placentus, Xylaria acuminata, Deodar oil, treatment

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Authors: Nivedita Sharma

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The global demand for fossil fuels is very high, but their use is not sustainable since its reserves are declining. Additionally, fossil fuels are responsible for the accumulation of greenhouse gases. The emission of greenhouse gases from the transport sector can be reduced by substituting fossil fuels by biofuels. Thus, renewable fuels capable of sequestering carbon dioxide are in high demand. Second‐generation biofuels, which require lignocellulosic biomass as a substrate and ultimately producing ethanol, fall largely in this category. Bioethanol is a favorable and near carbon-neutral renewable biofuel leading to reduction in tailpipe pollutant emission and improving the ambient air quality. Lignocellulose consists of three main components: cellulose, hemicellulose and lignin which can be converted to ethanol with the help of microbial enzymes. Enzymatic hydrolysis of lignocellulosic biomass in 1st step is considered as the most efficient and least polluting methods for generating fermentable hexose and pentose sugars which subsequently are fermented to power alcohol by yeasts in 2nd step of the process. In the present technology, a complete bioconversion process i.e. potential hydrolytic enzymes i.e. cellulase and xylanase producing microorganisms have been isolated from different niches, screened for enzyme production, identified using phenotyping and genotyping, enzyme production, purification and application of enzymes for saccharification of different lignocellulosic biomass followed by fermentation of hydrolysate to ethanol with high yield is to be presented in detail.

Keywords: cellulase, xylanase, lignocellulose, bioethanol, microbial enzymes

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Authors: P. Chaijak, M. Lertworapreecha, C. Sukkasem

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Extracellular laccases are copper-containing microbial enzymes with many industrial biotechnological applications. This study evaluated the ability of nutrients in coconut coir to enhance the yield of extracellular laccase of Galactomyces reesii IFO 10823 and develop a co-culture between this yeast and other filamentous fungi isolated from the fungus comb of Macrotermes sp. The co-culture between G. reesii IFO 10823 and M. indicus FJ-M-5 (G3) gave the highest activity at 580.20 U/mL. When grown in fermentation media prepared from coconut coir and distilled water at 70% of initial moisture without supplement addition, G3 produced extracellular laccase of 113.99 U/mL.

Keywords: extracellular laccase, production, yeast, natural inducer

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Authors: H. M. Takematsu, B. R. De Camargo, E. F. Noronha

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The use of hydrolyzing enzymes for degradation of lignocellulosic biomass is of great concern for the production of second generation ethanol. Although many hollocelulases have already been described in the literature, much more has to be discovered. Therefore, the aim of this study to evaluate hollocelulase production of P. polonicum grown in liquid media containing sugarcane bagasse as the carbon source. From a collection of twenty fungi isolated from Cerrado biome soil, P. polonicum was molecular identified by sequencing of ITS4, βtubulin and Calmodulin genes, and has been chosen to be further investigated regarding its potential production of hydrolyzing enzymes. Spore suspension (1x10-6 ml-1) solution was inoculated in sterilized minimal liquid medium containing 0,5%(w/v) of non-pretreated sugarcane bagasse as the carbon source, and incubated in shaker incubator at 28°C and 120 rpm. The supernatant obtained, was subjected to enzymatic assays to analyze xylanase, mannanase, pectinase and endoglucanase activities. Xylanase activity showed better results (67,36 UI/mg). Xylanases bands were indicated by zymogram and SDS-PAGE, and one of them was partially purified and characterized. It showed maximum activity at 50 °C, was thermostable for 72h at 40°C, and pH5.0 was the optimum observed. This study presents P. polonicum as an interesting source of hollocelulases, especially xylanase, for lignocellulose bio-conversion processes with commercial use.

Keywords: sugarcane bagasse, Cerrado biome , hollocelulase, lignocellulosic biomass

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Authors: Sukanya Devi Ramachandran, Vaishnavi Muralidharan, Kavya Chandrasekaran

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Polycaprolactone is polymer belonging to the polyester family that has noticeable characteristics of biodegradability and biocompatibility which is essential for medical applications. Polycaprolactone is produced by the ring opening polymerization of the monomer epsilon-Caprolactone (ε-CL) which is a closed ester, comprising of seven-membered ring. This process is normally catalysed by metallic components such as stannous octoate. It is difficult to remove the catalysts after the reaction, and they are also toxic to the human body. An alternate route of using enzymes as catalysts is being employed to reduce the toxicity. Lipase enzyme is a subclass of esterase that can easily attack the ester bonds of ε-CL. This research paper throws light on the extraction of lipase from germinating sunflower seeds and the activity of the biocatalyst in the polymerization of ε-CL. Germinating Sunflower seeds were crushed with fine sand in phosphate buffer of pH 6.5 into a fine paste which was centrifuged at 5000rpm for 10 minutes. The clear solution of the enzyme was tested for activity at various pH ranging from 5 to 7 and temperature ranging from 40oC to 70oC. The enzyme was active at pH6.0 and at 600C temperature. Polymerization of ε-CL was done using toluene as solvent with the catalysis of lipase enzyme, after which chloroform was added to terminate the reaction and was washed in cold methanol to obtain the polymer. The polymerization was done by varying the time from 72 hours to 6 days and tested for the molecular weight and the conversion of the monomer. The molecular weight obtained at 6 days is comparably higher. This method will be very effective, economical and eco-friendly to produce as the enzyme used can be regenerated as such at the end of the reaction and can be reused. The obtained polymers can be used for drug delivery and other medical applications.

Keywords: lipase, monomer, polycaprolactone, polymerization

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Authors: Lorena C. Cintra, Izadora M. De Oliveira, Amanda G. Fernandes, Francieli Colussi, Rosália S. A. Jesuíno, Fabrícia P. Faria, Cirano J. Ulhoa

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Xylan is the main component of hemicellulose and for its complete degradation is required cooperative action of a system consisting of several enzymes including endo-xylanases (XYN), β-xylosidases (XYL) and α-L-arabinofuranosidases (ABF). The recombinant hemicellulolytic enzymes an endoxylanase (HXYN2), β-xylosidase (HXYLA), and an α-L-arabinofuranosidase (ABF3) were used in hydrolysis tests. These three enzymes are produced by filamentous fungi and were expressed heterologously and produced in Pichia pastoris previously. The aim of this work was to evaluate the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of sugarcane bagasse (SCB). The interaction between the three recombinant enzymes during SCB pre-treated by steam explosion hydrolysis was performed with different concentrations of HXYN2, HXYLA and ABF3 in different ratios in according to a central composite rotational design (CCRD) 23, including six axial points and six central points, totaling 20 assays. The influence of the factors was assessed by analyzing the main effects and interaction between the factors, calculated using Statistica 8.0 software (StatSoft Inc. Tulsa, OK, USA). The Pareto chart was constructed with this software and showed the values of the Student’s t test for each recombinant enzyme. It was considered as response variable the quantification of reducing sugars by DNS (mg/mL). The Pareto chart showed that the recombinant enzyme ABF3 exerted more significant effect during SCB hydrolysis, with higher concentrations and with the lowest concentration of this enzyme. It was performed analysis of variance according to Fisher method (ANOVA). In ANOVA for the release of reducing sugars (mg/ml) as the variable response, the concentration of ABF3 showed significance during hydrolysis SCB. The result obtained by ANOVA, is in accordance with those presented in the analysis method based on the statistical Student's t (Pareto chart). The degradation of the central chain of xylan by HXYN2 and HXYLA was more strongly influenced by ABF3 action. A model was obtained, and it describes the performance of the interaction of all three enzymes for the release of reducing sugars, and can be used to better explain the results of the statistical analysis. The formulation capable of releasing the higher levels of reducing sugars had the following concentrations: HXYN2 with 600 U/g of substrate, HXYLA with 11.5 U.g-1 and ABF3 with 0.32 U.g-1. In conclusion, the recombinant enzyme that has a more significant effect during SCB hydrolysis was ABF3. It is noteworthy that the xylan present in the SCB is arabinoglucoronoxylan, due to this fact debranching enzymes are important to allow access of enzymes that act on the central chain.

Keywords: experimental design, hydrolysis, recombinant enzymes, sugar cane bagasse

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Authors: Flackson Tshuma, James Bennett, Pieter Andreas Swanepoel, Johan Labuschagne, Stephan van der Westhuizen, Francis Rayns

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Amongst other things, tillage and synthetic agrochemicals can be effective methods of seedbed preparation and pest control. Nonetheless, frequent and intensive tillage and excessive application of synthetic agrochemicals, such as herbicides and insecticides, can reduce soil microbial enzyme activity. A decline in soil microbial enzyme activity can negatively affect nutrient cycling and crop productivity. In this study, the effects of four tillage treatments; continuous mouldboard plough; shallow tine-tillage to a depth of about 75 mm; no-tillage; and tillage rotation (involving shallow tine-tillage once every four years in rotation with three years of no-tillage), and two rates of synthetic agrochemicals (standard: with regular application of synthetic agrochemicals; and reduced: fewer synthetic agrochemicals in combination with bio-chemicals/ or bio-stimulants) on soil microbial enzyme activity were investigated between 2018 and 2020 in a typical Mediterranean climate zone in South Africa. Four different bio-stimulants applied contained: Trichoderma asperellum, fulvic acid, silicic acid, and Nereocystis luetkeana extracts, respectively. The study was laid out as a complete randomised block design with four replicated blocks. Each block had 14 plots, and each plot measured 50 m x 6 m. The study aimed to assess the combined impact of tillage practices and reduced rates of synthetic agrochemical application on soil microbial enzyme activity in a dryland cropping system. It was hypothesised that the application of bio-stimulants in combination with minimum soil disturbance will lead to a greater increase in microbial enzyme activity than the effect of applying either in isolation. Six soil cores were randomly and aseptically collected from each plot for microbial enzyme activity analysis from the 0-150 mm layer of a field trial under a dryland crop rotation system in the Swartland region. The activities of four microbial enzymes, β-glucosidase, acid phosphatase, alkaline phosphatase and urease, were assessed. The enzymes are essential for the cycling of glucose, phosphorus, and nitrogen, respectively. Microbial enzyme activity generally increased with a reduction of both tillage intensity and synthetic agrochemical application. The use of the mouldboard plough led to the least (P<0.05) microbial enzyme activity relative to the reduced tillage treatments, whereas the system with bio-stimulants (reduced synthetic agrochemicals) led to the highest (P<0.05) microbial enzyme activity relative to the standard systems. The application of bio-stimulants in combination with reduced tillage, particularly no-tillage, could be beneficial for enzyme activity in a dryland farming system.

Keywords: bio-stimulants, soil microbial enzymes, synthetic agrochemicals, tillage

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Authors: M. B. Atui, A. M. Silva, M. A. M. Marciano, M. I. Fioravanti, V. A. Franco, L. B. Chasin, A. R. Ferreira, M. D. Nogueira

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Fungi attack large amount of fruits and those who have suffered an injury on the surface are more susceptible to the growth, as they have pectinolytic enzymes that destroy the edible portion forming an amorphous and soft dough. The spores can reach the plant by the wind, rain and insects and fruit may have on its surface, besides the contaminants from the fruit trees, land and water, forming a flora composed mainly of yeasts and molds. Other contamination can occur for the equipment used to harvest, for the use of boxes and contaminated water to the fruit washing, for storage in dirty places. The hyphae in tomato products indicate the use of raw materials contaminated or unsuitable hygiene conditions during processing. Although fungi are inactivated in heat processing step, its hyphae remain in the final product and search for detection and quantification is an indicator of the quality of raw material. Howard Method count of fungi mycelia in industrialized pulps evaluates the amount of decayed fruits existing in raw material. The Brazilian legislation governing processed and packaged products set the limit of 40% of positive fields in tomato pulps. The aim of this study was to evaluate the quality of the tomato pulp sold in greater São Paulo, through a monitoring during the four seasons of the year. All over 2010, 110 samples have been examined; 21 were taking in spring, 31 in summer, 31 in fall and 27 in winter, all from different lots and trademarks. Samples have been picked up in several stores located in the city of São Paulo. Howard method was used, recommended by the AOAC, 19th ed, 2011 16:19:02 technique - method 965.41. Hundred percent of the samples contained fungi mycelia. The count average of fungi mycelia per season was 23%, 28%, 8,2% and 9,9% in spring, summer, fall and winter, respectively. Regarding the spring samples of the 21 samples analyzed, 14.3% were off-limits proposed by the legislation. As for the samples of the fall and winter, all were in accordance with the legislation and the average of mycelial filament count has not exceeded 20%, which can be explained by the low temperatures during this time of the year. The acquired samples in the summer and spring showed high percentage of fungal mycelium in the final product, related to the high temperatures in these seasons. Considering that the limit of 40% of positive fields is accepted for the Brazilian Legislation (RDC nº 14/2014), 3 spring samples (14%) and 6 summer samples (19%) will be over this limit and subject to law penalties. According to gathered data, 82% of manufacturers of this product manage to keep acceptable levels of fungi mycelia in their product. In conclusion, only 9.2% samples were for the limits established by Resolution RDC. 14/2014, showing that the limit of 40% is feasible and can be used by these segment industries. The result of the filament count mycelial by Howard method is an important tool in the microscopic analysis since it measures the quality of raw material used in the production of tomato products.

Keywords: fungi, howard, method, tomato, pulps

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Authors: Dake Xiong, Ben Hankamer, Ian Ross

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The most urgent challenge of our time is to replace the depleting resources of fossil fuels by sustainable environmentally friendly alternatives. Hydrogen is a promising CO2-neutral fuel for a more sustainable future especially when produced photo-biologically. Hydrogen can be photosynthetically produced in unicellular green alga like Chlamydomonas reinhardtii, catalysed by the inducible highly active and bidirectional [FeFe]-hydrogenase enzymes (HydA). However, evolutionary and physiological constraints severely restrict the hydrogen yield of algae for industrial scale-up, mainly due to its competition among other metabolic pathways on photosynthetic electrons. Among them, a major challenge to be resolved is the inferior competitiveness of hydrogen production (catalysed by HydA) with NADPH production (catalysed by ferredoxin-NADP+-reductase (FNR)), which is essential for cell growth and takes up ~95% of photosynthetic electrons. In this work, the in vivo hydrogen production efficiency of mutants with ferredoxin-hydrogenase (Fd*-HydA1*) fusion protein construct, where the electron donor ferredoxin (Fd*) is fused to HydA1* and expressed in the model organism C. reinhardtii was investigated. Once Fd*-HydA1* fusion gene is expressed in algal cells, the fusion enzyme is able to draw the redistributed photosynthetic electrons and use them for efficient hydrogen production. From preliminary data, mutants with Fd*-HydA1* transgene showed a ~2-fold increase in the photosynthetic hydrogen production rate compared with its parental strain, which only possesses the native HydA in vivo. Therefore, a solid method of having more efficient hydrogen production in microalgae can be achieved through the expression of the synthetic enzymes.

Keywords: Chlamydomonas reinhardtii, ferredoxin, fusion protein, hydrogen production, hydrogenase

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Authors: Aleksandra Łochowicz, Daria Świętochowska, Loredano Pollegioni, Nazim Ocal, Franck Charmantray, Laurence Hecquet, Katarzyna Szymańska

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Multienzymatic cascade reactions are nowadays widely used in pharmaceutical, chemical and cosmetics industries to produce high valuable compounds. They can be carried out in two ways, step by step and one-pot. If two or more enzymes are in the same reaction vessel is necessary to work out the compromise to run the reaction in optimal conditions for each enzyme. So far most of the reports of multienzymatic cascades concern on usage of free enzymes. Unfortunately using free enzymes as catalysts of reactions accomplish high cost. What is more, free enzymes are soluble in solvents which makes reuse impossible. To overcome this obstacle enzymes can be immobilized what provides heterogeneity of biocatalyst that enables reuse and easy separation of the enzyme from solvents and reaction products. Usually, immobilization increase also the thermal and operational stability of enzyme. The advantages of using immobilized multienzymes are enhanced enzyme stability, improved cascade enzymatic activity via substrate channeling, and ease of recovery for reuse. The one-pot immobilized multienzymatic cascade can be carried out in mixed or coimmobilized type. When biocatalysts are coimmobilized on the same carrier the are in close contact to each other which increase the reaction rate and catalytic efficiency, and eliminate the lag time. However, in this type providing the optimal conditions both in the process of immobilization and cascade reaction for each enzyme is complicated. Herein, we examined immobilization of 3 enzymes: D-amino acid oxidase from Rhodotorula gracilis, commercially available catalase and transketolase from Geobacillus stearothermophilus. As a support we used silica monoliths with hierarchical structure of pores. Then we checked their stability and reusability in one-pot cascade of L-erythrulose and hydroxypuryvate acid synthesis.

Keywords: biocatalysts, enzyme immobilization, multienzymatic reaction, silica carriers

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Authors: Leonce Dusengemungu

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Filamentous fungi indigenous to heavy metals (HMs) contaminated environments have a considerable biosorption potential yet are currently under-investigated in developing countries. In the work presented herein, the biosorption potential of three indigenous filamentous fungi (Aspergillus transmontanensis, Cladosporium cladosporioides, and Geotrichum candidum) isolated from copper and cobalt mining wasteland sites in Zambia's Copperbelt province was investigated. In Cu and Co tolerance tests, all the fungal isolates were shown to be tolerant, with mycelial growth at HMs concentrations of up to 7000 ppm. However, exposure to high Cu and Co concentrations hindered the growth of the three strains to varying degrees, resulting in reduced mycelial biomass (evidenced by loss of the infrared bands at 887 and 930 cm-1 of the 1,3-glucans backbone) as well as morphological alterations, sporulation, and pigment synthesis. In addition, gas chromatography-mass spectrometry characterization of the fungal biomass extracts allowed to detect changes in the chemical constituents upon exposure to HMs, with profiles poorer in maltol, 1,2-cyclopentadione, and n-hexadecanoic acid, and richer in furaldehydes. Biosorption tests showed that A. transmontanensis and G. candidum showed better performance as bioremediators than C. cladosporioides, with biosorption efficiencies of 1645, 1853 and 1253 ppm at pH 3, respectively, and may deserve further research in field conditions.

Keywords: bioremediation, fungi, biosorption, heavy metal

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