Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases

Bacillus licheniformis 9945a α-amylase (BliAmy) has been described as potent enzyme for raw starch hydrolysis. Starch represents an inexpensive source for production of glucose, maltose syrups and fructose which are widely used in food industries. Regarding energy costs, effective utilization of natural resources and viscosity problems, direct hydrolysis of raw starch below the gelatinization temperature by using raw-starch-digesting enzymes, such as α-amylase is desirable. In spite of the extensive studies concerning the structure and thermal properties of B. licheniformis amylase and the numerous reports in the literature referring to the molecular mechanism of irreversible thermoinactivation, little attention has been paid to its enzymological characterisation. Detailed knowledge about subsite architecture of B. licheniformis amylase is scarce. No report on kinetics and mode of action of this industrially important enzyme can be found in the literature especially when raw starch is used as a substrate. For mechanistic studies enzyme preparations of high purity are required and improving downstream processing is very beneficial. BliAmy was produced using optimized fed-batch approach in defined media and significant overexpression of 1.2 g L-1 was achieved. These amylases have exposed tyrosine and tryptophan residues as part of their surface binding sites. Mixed mode Nuvia cPrime™ resin is tested as improvement of the downstream processing of raw starch digesting amylases aiming at exploiting hydrophobic patches at their surface. This resin combines hydrophobic interactions with cation exchange groups. Presence of salt facilitates hydrophobic interactions while ionexchange groups enable proper selectivity. Surface response methodology was used to optimize binding and eluting conditions of BliAmy. This single step procedure enables simultaneous concentration, pigments removal and purification of amylase with a yield of 96% directly from fermentation broth

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases

Bacillus licheniformis 9945a α-amylase (BliAmy) has been described as potent enzyme for raw starch hydrolysis. Starch represents an inexpensive source for production of glucose, maltose syrups and fructose which are widely used in food industries. Regarding energy costs, effective utilization of natural resources and viscosity problems, direct hydrolysis of raw starch below the gelatinization temperature by using raw-starch-digesting enzymes, such as α-amylase is desirable. In spite of the extensive studies concerning the structure and thermal properties of B. licheniformis amylase and the numerous reports in the literature referring to the molecular mechanism of irreversible thermoinactivation, little attention has been paid to its enzymological characterisation. Detailed knowledge about subsite architecture of B. licheniformis amylase is scarce. No report on kinetics and mode of action of this industrially important enzyme can be found in the literature especially when raw starch is used as a substrate. For mechanistic studies enzyme preparations of high purity are required and improving downstream processing is very beneficial. BliAmy was produced using optimized fed-batch approach in defined media and significant overexpression of 1.2 g L-1 was achieved. These amylases have exposed tyrosine and tryptophan residues as part of their surface binding sites. Mixed mode Nuvia cPrime™ resin is tested as improvement of the downstream processing of raw starch digesting amylases aiming at exploiting hydrophobic patches at their surface. This resin combines hydrophobic interactions with cation exchange groups. Presence of salt facilitates hydrophobic interactions while ionexchange groups enable proper selectivity. Surface response methodology was used to optimize binding and eluting conditions of BliAmy. This single step procedure enables simultaneous concentration, pigments removal and purification of amylase with a yield of 96% directly from fermentation broth

Efficiency of ultrasonic Vertex III hypsometer compared to the most commonly used hypsometers in Croatian forestry

The article investigates the efficiency of the ultrasonic Vertex hypsometer in tree height measurements in relation to some of the most commonly used hypsometers in Croatian forestry. The whole measurement process consists of three steps: time needed to reach the measurement zone, time needed to determine the distance to a tree, and time needed to measure and read the height of a tree. Measurements were conducted with four hypsometers: Vertex, Blume-Leiss, Bitterlich\u27s Spiegel Relaskop with a standard scale (RO) and Bitterlich\u27s Spiegel Relaskop with a CP scale (with the horizontally /RCPH/ or vertically /RCPV/ positioned staff). Research results do not show any statistically important differences among the hypsometers in terms of time needed to reach the measurement zone. In measuring tree heights in a stricter sense (without the element of reaching the measurement zone), the least amount of time was required by the Vertex (28.4 cmin) on average. The Vertex is followed by the relaskop with a CP scale, the horizontally (57.4 cmin) or vertically positioned staff (86.1 cmin), then the Blume-Leiss (84.0 cmin) and finally the relaskop with a standard scale (106.8 cmin). The differences between the Vertex and other hypsometers are statistically significant. Despite a slightly higher price of the Vertex, its speed, precision, accuracy and simplicity of use, as well as the possibility of simple circular plot forest inventories rank it above all the other instruments

Supplementary data for article: Lončar, N.; Slavić, M. Š.; Vujčić, Z.; Božić, N. Mixed-Mode Resins: Taking Shortcut in Downstream Processing of Raw-Starch Digesting α-Amylases. Scientific Reports 2015, 5. https://doi.org/10.1038/srep15772

Supplementary material for: [https://doi.org/10.1038/srep15772]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1985

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting alpha-amylases

Bacillus licheniformis 9945a alpha-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime (TM) resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. alpha-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L-1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3384

Osetljivost Chenopodium album i Abutilon theophrasti na mezotrion u zavisnosti od fenofaze razvoja

Growth stage of weeds can significantly affect the level of control at the moment of herbicide application. Maturation of plants usually lead to decreased sensitivity of plants. The aim of this research was to determine whether growth stage of weed species can affect the sensitivity of Chenopodium album and Abutilon theophrasti to mesotrione at the moment of application. Seeds of these species were sown in 5 days interval to ensure that at the moment of herbicide application Ch. album would be in stages of 2, 4, 6, 8 and 10 leaves and A. theophrasti in stages of 1, 2, 3, 4 and 5 leaves. When plants reached this stages herbicide Skaut (mesotrione 480 g/l) was applied in recommended dose rate 0.25 l/ha in tank-mix with adjuvant Alteox wet 40 (mineral oil (500 g/l) and polyoxyethylene alcohol (500 g/l)) in the dose rate 1 l/ha. The results showed that growth stage at the moment of application of mesotrione can significantly affects the sensitivity of both species to herbicide. The application of herbicides in the early growth stages (A. theophrasti - 1 leaf, Ch. Album - 2 and 4 leaves) resulted in total control of plants. With further maturation, sensitivity has decreased, in terms that the lowest effect was noted with A. theophrasti in growth stage of 5 leaves and Ch. album in growth stage of 10 leaves.Fenofaza razvoja korovskih biljaka u momentu primene herbicida može značajno uticati na njihovu osetljivost. Sa starenjem biljke najčešće dolazi do smanjenja osetljivosti biljaka. Obavljena su istraživanja kako bi se utvrdilo da li fenofaza razvoja biljaka u momentu primene mezotriona utiče na osetljivost Chenopodium album i Abutilon theophrasti. Setva semena ovih vrsta je obavljena u razmacima od po 5 dana kako bi se obezbedilo da u momentu primene herbicida biljke Ch. album budu u fazi 2, 4, 6, 8 i 10 listova, odnosno A. theophrasti u fazi 1, 2, 3, 4 i 5 listova. Kada su biljke dostigle navedene fenofaze razvoja primenjen je preparat Skaut (mezotrion 480 g/l) u količini primene 0,25 l/ha, kome je dodat ađuvant Alteox wet 40 (mineralno ulje (500 g/l) + polioksi alkohol (500 g/l)) u količini 1 l/ha. Dobijeni rezultati su pokazali da fenofaza razvoja u vreme primene mezotriona značajno utiče na osetljivost obe ispitivane vrste. Primena herbicida u najranijim fazama razvoja (A. theophrasti - 1 list; Ch. album - 2 i 4 lista) dovela je do potpunog propadanja biljaka. Sa daljim starenjem biljaka osetljivost se smanjivala, tako da je najslabiji efekat utvrđen kod biljaka A. theophrasti u fenofazi 5 listova, a kod biljaka Ch. album u fenofazi 10 listova

DOPRINOS RAZVIJENOSTI MOTORIČKIH SPOSOBNOSTI KOD SAVLADAVANJA SKIJAŠKE TEHNIKE

Ovim istraživanjem utvrđen je uticaj motoričkih sposobnosti na proces učenja alpskog skijanja. Takođe, cilj ovog istraživanja bio je utvrditi uticaj određenih morfoloških karakteristika na savladavanje skijaških znanja. Na početku istraživanja ispitanici su bili testirani pomoću testova za procjenu: ravnoteže, agilnosti, eksplozivne i statičke snage, frekvencije pokreta, te fleksibilnosti. Nakon procjene motoričkih sposobnosti, ispitanicima su utvrđene osnovne morfološke karateristike, te su pristupili školi alpskog skijanja u trajanju od sedam dana. Po završetku učenja alpskog skijanja svi ispitanici demonstrirali su sedam elemenata skijaške tehnike ispred pet nezavisnih ispitivača. Analiza rezultata pokazala je kako agilnost pozitivno utiče pri učenju svih ispitanih elemenata skijaške tehnike (kosi spust, β = 0,38; β = 0,26; zavoj ka padini, β = 0,33; β = 0,23; osnovni zavoj, β = 0,18; paralelni zavoj, β = 0,32; vijuganje, β = 0,34). Rezultati ispitanika u testu izdržaj u čučnju kojim se procjenjuje statička snaga nogu pozitivno utiče kod učenja zavoja ka padini (β = 0,19), osnovnog zavoja (β = 0,35), te paralelnog zavoja (β = 0,27). Test za procjenu ravnoteže (ravnotežni položaj lijeve noge uzdužno) značajno utiče pri učenju osnovnih elemenata skijaške tehnike (kosi spust u desnu stranu, β = 0,23; kosi spust u lijevu stranu, β = 0,28; zavoj ka padini u lijevu stranu, β = 0,24). Tjelesna visina skijaša početnika negativno utiče na efikasnost usvajanja skijaških znanja (β = -0,5), što znači da će višim skijašima biti teže usvajati skijaška znanja u odnosu na niže skijaše. S obzirom na rezultate ovog istraživanja moguće je istaknuti agilnost, statičku snagu, te ravnotežu kao one motoričke sposobnosti koje najviše doprinose skijaškim početnicima prilikom učenja skijaške tehnike

Supplementary data for the article: Loncar, N.; Bozic, N.; Vujcic, Z. Expression and Characterization of a Thermostable Organic Solvent-Tolerant Laccase from Bacillus Licheniformis ATCC 9945a. J. Mol. Catal. B-Enzym. 2016, 134, 390–395. https://doi.org/10.1016/j.molcatb.2016.06.005

Supplementary material for: [https://doi.org/10.1016/j.molcatb.2016.06.005]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2375]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3517

Uporedna analiza izoformi α-amilaze iz srednjeg creva larvi Cerambyx cerdo L. (Coleoptera: Cerambycidae) iz prirode i gajenih na veštačkoj podlozi

alpha-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL) and reared in the laboratory (ADL) were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF). All C. cerdo amylase isoforms were acidic proteins (pI lt 3.5). Seven amylase isoforms (ACC 1-7) from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa.Upoređene su izoforme α-amilaze larvi Cerambyx cerdo sakupljenih iz prirode (ML i SL) i gajenih na veštačkoj podlozi u laboratoriji (ADL). Zimogramskom detekcijom posle IEF-a po tri izoforme su detektovane u ML i SL ekstraktima, a u ADL dve izoforme. Sve amilazne izoforme iz C. cerdo su bile kisele (pI lt 3.5). Zimogramskom detekcijom posle nativne elektroforeze sedam izoformi je detektovano u ML ekstraktu, šest u SL ekstraktu i četiri u ADL ekstraktu. Najveća amilazna aktivnost je detektovana u ADL ekstraktu. Svi ekstrakti srednjih creva larvi C. cerdo su frakcionisani na koloni Superose 12 HR. Molekulska masa ACC-a je bila 34 kDa