Characterization, rational design and application of highly efficent raw starch degrading α-amylase from Bacillus licheniformis.

Složeni ugljeni hidrati kao što je skrob, od velikog su industrijskog značaja kako uoblastima bioenergije i bioprocesovanja, tako i u ljudskoj ishrani. Enzimi koji mogu direktnoda hidrolizuju sirovi (nativni, granularni) skrob na temperaturama nižim od temperaturegeliranja privlače sve veći interes jer bi njihovom upotrebom mogao da se smanji utrošakvelike količine energije u zagrevanju skroba prilikom geliranja, a time i pojeftini proces.Rekombinantna α-amilaza iz Bacillus licheniformis ATCC 9945a (BliAmy), efikasna uhidrolizi granularnog skroba ispod temperature geliranja, proizvedena je ekstracelularnokorišćenjem Escherichia coli kao domaćina. Razvijeni su efikasni i reproduktivni procesifermentacije sa ćelijama E. coli BL21 (DE3) i E. coli C43 (DE3) kroz dizajn medijuma zarast mikroorganizma i optimizaciju koraka u proizvodnji amilaze. Dobijene su velike količineBliAmy i to, u fermentaciji sa ćelijama E. coli BL21 (DE3) ukupna amilazna aktivnost od250,5 IU mL-1 (0,7 g L-1 ), a sa ćelijama E. coli C43 (DE3) 500 IU mL−1 (1,2 g L−1).Primenjen je eksperimentalni dizajn u optimizaciji hidrolize skroba, prečišćavanjuproteina i dobijanja bioetanola. Korišćenjem statističke metode odgovornih površinaoptimizovan je proces hidrolize koncentrovane suspenzije sirovog kukuruznog skroba.Pokazano je da je BliAmy veoma efikasna jer je hidrolizovala 91 % prilikom hidrolize 30 % -ne suspenzije skroba nakon 24 sata.Na osnovu promena u kristalnoj strukturi analiziranjem rezidualnog skroba kojizaostaje nakon hidrolize 30%-ne susupenzije nakon 5 i 24 sata pokazano je da kukuruzniskrob ima A-tip i Vh-tip kristaliniteta. Upoređivanjem difrakcionih intenziteta pikovakristalne strukture i SEM mikrografije nativnog i rezidualnog skroba može se zaključiti da seistom brzinom hidrolizuju i kristalni i amorfni regione u granulama skroba, što je u skladu sanačinom hidrolize skrobnih granula tzv. „granula po granula“...Complex carbohydrates such as starch are of great industrial interest, especially inthe areas of bioenergy and bioprocessing and increasingly in human nutrition. Theimportance of enzymes capable of direct hydrolysis of raw (native, granular) starch belowthe temperature of gelatinization has been well recognized due to the energy savings andthe effective utilization of biomass, which reduces the overall cost of starch processing.Recombinant α-amylase from Bacillus licheniformis ATCC 9945a (BliAmy), highlyefficient in raw starch hzdrolysis was produced extracellularly in Escherichia coli. Anefficient and reproducible fermentation approach was developed with cells E. coli BL21(DE3) and E. coli C43 (DE3) through the smart design of defined growth medium andoptimization of process steps. Significant overexpression of BliAmy was achieved. UsingE. coli BL21 (DE3) total of 250.5 IU mL-1 (0.7 g L-1 ) was obtained while using E. coliC43 (DE3) total of 500 IU mL−1 (1.2 g L−1) was achived.Experimental design was applied to optimize the hydrolysis of starch, proteinpurification and production of bioethanol. The hydrolysis of concentrated raw starch wasoptimized using response surface methodology. BliAmy was very effective, achieving thefinal hydrolysis degree of 91 % for the hydrolysis of 30 % starch suspension after 24 h.Cystalline structure of starch residues analysed by X-ray diffraction on the samplesoriginating from 30 % starch suspensions after 5 and 24 h shown A-type and a Vh-typecrystallinity of corn starch. Constant diffracted intensities and scattering backgroundindicated that BliAmy most likely degraded both amorphous and crystalline areas at thesamerates. This is in agreement with a granule by granule mode of attack and can be concludedfrom the SEM micrographies..

Characterization, rational design and application of highly efficent raw starch degrading α-amylase from Bacillus licheniformis.

Složeni ugljeni hidrati kao što je skrob, od velikog su industrijskog značaja kako uoblastima bioenergije i bioprocesovanja, tako i u ljudskoj ishrani. Enzimi koji mogu direktnoda hidrolizuju sirovi (nativni, granularni) skrob na temperaturama nižim od temperaturegeliranja privlače sve veći interes jer bi njihovom upotrebom mogao da se smanji utrošakvelike količine energije u zagrevanju skroba prilikom geliranja, a time i pojeftini proces.Rekombinantna α-amilaza iz Bacillus licheniformis ATCC 9945a (BliAmy), efikasna uhidrolizi granularnog skroba ispod temperature geliranja, proizvedena je ekstracelularnokorišćenjem Escherichia coli kao domaćina. Razvijeni su efikasni i reproduktivni procesifermentacije sa ćelijama E. coli BL21 (DE3) i E. coli C43 (DE3) kroz dizajn medijuma zarast mikroorganizma i optimizaciju koraka u proizvodnji amilaze. Dobijene su velike količineBliAmy i to, u fermentaciji sa ćelijama E. coli BL21 (DE3) ukupna amilazna aktivnost od250,5 IU mL-1 (0,7 g L-1 ), a sa ćelijama E. coli C43 (DE3) 500 IU mL−1 (1,2 g L−1).Primenjen je eksperimentalni dizajn u optimizaciji hidrolize skroba, prečišćavanjuproteina i dobijanja bioetanola. Korišćenjem statističke metode odgovornih površinaoptimizovan je proces hidrolize koncentrovane suspenzije sirovog kukuruznog skroba.Pokazano je da je BliAmy veoma efikasna jer je hidrolizovala 91 % prilikom hidrolize 30 % -ne suspenzije skroba nakon 24 sata.Na osnovu promena u kristalnoj strukturi analiziranjem rezidualnog skroba kojizaostaje nakon hidrolize 30%-ne susupenzije nakon 5 i 24 sata pokazano je da kukuruzniskrob ima A-tip i Vh-tip kristaliniteta. Upoređivanjem difrakcionih intenziteta pikovakristalne strukture i SEM mikrografije nativnog i rezidualnog skroba može se zaključiti da seistom brzinom hidrolizuju i kristalni i amorfni regione u granulama skroba, što je u skladu sanačinom hidrolize skrobnih granula tzv. „granula po granula“...Complex carbohydrates such as starch are of great industrial interest, especially inthe areas of bioenergy and bioprocessing and increasingly in human nutrition. Theimportance of enzymes capable of direct hydrolysis of raw (native, granular) starch belowthe temperature of gelatinization has been well recognized due to the energy savings andthe effective utilization of biomass, which reduces the overall cost of starch processing.Recombinant α-amylase from Bacillus licheniformis ATCC 9945a (BliAmy), highlyefficient in raw starch hzdrolysis was produced extracellularly in Escherichia coli. Anefficient and reproducible fermentation approach was developed with cells E. coli BL21(DE3) and E. coli C43 (DE3) through the smart design of defined growth medium andoptimization of process steps. Significant overexpression of BliAmy was achieved. UsingE. coli BL21 (DE3) total of 250.5 IU mL-1 (0.7 g L-1 ) was obtained while using E. coliC43 (DE3) total of 500 IU mL−1 (1.2 g L−1) was achived.Experimental design was applied to optimize the hydrolysis of starch, proteinpurification and production of bioethanol. The hydrolysis of concentrated raw starch wasoptimized using response surface methodology. BliAmy was very effective, achieving thefinal hydrolysis degree of 91 % for the hydrolysis of 30 % starch suspension after 24 h.Cystalline structure of starch residues analysed by X-ray diffraction on the samplesoriginating from 30 % starch suspensions after 5 and 24 h shown A-type and a Vh-typecrystallinity of corn starch. Constant diffracted intensities and scattering backgroundindicated that BliAmy most likely degraded both amorphous and crystalline areas at thesamerates. This is in agreement with a granule by granule mode of attack and can be concludedfrom the SEM micrographies..

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

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

Growth Temperature of Different Local Isolates of Bacillus Sp in the Solid State Affects Production of Raw Starch Digesting Amylases

Natural amylase producers, wild type strains of Bacillus sp., were isolated from different regions of Serbia. Strains with the highest amylase activity based on the starch-agar plate test were grown on solid-state fermentation (SSF) on triticale. The influence of the substrate and different cultivation temperature (28 and 37 degrees C) on the production of amylase was examined. The tested strains produced alpha-amylases when grown on triticale grains both at 28 and at 37 degrees C, but the activity of amylases and the number and intensity of the produced isoforms were different. Significant hydrolysis of raw cornstarch was obtained with the Bacillus sp. strains 2B, 5B, 18 and 24B. The produced alpha-amylases hydrolyzed raw cornstarch at a temperature below the temperature of gelatinization, but the ability for hydrolysis was not directly related to the total enzyme activity, suggesting that only certain isoforms are involved in the hydrolysis

Production of polyglutamic acid by Bacillus sp: Strains selection, optimization, batch fermentation and characterization

Polyglutamic acid (PGA) is an anionic, non-toxic natural polymer that consists of D- and L-glutamic acid. Glutamic acids that makeup PGA create bonds between α-amino and γ-carboxyl groups. PGA can be found on the surface of many different bacteria, usually as a part of their capsule. Bacteria use polyglutamic acid to survive adverse environmental conditions. PGA has found multiple potential applications as a thickener, drug carrier, biological adhesive, heavy metal and basic dye adsorber, etc. Its biodegradability is especially useful in the fields of food, cosmetics, medicine and water treatment1. The aim of this study was to find the best polyglutamic acid producer from the selection of 50 different Bacillus sp strains originating from Serbia, as well as the optimal medium composition. It was discovered that the best PGA producing strain was 17B and it was selected for further fermentation medium optimization. Optimization was carried out using Design of Experiment, specifically Box Behnken design. Results were analyzed using response surface methodology. PGA that was produced during this process was analyzed using SDS PAGE and basic dye adsorption was attempted as well. The best PGA producer, Bacillus sp strain 17B, was used for PGA production in batch fermenter 2. PGA isolated from the fermentation broth was purified using gel filtration and further characterized using SDS PAGE, FTIR spectroscopy and direct-infusion MS

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

A search for nature’s robust proteases with zein as a substrate

Zein is produced in large quantities as a byproduct of corn starch manufacturing since it constitutes a majority of the total protein of maize seed (44–70%). Enzymatic treatment of zein significantly improves its aqueous solubility and provides peptides that are used as animal feed, functional food, or biologically active carriers for other bioactive molecules. Moreover, zein-derived peptides exhibit antioxidant, anti-inflammatory, antihypertensive, anticancer, and antimicrobial activities in human organisms 1. Few attempts up to this day have been made to screen for microorganisms that are capable of zein degradation. Available protocols for proteases identification almost exclusively rely on screening on casein, skim milk, and gelatin agar in limited experimental conditions. We have screened different Bacillus sp strains isolated from across Serbia for zein-degrading proteases. To do so we developed an inexpensive, simple, and reproducible way of high throughput functional screening of zein-degrading proteases on zein-containing gels. Besides detecting proteases with specificity towards zein, a developed diffusion assay was designed to support screening for naturally occurring robust proteases with high potential for industrial application. By using classical methods of protein purification, we isolated an alkaline thermostable protease from Bacillus amyloliquefaciens strain 12B that is resistant to the presence of detergents, organic solvents, and high salt concentrations