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

Abstract

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..