Optimization Of Fermentation Conditions Of Low Temperature Cellulase By Psychrotrophic Bacteria And Analysis Of Active Enzyme Spectrum

Compared with normal temperature cellulase, low temperature cellulase has a lower action temperature of 20℃-30℃, which has a great advantage for long cold period northern regions in our country. Endo glucanase (CMCase) is a key group of cellulase system. In this paper, to optimize fermentation conditions of low temperature CMCase produced by a psychrotrophic bacterium (HQ) systematically and active enzyme spectrum analysis, which lays the theoretical basis for itsl application in practical work.The main results obtained in this paper as follows:(1) To measure CMCase enzyme activity of cell fermentation broth and ultrasonic cell-break liquid of strain HQ respectively, to determine CMCase produced by HQ is mainly secreted in the extracellular.(2) Via 16S rDNA sequence determination and GenBank sequence similarity analysis, according to "Bergey’s bacteria identification Manual" Eighth Edition, combined with physiological and biochemical characteristics, HQ is identified as Bacillus subtilis subsp..(3) CMC-Na as substrate, to study enzymatic properties, the results showed:the optimum reaction temperature is 30℃, enzyme activity gets higher during 30-50℃; the optimal pH is 5.0, stability is better during pH 3.0-5.0; Mn2+ and Cu2+ can improve CMCase enzyme activity remarkably, the inhibitory effect of Zn2+, Mg2+ and Fe3+ on CMCase enzyme activity is more obvious.(4) CMCase enzyme activity as index, using P-B (Plackett-Burman) and Response Surface Method (RSM) to optimize fermentation conditions of CMCase produced by HQ. Ultimately the optimal fermentation conditions obtained as:30.0 g·L-1 CMC-Na as carbon source,40.0 g·L-1 1:1 peptone beef extract as nitrogen source; 0.11% Tween-80; initial pH 5.35, temperature is 40℃, inoculation dose is 6.42%, then to culture shakily under the optimum conditions, CMCase enzyme activity can reach 32.5 U·mL-1 after 4 days, is 2.96-fold than before the optimization.(5) Detection of crude enzyme solution by SDS-PAGE, about 14 kinds of protein were obtained; to test CMCase activity using CMC-Native PAGE, electrophoresis showed three active bands, indicating that low temperature cellulase produced by HQ containing three enzyme groups which has CMCase activity; separation and purification by DEAE-Cellulose 52 ion exchange column chromatography furtherly, two obvious CMCase active peaks is obtained by DEAE-Cellulose 52 ion exchange column chromatography, showed that low temperature cellulase system of HQ contains two kinds of CMCase isoenzymes at least.