Gene Cloning And Expression Of A β-mannanase From Enterobacter Aerogenes B19 And Characterization Of The Recombinant Enzyme

β-mannanase（endo-1,4-d-mannanase,EC 3.2.1.78）is a hydrolase that catalyzes the random hydrolysis ofβ-1,4-mannosidic linkages in mannan,galactomannan,glucomannan,and galactoglu-comannan.Due to the value and demand ofβ-mannanase for mannan degradation,β-mannanases from microorganisms have been widely studied for their biochemical characteristics and structural properties,as well as for their use in various applications.β-mannanases have been used in pulp bleaching,for reducing viscosity of coffee,as detergent formulations and animal feed industries,and endo-β-mannanases from different sources are continually being discovered and studied for their use in various industrial applications.Thus,it is important significance to research the beta-mannanase related to this enzyme heterologous expression,purification and enzymatic properties analysis.In this study,Enterobacter aerogenes B19 was screened and preserved by our laboratory and founded its 16S r DNA is highly homologous with Enterobacter cloacae p101 through blast analysis in the web of the National Center for Biotechnology Information（NCBI）.The full length gene of beta-mannanase was amplified and obtained successfully using the beta-mannanase gene sequence of Enterobacter cloacae p101 as a template by constructed cloning vector pMD19-T-ManE.The recombinant beta-mannanase was expressed successfully after expression vector pET-28a（+）-ManE transformed into Escherichia coli BL21（DE3）.The crude enzyme was purified by HisTrap HP Ni-affinity chromatography and the molecular weight of the purified recombinant enzyme was detected about 82.5 kDa by SDS-PAGE protein electrophoresis.The heterologousexpression of beta-mannanase ofrecombinant strain BL21（DE3）-pET-28a（+）-ManE was optimised showed that 0.6 mmol/L IPTG was added when OD₆₀₀ is about 0.6,under the condition of 20℃,160 rpm induced culture 12¹6 h.The enzymatic properties analysis showed that the optimal reaction temperature of this enzyme were 55℃,and the recombinant enzyme exhibited high thermal stability when the temperature ranged from 50 to 55℃,and it relative enzyme activity was retained 85%for 1h.Meanwhile,the optimal pH of the recombinantβ-mannanase was around 6.5,and the pH value ranged from 4.0 to 7.0.The recombinantβ-mannanase activity was activated in different degree using Co²⁺,Mn²⁺,Zn²⁺,Ba²⁺and Ca²⁺with low concentration.But was inhibited by K⁺,Mg²⁺and Cu²⁺.In addition,Cu²⁺was inhibitor of enzyme activity,had obviously inhibitory effect on the recombinant enzyme and the relative enzymatic activity only retained 65.3%.