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

?-mannanase?endo-1,4-d-mannanase,EC 3.2.1.78?is a hydrolase that catalyzes the random hydrolysis of?-1,4-mannosidic linkages in mannan,galactomannan,glucomannan,and galactoglu-comannan.Due to the value and demand of?-mannanase for mannan degradation,?-mannanases from microorganisms have been widely studied for their biochemical characteristics and structural properties,as well as for their use in various applications.?-mannanases have been used in pulp bleaching,for reducing viscosity of coffee,as detergent formulations and animal feed industries,and endo-?-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 recombinant?-mannanase was around 6.5,and the pH value ranged from 4.0 to 7.0.The recombinant?-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%.