Cloning And Expression Of Genes Encoding β-Mannanase And Endo-β-1,3-Glucananse

Enzymes, which were applied in feed in 1950s, nowadays have been believed as a kindof "green" feed addictive of high efficiency, poison free, side-effect free and friendliness toenvironment. There have been a lot of reports on strain screening,mutagenesis and theprocess of fermentation and inducement about β-mannanase to apply in feed, whereas moreand more research work was focused on the gene cloning and heterologous expression ofthis enzyme nowadays. Endo-1, 3-glucanase is expected to be a new type of feed addictivebecause of its potential to decrease the viscosity of β-glucan although there has no report onthis enzyme used in feed. In this paper, purification,enzymatic characterization,genecloning and expression in E. coli of β-mannanase from B. licheniformis 2004 and cloning,splicing and expression in E. coli of endo-β-1, 3-glucanase gene from Neurospora crassawere investigated.The β-mannanase was extracted and purified by (NH4)2SO4 precipitation, Superdex 75column gel filtration and DEAE–Cellulose FF ion-exchange chromatography. The purifiedenzyme showed a specific activity of 2826.4 u/mg with purification fold of 16.17 and theyield of 10.56%.Enzymatic characterization was done with purified enzyme. Optimum temperature andpH were 75℃ and 5.5 respectively. The enzyme was stable from pH 4.0 to 9.0. The activityof enzyme was enhanced by K+,and strongly inhibited by Al³⁺ and Cu²⁺ , and wasmoderately inhibited by Rb⁺,Mn²⁺,Fe²⁺ and Co²⁺. Na⁺ and Li⁺ had no significant effect onthe activity of enzyme. Under a certain concentration, Mg²⁺ enhanced the activity of thepurified enzyme and showed protective effect from thermo-denaturation. The Km values ofthe purified enzyme were 6.03 mg/ml for LBG and 2.99 mg/ml for Konjac gum. Maximumvelocities (Vmax) were 668.6 mg/min-mg for LBG and 1173.7 mg/min-mg 0.115mg/min forKonjac powder. A lot of oligosaccharide and a little manose were produced in hydrolysisexperiment LBG as substrate.PCR technique was used to clone the gene fragment coding the mature peptide ofβ-mannanase which contained 336 of amino acid and with a deduced molecular weight of 38kDa. β-mannanase gene fragment was inserted into a new plasmid pBL-WZX by molecularcloning techniques, and was expressed in E. coli JM109. The activity of β-m-annanase fromrecombinant strains was 8 times higher (325 u/ml) as compared to that of the native strains(42.19 u/ml). Analysis of amino acid indicate that β-m-annanase from B. licheniformis 2004 isclarified in glycosyl hydrolase family 26.Endo-beta-1, 3-glucanase gene named glu without signal sequences but two introns wasamplified, genome DNA from Neurospora crassa being template. In order to be functionallyexpressed the target gene in E. coli, glu, in which the introns was removed by cross-over PCR,was cloned into expression vector pET28a and then the recombinant vector was transformedinto E. coli BL21. The band about 80 kDa in size was clearly detected on SDS-page, the samehost holding empty pET28a as control.