| Mannan and mannan oligosaccharide which hydrolyzed byβ-mannanase are widely employed in many industry fields,such as drug,feed,biodiesel,paper and pulp,detegent and textile industry.However,biotechnological process of industrial production experiences many harsh conditions such as high temperature and different p H environment.Therefore,many effort has been put into exploring properties ofβ-mannanase via diffenrent methods such as genetic and protein engineering.Acidicβ-mannanase 1013 was engineered due to its low specific activity in Bacillus subtilis.In this work,in order to improve enzyme expression and specific activity,original signal peptide（SP）of acidicβ-mannanase 1013 was substituted to30 SPs.Finally,one SP（Lip A）was selected for its excellent improvement in the specific activity of aidicβ-mannanase 1013 for almost 1.89-fold enhancement.Further sequence investigation and optimization of the selected peptide will potentially obtainβ-mannanase with better performance.In addition,β-mannanase 3118,3119,3120,3127,and 3128 were heterologously expressed in Pichiap pastoris.Enzyme properties of these fiveβ-mannanases were explored for further use.It is discovered thatβ-mannanase 3127 has high enzyme specific activity.However,its thermosability was the worst among the fiveβ-mannanases.Therefore,we intended to optimizing its thermostability via rational design.β-mannanase 3127 was compared with all sequences in PDB database,three crystal structures were selected with has more than 90%similarity.Based on analysis of B-factor of the three crystal structures,aimo acid 297-301 were determined with enhanced flexibility.Conserved amino acids were analyzed by multiple sequence alignment between thermostable/thermophilicβ-mannanase,and two combinations of amino acids in position 297-301 were identified.Subsequently,we conducted seven single-point mutations and two multi-point mutations using side-directed mutagenesis method.Specific activity ofβ-mannanase mutant 297-IAAAE-301 was highest among all mutants and wild type,raised almost 1.40-fold.Specific activities of T297V,T297I,and S301E all improved compared with that of wild typeβ-mannanase 3127.Besides,mutants showed enhanced acid-resistivity compared with that of wild type.T297I was revealed with better resistance to Hg2+compared with that of wild type enzyme.Thermostability of all the mutants were not improved,this phenomenon may due to residues 297-301 are quite closed to the active site ofβ-mannanase.Although our result was not as expected,it still can direct furtherβ-mannanase 3127 engineering.Enzyme activities of two kinds ofβ-mannanase were evaluated based on in vitro simulated digestion.Acid mannanase and neutral mannanase were mixed according to the enzyme activity ratio of 1:0,7:3,5:5,3:7 and 0:1,it is discovered that the two mannanases had the best synergistic effect when simulated pig gastric juice and small intestine environment with ratio 1:1.This conclusion was useful for the application and selection of feed compound mannanase in the future,especially feed industry. |
