Breeding Of ?-mannanase High-yielding Strains And Optimization Of Enzyme Production Conditions By Immobilized Cells

The research and application of ?-mannanase has been concerned by researchers in recent years.Screening the microbes with high ?-mannanase-producing level and apply them into productive practice are of great importance.This study utilized mutation breeding to screen a ?-mannanase-producing strain.The result of strain immobilization suggested the potential of its application in productive practice.This research provided the theoretical basis for the usage of the strain in the production.The mutant was obtained from original Bacillus licheniformis HDYM-04 by mutagenesis treatment of UV mutation,He-Ne mutation and NTG mutation.?-mannanase activity was assayed as index.The best conditions of UV mutation were as followed:the irradiate distance 50 cm,the irradiate time 10 secomds.The enzyme acticity of mutant U2-12(1103±32.19 U/mL)had an increase of 241.84%.The?-mannanase activity of original Bacillus licheniformis HDYM-04 was 322.67±9.88 U/mL.The best conditions of He-Ne mutation were as followed:the irradiate distance 5 cm,the irradiate time 30 minutes.The enzyme activity of the mutant N2-10(1103±32.19 U/mL)had an increase of 273.63%compared with the original strain and an increase of 10.52%compared with the mutant U2-12.The best conditions of NTG mutation were as followed:the concentration of NTG 5 cm,the impact time 16 minutes.The enzyme activity of mutant G2-3(1259.33±39.37 U/mL)increased 290.70%,14.17%and 9.86%compared to the original strain,the mutant U2-12 and N2-10,respectively.In order to enhance the fermentation capacity of mutant G2-3,this study optimized the fermentation conditions in flasks.After investigating the effects of single factors on ?-mannanase production,orthogonal tests were carried out to obtain the optimal combinations of those factors.The optimum conditions were:cultivation temperature 36?,inoculum size 2%,konjac flour 1%,initial pH value 7.0,rotation speed 150 r/min.The ?-mannanase activity reached 1579.58±59.41 U/mL which was increased by 32.91%.This research cloned the ?-mannanase gene from Bacillus licheniformis HDYM-04 and mutants,including the high-yield mutants U2-12,N2-10,G2-3 together with the low-yield mutants UI-36,N1-41,G1-10.The above-mentioned procedure aimed to(1)clarify the mutagenesis impact on the nucleotide sequences and amino acid sequences of the original strain from the molecular point of view and(2)explore the sites associated with the changes of enzyme acticity in ?-mannanase gene sequence.The results derived from bioedit showed that(1)the active changing site located in 1?13 bp,16?18 bp,30 bp,187 bp,202?203 bp,213 bp,226?227 bp,255?256bp,264?265 bp,301 bp,445 bp and 469?471 bp on the ?-mannanase gene;(2)the conserved region located in 31 bp to 202 bp and 302 bp to 444 bp.?-mannanase can be applied in many industrial fields such as actual production and dye bleaching.This study explored the applicable possibility by immobilization technology of ?-mannanase.The retention of ?-mannanase was as used as indicator to obtain the best immobilization agent,i.e.polyvinyl alcohol.Confirm the best fermented conditions then put it into production performance verification.Single factor experiments were conducted to investigate the effect of retention on ?-mannanase activity in immobilized pellet.In conclusion,the optimum immobilization conditions were:concentration of polyvinyl alcohol 2%,density of entrapped cells 1:1.Under this condition the retention of ?-mannanase was up to 41.93±0.90%with an increase of 18.60%.The optimum conditions of ?-mannanase fermentation based on the optimum immobilization condition were:konjac flour concentration 0.7%,inoculums size 1%,proliferated time 60 hours.Under this condition,the ?-mannanase activity was 838.96±25.79 U/mL.Based on these parameters,the ?-mannanase activity reached 962.37±15.61 U/mL within 32 h and the activity didn't significantly change after repeated usage for six times.The decolorization of 32 various dyes by HDYM-04 ?-mannanase was tested.Thses dyes belonged to different structural types,including azo dyes,cyanine dyes,anthraquinone dyes and riaromatic methane dyes,three aryl methyl dyes and heterocyclic dyes.The absorption luminosity values were detected as the indicator of decolorization effect.The results showed that HDYM-04 ?-mannanase has stronger ability to decolorize triaromatic methane dyes compared with other types of dyes.The decolorization rate of aniline blue and water soluble aniline blue reached more than 70%in 24 h.For malachite green,the decolorization rate was 49.14%in 12 h and 83.02%in 24 h.The decolorization rate of phenol red reached 95.97%in 24 h.This research screened a bacterial mutant with high ?-mannanase-producing ability by mutation breeding and optimized the fermentation and immobilization conditions.The {-mannanase production was enhanced and the molecular mechanism of enzyme change was discussed.Moreover,the applicable potential in dye decolorization was investigated.These achievements suggested the industrial application of this strain in enzyme production and bleaching.