Protein Engineering,High Level Expression,and Application Of The β-mannanases From Rhizomucor Miehei And Rhizopus Microsporus Var.Rhizopodiformis

β-Mannanase(EC 3.2.1.78)is the foremost enzyme for the degradation of mannans,which cleaves the β-1,4-linked backbone of mannans randomly to produce manno-oligosaccharides and dietary fibers with various beneficial functions.In recent years,β-mannanase has been widely used in many industrial applications,such as food and feed.However,due to the low expression level and the limited biochemical properties,many wild type β-mannanases are still defective for some industries.Thus,engineering and high level expresiing β-mannanases to make them suitable for industrial application is very important and necessary for the development of β-mannanase.In the present dissertation,theβ-mannanases from Rhizomucor miehei CAU432 and Rhizopus microsporus var.rhizopodiformis F518 were engineered by rational and irrational design.These β-mannanase genes were then successfully expressed in Pichia pastoris for high level expression.Furthermore,the β-mannanases were employed for the hydrolysis of mannan-rich plant gums and agro-industrial biomass,such as guar gum,cassia gum,and palm kernel cake.The main results are as below:(1)A novel β-mannanase(RmMan5A)from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for partially hydrolyzed guar gum(PHGG)production.Enzyme activity of fermentation supernatant reached 85,200 U/mL after 168 h high cell density fermentation.The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65 ℃.RmMan5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight(Mw)of 2.5×104 Da.Total dietary fiber accounted 90.6%of PHGG and 24.9%(w/w)of PHGG were identified as manno-oligosaccharides with degree of polymerization<7.PHGG was further fractionated(F1-F4)by gradual ethanol precipitation.According to the structure analysis,the distribution of α-D-galactose of PHGG F1 was compact and regular,and that of other fractions was more random.(2)The β-mannanase(RmMan5A)from R.miehei was successfully engineered by directed evolution.Through two rounds of screening,a mutated β-mannanase(mRmMan5 A)with high catalytic activity in acidic and thermophilic conditions was obtained,and then characterized.The mutant displayed maximal activity at pH 4.5 and 65 ℃,corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 ℃ in optimal temperature.The catalytic efficiencies(kcat/Km)of mRmMan5A towards many mannan substrates were enhanced more than threefold in its optimal condition.According to the result of sequence analysis,three amino acid residues were substituted in mRmMan5A,namely Tyr233His,Lys264Met,and Asn343Ser.The mutated β-mannanase(mRmMan5A)was then successfully expressed in Pichia pastoris.Through high cell density fermentation,the expression level of mRmMan5A reached 79,680 U/mL.To produce manno-oligosaccharides,palm kernel cake(PKC)was pretreated by steam explosion at 200 0C for 7.5 min,and then hydrolyzed by mRmMan5A.As a result,the total manno-oligosaccharide yield reached 34.8 g/100 g dry PKC,indicating that 80.6%of total mannan in PKC was hydrolyzed.Moreover,the kilo-scale production of manno-oligosaccharides was carried out to verify the feasibility of mass production.A total of 261.3 g manno-oligosaccharides were produced from 1.0 kg of dry PKC.(3)According to the sequence alignment of RmMan134A from R microsporus var.rhizopodiformis with other GH family 134 β-mannanases,seven amino acid residues were selected for site-directed mutagenesis.The substitutions at Glu25,Gln36,Ser71,and Asp159 could significantly affect the biochemical property of RmMan134A.Among all mutated β-mannanases,the mutant RmMan134AM36 showed increase by 108.1%in specific activity and inherited other excellent properties of RmMan134A.The mutated β-mannanase(RmMan134AM36)was then successfully expressed in Pichia pastoris.Enzyme activity of fermentation supernatant reached 3,680 U/mL after 168 h high cell density fermentation.Then,RmMan 134AM3 6 was employed for hydrolyzing cassia gum to produce manno-oligosaccharides.The total yield of manno-oligosaccharides reached 70.6%,and the hydrolysis ratio of galactomannan in cassia gum was 81.6%.86.4%(w/w)of the product were identified as manno-oligosaccharides with degree of polymerization<6.Through activated-charcoal column and silica gel column,seven fractions(P1-P7)were purified from the manno-oligosaccharide products,which were identified as mannose,mannobiose,galactose,mannotriose,mannotetraose,α-1,61-D-galactosyl mannobiose,and mannopentaose,respectively.