| ?-Mannanase(EC 3.2.1.78)is the foremost enzyme for the degradation of mannans,which cleaves the ?-1,4-linked backbone of mannans randomly to produce manno-oligosaccharides and dietary fibers with various beneficial functions.In recent years,?-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 ?-mannanases are still defective for some industries.Thus,engineering and high level expresiing ?-mannanases to make them suitable for industrial application is very important and necessary for the development of ?-mannanase.In the present dissertation,the?-mannanases from Rhizomucor miehei CAU432 and Rhizopus microsporus var.rhizopodiformis F518 were engineered by rational and irrational design.These ?-mannanase genes were then successfully expressed in Pichia pastoris for high level expression.Furthermore,the ?-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 ?-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 ?-D-galactose of PHGG F1 was compact and regular,and that of other fractions was more random.(2)The ?-mannanase(RmMan5A)from R.miehei was successfully engineered by directed evolution.Through two rounds of screening,a mutated ?-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 ?-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 ?-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 ?-mannanases,the mutant RmMan134AM36 showed increase by 108.1%in specific activity and inherited other excellent properties of RmMan134A.The mutated ?-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,?-1,61-D-galactosyl mannobiose,and mannopentaose,respectively. |
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