Gene Cloning, Molecular Modification Of β-Mannanase And Preparation Of The Manno-Oligosaccharides By Enzymatic Hydrolysis

β-Mannanases (EC3.2.1.78), abbreviated from β-1,4-D-mannan mannohydrolases, canrandomly hydrolyze the internal β-1,4-D-mannosidic linkages of mannans to form varyinglength mannooligosaccharides, which have important physiological activities and can be usedas dietary fiber and prebiotics. The traditional production method that high temperature andacid or alkaline were used to hydrolyze konjac gum to mannooligosaccharides needs a largeamount of acid or alkaline. That can cause complex post-processing, high cost and severeenvironmental pollution, while enzymatic production has the advantages of ambienttemperature and environmentally friendliness. The key biocatalyst is β-mannanase in theenzymatic production of mannooligosaccharides, which was paid more concern and attentionby the researchers. Although there are a lot of reports about the-mannanases, theirapplications are still limited by their low activities and poor stability in an extremeenvironment.To obtain excellent engineered β-mannanases that have good thermostability and/or highcatalytic activity, and realize the biosynthesis of glucomannan oligosaccharides, we cloned thefull-length cDNA and partial DNA sequences of A. usamii YL-01-78-mannanase(abbreviated as AuMan5A) and made its mature peptide-encoding gene (Auman5A) expressedin P. pastoris GS115. On the basis of that, it was done that the preliminary research on therational modification and application of this-mannanase. The main results are listed asfollows:(1) The full-length cDNA and partial DNA sequences of AuMan5A gene were amplifiedby3,5rapid amplification of cDNA ends (RACE) and T vector-mediated PCR techniques.The DNA sequence is2168bp in length, harboring275bp5′flanking regulatory regions,343bp3′flanking regulatory regions and the1427bp full-length cDNA sequence in which twoshort introns with63and60bp are inserted, respectively. Among which, the cDNA includes51bp5′non-coding region,224bp3′non-coding region and an1152bp open reading frame(ORF). The ORF encodes a21-aa signal peptide, a17-aa propeptide, and a345-aa maturepeptide (AuMan5A) with two putative N-glycosylation sites. Then, its secondary andthree-dimensional (3-D) structures were predicted. The modelled3-D structure of AuMan5Aconsists principally of the (/)8barrel fold. One Glu catalytic-residue is located at theC-terminus of4and7, respectively.(2) The Auman5A was integrated into P. pastoris GS115genome by pPIC9K vecotor,and one strain labeled as GSKM4-8having the highest recombinant-mannanase activity of54.6U mL-1was chosen from the first-batch P. pastoris transformants. Then, the double P.pastoris transformant GSKZαM4-2with the highest-mannanase activity of78.1U mL-1andfavorable genetic stability was obtained by pPICZαAvecotor, and used to optimize expressionconditions. As GSKZαM4-2was induced under the optimized conditions (initial pH value6.5,induction period120h, methanol concentration1.5%and induction temperature32℃),-mannanase activity reached162.8U mL-1, being2.08times as high as that expressed using the standard protocol (Invitrogen, USA), and1.98-fold higher than that expressed byGSKM4-8. In addition, mutation analyses were carried out on the Glu168and Glu276residuesof AuMan5A, these results confirmed that the Glu168and Glu276residues of AuMan5A playedimportant roles in keeping its catalytic activity. The reAuMan5A is an N-glycosylated protein,and its carbohydrate content was determined to be21.3%. It displayed the maximum activityat pH3.0and70℃, and was stable at a pH range of3.0~7.0and at60℃or below. Its activitywas not significantly affected by metal ions tested and EDTA, but inhibited by Ag+and Hg2+.The reAuMan5A showed the highest activity towards locust bean gum, but lower activitiestowards konjac gum and guar gum. No activity was detected towards birchwood xylan,soluble starch or carboxyl methyl cellulose. The kinetic parameters, Kmand Vmax, of thereAuMan5A towards locust bean gum were1.36mg mL-1and415.8U mg-1, respectively.(3) The AuMan5A-CBM was designed based on the binding free energy, then theAuMan5A-CBM encoding gene was constructed by overlapping PCR and expressed in P.pastoris GS115. The GSAuMC4-5with the highest-mannanase activity of40.6U mL-1possesses favorable genetic stability. The temperature optimum of the reAuMan5A-CBM was75℃, being5℃higher than that of the reAuMan5A. And it was stable at68℃, being8℃higher than that of the reAuMan5A. The kinetic parameters, Kmand Vmax, of thereAuMan5A-CBM towards locust bean gum were0.66mg mL-1and389.1U mg-1, showingan obvious decrease in Km. In addition, the reAuMan5A-CBM possesses favorablecellulose-binding capacity.(4) Based on the3-D structure of AuMan5A, a truncated mutant AuMan5AN3C3with thelowest RMSD value and binding free energy was designed by in silico design. TheAuMan5AN3C3encoding gene was integrated into P. pastoris GS115genome by pPIC9KMvecotor and expressed, the β-mannanase activity of P. pastoris GSNC4-7expressionsupernatant was73.4U mL-1, which increased by39.0%as compared with that (52.8U mL-1)of reAuMan5A. Compared with the reAuMan5A, the reAuMan5AN3C3showed an obviousincrease in the specific activity and no significant change in the other enzymatic properties. Adisulfide bond was rationally introduced into AuMan5A forming a mutant AuMan5Ads324330by disulfide bond design, molecular docking and molecular dynamics simulation softwares,and its encoding gene was expressed in P. pastoris GS115. Unfortunately, there were somedifferences between the expected result and experimental result, which indicated that therewere still some room for improvement in the conditions and parameters of the in silico design.(5) The optimal hydrolytic condition of konjak gum for producing glucomannanoligosaccharides were as follows:30g L-1konjak gum solution (with deionized water),60U g-1konjak gum β-mannanase dosage,60℃hydrolytic temperature and6h hydrolytictime. Under this condition, the hydrolytic rate of konjak gum can reach36.6%. In addtion, theβ-endo-glucanase can play a synergistic role in the enzymatic hydrolysis of konjak gum byβ-mannanase, and its hydrolytic rate can reach65.4%. Analysis of oligosaccharide productsobtained by enzymatic hydrolysis of konjak gum using thin layer chromatography revealedthat these enzymes yielded mixture of mannobiose to mannohexose as their main products,and no trace of mannose could be detected in these hydrolysis experiments. These studies can lay the foundation for industrialized production of glucomannan oligosaccharides bybiocatalysis.