Molecular Cloning and Characterization of Fruit Softening Related Gene β-Mannanase from Banana Fruit

A 1 250 bp cDNA fragment encoding β-mannanase, named MaMAN, was cloned from banana （Musa spp cv. Baxi） fruit using degenerate primers designed with reference to the conserved nucleic acid sequences of known β-mannanase genes by RT-PCR. Sequence analysis showed that MaMAN cDNA encompassed a 1 085 bp open-reading frame （ORF）, encoding a predicted polypeptide of 395 amino acids. Alignment of the deduced amino acid sequence of MaMAN and other putative β-mannanases showed that MaMAN has an identity of 86, 70, 69, 54, and 57%, respectively, to β-mannanases from tomato, lettuce, arabidopsis, carrot and oryza sativa. The catalytic residues： Asn203, Glu204, Glu318 and the active site residues： Arg86, His277, Tyr279, and Trp360, which were strictly conserved in the glycoside hydrolase family 5 to which all β-mannanases belonged, were found in MaMAN. Semi-quantitative RT-PCR revealed that the level of MaMAN transcript in the pulp increased during banana fruit ripening, suggesting that MaMAN was likely to be involved highly in banana fruit softening.

Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.

Development of two methods for rapid screening of recombinant Pichia pastoris strains with high-level expression of β-mannanase

The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.

Comparative standardized ileal amino acid digestibility and metabolizable energy contents of main feed ingredients for growing pigs when adding dietary β-mannanase

The present study was conducted to test whether the dietary supplementation of β-mannanase affects amino acids(AA) digestibility,metabolizable energy(ME) contents of corn,wheat,soybean meal,distillers dried grains with solubles,and palm kernel meal(PKM),nutrient digestibility,and growth performance of pigs.In Exp.1,22 cannulated pigs were used for 10 dietary treatments including 5 feed ingredients and 2 β-mannanase concentrations(0 and 0.5 g/kg of the diet) in 6 periods in an incomplete Latin square design to determine the AA and energy digestibility.In Exp.2,200 growing pigs were randomly allotted to 4 treatments with 2 nutrient levels(high and low) and 2 concentrations of β-mannanase(2×2 factorial arrangement) in 2 phases(phase 1,d 0 to 21;and phase 2,d 22 to 42).In Exp.1,β-mannanase increased the mean standardized ileal digestibility(SID) of AA in all feed ingredients.The amount of digestible energy was increased(P <0.05) in β-mannanase-treated PKM.Pigs fed β-mannanase showed a greater(P <0.05) digestibility of gross energy(GE).The feed-to-gain(F:G) ratio was improved(P <0.01) in pigs fed high-nutrient diets.Pigs fed β-mannanase in the diets had greater(P <0.05) average daily gain and F:G.In phase 2,the concentration of fecal ammonia was decreased(P <0.05) in pigs fed β-mannanase.Considering the 2 experiments,it can be concluded that β-mannanase increases the SID of AA,which has to be considered in balancing the rations.

Changes in Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination

To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and a-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and a-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.

Establishment of Kinetics Models for Batch Fermentation Process of β-mannase with Bacillus licheniformis HDYM-04

In order to improve the yield of β-mannase and to investigate the rules of fermentation production, a high-yield β-mannase producing strain, Bacillus licheniformis HDYM-04, was used to investigate the kinetics models based on the optimal fermentation conditions： HDYM-04 strain was fermented at 37℃ for 30 h with agitation speed at 300 r/min and aeration rate at 3 L/min in a 5 L fermenter, the initial addition amount of konjac flour was 2%（w/v）, the initial pH of medium was 8.0, and the inoculum concentration was 6.7%（v/v）. Three batch fermentation kinetic models were established （cell growth kinetic model, substrate consumption kinetic model, product formation kinetic model） bases on Logistic and Luedeking-Piret equations. To be specific, cell growth kinetic model was dX/dt =0.431X （1- X/ 15.522 ）, substrate consumption kinetic model was -ds/dt =1.11 dX/dt ＋0.000 2 dP/dt ＋0.000 8X, and product formation kinetic model was dP/dt=133.1 dX ＋222.87X. The correlation coefficients R^2 of the three equations were 0.990 21, 0.989 08 and 0.988 12, respectively, which indicated a good correlation between experimental values and models. Therefore, the three equations could be used to describe the processes of cell growth, enzyme synthesis and substrate consumption during batch fermentation using B. licheniformis strain HDYM-04. The establishment of batch fermentation kinetic models （cell growth kinetic model, substrate depletion kinetic model, product formation kinetic model） could lay the theoretical foundation and provide practical reference for the applica- tion of HDYM-04 in fermentation industry.

Characterization of Novel Alkaliphilic Isolate of <i>Bacillus mannanilyticus</i>, Strain IB-OR17, Displaying Chitinolytic and Antifungal Activities

The novel alkaliphilic strain, designated as Bacillus sp. IB-OR17 and isolated from soda lake sediments, was identified and characterized. Isolated strain demonstrated slight antifungal activity against some plant pathogen fungi that are capable to grow under alkaline conditions. Based on its morphological, physiological and biochemical characteristics as well as on 16S rRNA gene analysis data, Bacillus sp. IB-OR17 were related to alkaliphilic species B. mannanilyticus. Such as type species, Bacillus sp. IB-OR17 produced extracellular β-mannanase but additionally it displayed also chitinolytic activity which is a new property reported for this species. Bacillus sp. IB-OR17 grew in pH range 8.0 - 11.0 with maximal intensity under 9.0 - 10.0 but not showed halophilic properties (growth limit under NaCl concentrations < 5%). Maximal production of chitinase is observed at the same pH interval after 96 h of submerged cultivation of the strain. Bacillus sp. IB-OR17 produced chitinase(s) in presence of colloidal chitin as main carbon source and sodium carbonate (0.25% - 1.0%) demonstrating high enzyme yield under enough low concentrations of the substrate (0.20%). Unlike chitinase, β-mannanase was constitutively produced by Bacillus sp. IB- OR17 in presence of various substrates including crab shell chitin. Probable involvement of the enzymes in antifungal activity of Bacillus sp. IB-OR17 is discussed shortly in terms of further researches and application of this strain.

Heterologous expression and characterization of man gene from Bacillus Subtilis in Pichia Pastoris

β-Mannanase catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannan,which are abun-dant in the cell wall structure of ungerminated leguminous seeds.The mature β-mannanase originated from Bacillus subtilis was expressed in Pichia pastoris,a methylotrophic yeast,using the leader peptide sequence of Saccharomyces cerevisiae α-factor.The cultivation of β-mannanase express-ing Pichia pastoris yields up to 1.8 g/L protein.In the super-natant the activity of the 40 kDa—total mannanase attained a level of 1102.0 IU/mL.The properties of the β-mannanase were characterized.Optimum pH and temperature for the recombinant enzyme were 5.5 and 50℃ respectively.The enzyme was stable at pH 5.0-10.0 and maintained over 30%original activity after incubating at 70℃ for 30 min.