【作者】 张益波；

【导师】 滕利荣； 林凤；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2011， 博士

【摘要】 本研究从腐败的玉米秸秆上分离得到一株能够发酵生产耐热耐酸性的β-葡萄糖苷酶的菌株,经过形态学和分子生物学方法的鉴定为Tolypocladium cylindrosporum syzx4。然后利用计算机辅助的方法对此菌株的发酵培养基进行了优化,菌株的筛选过程和酶的发酵过程申请了国家专利。首先,使用Plackett-Burmans实验设计法对碳源、氮源和无机盐进行产酶显著性的分析,培养基组分对β-葡萄糖苷酶生产影响排序为：TCS>KH2PO4> SM=(NH4)2SO4;然后应用计算机辅助模型拟合的方法对培养基进行进一步的优化,建立的模型包括响应面法和人工智能,人工智能模型表现的非常优异,得到的最佳培养基组成为：TCS 26.274 g/L,SM 7.066 g/L, KH2PO41.991 g/L, (NH4)2SO42.328 g/L,其它成分保持原始值,30℃,初始pH值为5时,发酵8天,此时发酵生产的β-葡萄糖苷酶活力可以达到2.714U/mL。接下来对Tolypocladium cylindrosporum syzx4发酵生产的β-葡萄糖苷酶进行纯化、性质分析、糖化和同步糖化发酵气爆玉米秸秆的应用研究。酶的纯化过程采用的是硫酸铵沉淀法,DEAE-52纤维素离子交换法和Sephadex G-100凝胶层析法。纯化后相对于纯化前的粗酶液活力提高了9.47倍,回收率为12.27%,得到的酶活力为40.50U/mL。SDS-PAGE结果显示为分子量为58.6 kDa的一条带,表明达到了电泳纯,Native-PAGE的结果表明此β-葡萄糖苷酶表现出良好的生物活性。酶学性质分析以β-葡萄糖苷酶对常用的三种水解底物为对象即纤维二糖,水杨素和p-NPG,研究了酶的水解性质,水杨素为底物时Km为8.8 mmoL/L Vmax为25.6 mmoL/s和Kcat为53.89 s-1；以纤维二糖为底物时得到Km为2.59mmoL/L、Vmax为45.3 mmoL/s和Kcat为95.37s-1；对硝基苯-β-D葡萄糖苷(p-NPG)为底物时得到Km为0.85 mmoL/Lmax为85.23 mmoL/s和Kcat为179.43s-1；以p-NPG为底物时,酶对葡萄糖的抑制常数Ki为3.95 mmoL/L,对葡萄糖醛酸内酯的抑制常数Ki为13.29μmol/L,β-葡萄糖苷水解酶具有较强的耐受葡萄糖和葡萄糖醛酸内酯的的性能,温度和pH值对酶活性的影响分析表明广泛的温度适应性在35到70℃反应时酶活性保留了85%以上；该酶耐酸性很强,在pH值为3.0时活力比标准测定方法提高了20%,在酸性条件下比中性条件下稳定。最后,研究了此β-葡萄糖苷酶和其它商业化的纤维素酶复配对气爆秸秆糖化和同步糖化发酵方面的应用,经过单因子方法设计和统计学分析优化,确定糖化条件为：基质浓度为3.05%、糖化pH值为3.73、糖化温度为43.38℃和酶配比中β-葡萄糖苷酶和纤维素酶的比例为0.91时,糖化率达到88.41%；同步糖化发酵结果证明,添加Novo-188商业化β-葡萄糖苷酶的乙醇产量比不添加β-葡萄糖苷酶的提高了50.4%,比添加同等酶活力的商业化Novo-188β-葡萄糖苷酶的乙醇产量提高了32.9%。添加Syzx4苷酶比不添加苷酶乙醇产量提高了一倍。添加Syzx4生产乙醇的速率方面也明显的优于其它两种反应体系。48h乙醇产量已经接近了17.5g/L,120h连续发酵乙醇产量可以达到23.8g/L。本研究结果表明无论是在气爆秸秆的糖化过程还是在同步糖化发酵过程中,此β-葡萄糖苷酶都表现出优异的性能,具有广阔的应用前景,证实了从腐败玉米秸秆中分离得到的Tolypocladium cylindrosporum syzx4菌株为开发新型β-葡萄糖苷酶制剂提供了应用基础和理论研究。更多还原

【Abstract】 A thermoacidophilic P-glucosidase from a newly isolated strain Tolypocladium cylindrosporum syzx4 was purified, characterized and applied to saccharify the corn stover. The enzyme was purified to homogeneity by sulfate precipitataion, diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and 12.27 % recovery. The Mw of the P-glucosidase was 58.6 kDa. Then the following physicochemical and kinetic parameters of theβ-glucosidase were studied respectively:Km, Vmax, Kcat with high affinity p-NPG as the substrate, Ki with the tolerance of glucose and Gluconolactone. Theβ-glucosidase showed highly activity over a wide range of temperature from 35 to 70℃and it was more stable at acidic than neutral pH. The P-glucosidase was activated by Zn2+, Cu2+and Mn2+, while, Ag+and Hg2+inhibited it. The unique enzymatic properties make theβ-glucosidase more effective in the saccharification of steam explosion pretreated corn stover than that from Aspergillus sp. (Novo-188).1. Production ofβ-glucosidase by a novel isolated strain Tolypocladium cylindrosporum syzx4 and optimization of fermentation mediumA novel fungus, named T. cylindrosporum syzx4, which can efficiently produce extracellularβ-glucosidase, was isolated from naturally rotten corn stover. It is first time to report theβ-glucosidase produced by T. cylindrosporum gams using agro-industrial residues in SmF. The fermentation variables optimized by Single-factor experiment approach were further optimized by statistical optimization. Results of Plackett-Burman design indicated the evaluation of the medium components could be ranked as:TCS> KH2PO4> SM= (NH4)2SO4 With the optimum medium with viz. TCS 26.274 g/L, SM 7.066 g/L, KH2PO4 1.991 g/L, (NH4)2SO4 2.328 g/L predicted by GA-ANN model with others as the original,2.662U/mLβ-glucosidase activity was obtained. With the optimal condition,2.714U/mL P-glucosidase activity was obtaind. Results suggest that the P-glucosidase can be used for various biotechnological applications.2. Purification ofβ-glucosidase from T. cylindrosporumAll purification processes were conducted at room temperature unless otherwise specified. A clean supernatant with theβ-glucosidase activity of 3300 U was adjusted to 80% saturation by adding solid ammonium sulfate over night. Precipitate was collected by centrifugation at 10000xg for 30min at 4℃and dialyzed against the citrate buffer. Insoluble precipitates were removed by centrifugation, and the supernatant was used for further purification. This step resulted in a 70.3% recovery and 1.38-fold purification. The enzyme was loaded onto a DEAE cellulose column equilibrated with citrate buffer. The elution result of the DEAE cellulose column with a linear gradient of 0-1 moL/L sodium chloride. Two peaks were obtained with one of them showing the P-glucosidase activity. Activity fractions were pooled, concentrated with PEG4000 and dialyzed against citrate buffer. The result showed a total activity of 1488 U,45.1% recovery,5.33-fold purification with the specific activity 19.8 U/mg. The disposed fractions with P-glucosidase activity were then chromatographed on a Sephadex G-100 column pre-equilibrated with citrate buffer. Elution was done with the same buffer, and fractions withβ-glucosidase activity were pooled, dialyzed and concentrated. Theβ-glucosidase ctivity revealed as a single peak through elution. The results showed 12.3 % recovery,9.47-fold purification, a total activity of 405 U and the specificβ-glucosidase activity reached 35.3 U/mg.3. Characterization of the purified P-glucosidase from T. cylindrosporumThe purified P-glucosidase preparation was examined by SDS-PAGE and Native-PAGE. In SDS-PAGE examination, the purified P-glucosidase was represented by a single band of Mw 58.6 kDa strained with Coomassi blue. The result of Native-PAGE showed that the enzyme reversed the activity of dark halo.The optimum temperature of the purified enzyme was found to be 60℃, and the purified P-glucosidase activity left 81% and 90% even if it was incubated at 35℃and 70℃, respectively. It is first time for us to report the P-glucosidase with wide temperature adaptability (35-70℃). The purified P-glucosidase had the potential to widely use in various biotechnological applications. Moreover, its maximal activity is at 60℃, which is higer than known P-glucosidases in microorganism in previous reported. The thermal stability results were showed at pH 4.5 incubation in different temperatures. The results indicate that the enzyme with half life about 50 min at 70℃was much more stable than any other (3-glucosidases reported. The enzyme is not stable above 80℃and only 20% activity was reserved for 20min.The purifiedβ-glucosidase was incubated for various times in buffer with different pH (from 2.0 to 9.0) for 30 min at 37℃. The purified P-glucosidase had an optimum pH around 2.4. The P-glucosidase activity increased markedly with the pH from 1.8 to 2.4, however, it decreased observably while pHs above 2.6. The optimum pH of purifiedβ-glucosidase is lower than that of reported. Interesting, the optimum pH is according to the sulfuric acid for the pretreatment cellulose and steam explosion pretreated cellulose. In addtion, according to the pH stability data, theβ-glucosidase activity retained more than 80 % after being incubated for 12 h at different pH ranged from 3 to 6. The result indicated that P-glucosidase was more stable at acidic pH than neutral pH. The P-glucosidase from T. cylindrosporum, therefore, exhibits a number of highly appealing and promising features, which make it a strong candidate for various biotechnological applications and industrial processes.The effects of various mental ions and chemical reagents (10 mmol/L) on enzyme activity were examined on p-NPG at pH 4.5 and 37℃. No significant effect was examined in the presence of Mg2+, Ca2+, K+, Fe2+and Ba2+. The P-glucosidase was activated by Zn2+, Cu2+and Mn2+; on the other hand, Ag+and Hg2+had a strong inhibitory effect on the enzyme activity. The P-glucosidase was resistant to 10 mmmol/L SDS with all its activity. However, EDTA, a chelating reagent, and DTT, a disulfide-reducing reagent, were observed to inhibit the enzyme activity, which suggested that the moderate mental ions and the disulfide bonds are essential to maintain the P-glucosidase activity.Kinetic parameters of the P-glucosidase for p-NPG, salicin or cellobiose were estimated at pH 4.5 and 37℃, respectively.The Km indicated that theβ-glucosidase had greatest affinity towards p-NPG than others. And the Vmax for p-NPG was much higher than salicin and cellobuose. It may be caused by the structural difference of substrate, as p-NPG and salicin are synthetic aryl P-glucosidase, and cellobiose is the natural substrate. In addition, the Km of salicin is about ten times than that of p-NPG, the reason of which may due to the structural difference of the two arylβ-glucosidases. The Vmax(85.23 mmoL/s) was much higher and the Km (0.85mmol/L) were much lower than P-glucosidase (Novo-188) from A. niger and others when p-NPG was used as a substrate. The effects of glucose (0-100 mmol/L) on p-NPG hydrolyzing activity of theβ-glucosidase were determinated. The inhibition pattern was of the competitive type which was accorded to previous reportes. The hydrolyzing activity decreased markedly with the concentration increasing of glucose. It had an obviously inhibitory effect on p-NPG hydrolyzing activity while the concentration of glucose was above 25.0 mmol/L.Furthermore, the inhibition constant (Ki) of theβ-glucosidase for glucose was 39.5 mM with p-NPG as a substrate and Ki of Novo-188 with the same condition was 3.0 mM. The Ki of glucose to the purifiedβ-glucosidase was much higher than others reported in literates. The P-glucosidase with the higher concentration tolerance of glucose was very useful for saccharification of lignocellulose.The effects of gluconolactone (0-200μmol/L) on p-NPG hydrolyzing activity of theβ-glucosidase were determinated. The inhibition pattern was of the competitive type which was accorded to previous reportes. The hydrolyzing activity decreased markedly with the concentration increasing of glucose. It had an obviously inhibitory effect on p-NPG hydrolyzing activity while the concentration of glucose was above 40μmol/L. The inhibition constant (Ki) of theβ-glucosidase for gluconolactone was 13.29μmol/L.4. Application of P-glucosidase from T. cylindrosporumAfter purification and characterization the P-glucosidase, it was applied to saccharify steam explosion pretreated corn stover(SCS) with commercial Celluclast from Trichoderma reesei comparison with theβ-glucosidase(Novo-188) from Aspergillus niger supplementation. Based on the results of single factor tests, further studies with Response Surface Methodology(RSM) with a five level four-factor Central Composite Design(CCD) were used to optimal the hydrolysis parameters such as the concentration of substrate, pH, temperature, the ratio of Celluclast to Syzx4 to achieve the highest saccharification yield. The concentration of substrate and the ratio of Celluclast to Syzx4 were identified as the limiting factor for the saccharification yield. A maximum saccharification yield of 88.4 % was obtained at an the optimal hydrolysis condition as follows:the concentration of substrate 3.05 %, pH 3.73, temperature 43.4℃and the ratio of Syzx4 to Celluclast 0.91(18.2 BGU/g substrate): 1 (20 FPU/g substrate). The results of a confirmation experiment under the optimum conditions agreed well with model predictions and got the maximum value of 88.41 % saccharification yield. The results suggested that theβ-glucosidase(Syzx4) from T. cylindrosporum is a good supplementation for the production of reducing sugars from cellulosic biomass.In SSF of SCS, three kinds of cellulase mixtures were added to the reaction mixture, respectively,1:Cellulast 20U/g substrate; 2:Cellulast 20U/g substrate and Novo-188 18 U/g substrate; 3:Cellulast 20U/g substrate and Syzx4 18 U/g substrate. The extra 50.4% enthal yield was obtained compared Cellulast 20U/g with the Cellulast 20U/g and Novo-188 18 U/g. The enthal yield of Cellulast 20U/g and Syzx4 18 U/g was 32.9% higher than Cellulast 20U/g and Novo-188. The result indicated that theβ-glucosidase (Syzx4) from T. cylindrosporum was more effective than Novo-188 in the SSF process. After 48h formenation, the ethanol concentration was 17.5%. With the help of the high concentration tolerance of glucose forβ-glucosidase from T. cylindrosporum and other excellent enzymatic parametics make the P-glucosidase (Syzx4) potential application in the saccharification of steam explosion pretreated corn stover for ethanol production.更多还原