一步法发酵菊芋产乙醇菌种的筛选及产酶条件、酶学性质的研究

从腐烂的菊芋及实验室保存的菌种中,选育到一株发酵菊芋产乙醇的菌株克鲁维酵母Kluyveromyces marxianus Y1。利用正交实验法对克鲁维酵母产菊粉酶的培养基组成及培养条件进行优化,确定培养基组成（g/L）为：菊粉40,酵母粉4,蛋白胨4,尿素1;初始pH5．0,温度30℃,150r／min条件下培养达到最佳产酶效果（57U／mL）。该菌株所产菊粉酶的性质测定结果表明：以菊粉为底物,该菊粉酶最适反应温度为55℃,在60℃以下稳定性很好,高于60℃时酶迅速失活;最适pH为5．0,pH4．6—5．2范围内酶稳定性很好;该酶属于外切型菊粉酶,体积分数为8％的乙醇对酶活力基本没有影响。     

一个外切菊粉酶基因的克隆表达及酶学性质

从马克斯克鲁维酵母（Kluyveromycesmarxianus）DSM5418中克隆出外切菊粉酶（INU）的成熟肽编码区域,在毕赤酵母（Pichiapastoris）GS115中实现了高效分泌表达,体积酶活力达到15．27U／mL,进一步对重组酶进行了纯化与表征。经过（NH4）2SO4沉淀、透析和分子筛过滤后,得到了纯度大于95％的纯化重组酶,SDS-PAGE分析发现INU的表观相对分子质量为9．0×10^4,大于理论预测值6．0×10^4。纯化酶液的表征结果表明,INU的最适温度和最适pH分别为55℃和5．0,在此条件下INU对菊粉的K。值和比酶活分别为1．90mmol／L和433．86U／mg,对蔗糖的K。值和比酶活分别为27．81mmol/L和1249．49U／mg,I/S值为0．34;HPLC分析表明,INU酶解菊粉的产物由果糖和葡萄糖组成;金属离子Mn2＋、Fe3｜、K｜和Co2＋对酶有促进作用,而Zn2＋、Cu2＋、Ni2＋、SDS和EDTA对酶活力有不同程度的抑制作用。     

4株海洋酵母菌胞外酶活力及其对水产养殖有机污染物的降解

研究利用平板鉴别培养基法观察比较4株海洋酵母菌生淀粉酶、蛋白酶、脂肪酶和纤维素酶等胞外水解酶的能力,并进一步分析测定其酶活性。结果表明, 圆丘假丝酵母菌 (Candida.colliculosa)DY11-1能产淀粉酶、蛋白酶和脂肪酶, 其酶活力分别为181.4 U/mL、123.1 U/mL和36.7 U/mL;毕赤酵母(Pichia sp)DY11-6和近平滑假丝酵母(Candida parapsilosis)DY07-1都能产生淀粉酶和蛋白酶, 它们淀粉酶的活力分别为108.1 U/mL和96.5 U/mL, 蛋白酶的活力分别是35.1 U/mL和128.4 U/mL;粘红酵母(Rhodotorula glutinis)DY02-3四种胞外酶皆无。以罗非鱼(Tilapia mossambica)饲料浸出液作为养殖有机污染物, 分析比较4株酵母菌对其的降解率。结果显示, 圆丘假丝酵母DY-11-1菌株对饲料浸出液的COD、总氮、总磷降解能力最高, 9 d后分别由27.60 mg/mL、12.22 mg/mL和2.67 mg/mL下降到15.73 mg/mL、7.59 mg/mL和1.40 mg/mL, 降解率分别为43.0%、37.8%和47.5%。表明DY11-1菌株对养殖有机污染物有较强的分解作用, 在改善养殖水质方面具潜在的应用价值。     

马克斯克鲁维酵母菌菊粉酶的异源表达及低聚果糖制备工艺优化

【目的】低聚果糖是新型的食品和保健品原料,具有广阔的市场需求。以菊粉酶水解菊粉制备低聚果糖的酶法工艺是先进的绿色制造。本研究旨在获得高产的菊粉酶菌株及以菊粉为原料酶法制备低聚果糖的优化工艺。【方法】采用基因工程手段克隆马克斯克鲁维酵母菌(Kluyveromyces marxianus)的菊粉酶基因,实现其在毕赤酵母中的高效表达;测定菊粉酶在不同p H、温度、金属离子和底物浓度等条件下的酶活变化趋势,获得最佳的反应参数;通过高效液相色谱法检测水解产物,获得不同酶量水解产物各组分分布。【结果】菊粉酶工程菌株在10 L发酵罐中的产菊粉酶活达1 570 U/m L、蛋白质含量为2.75 g/L发酵液;菊粉酶最适反应参数为:在体积为1 L的反应体系中,p H 5.0、反应温度50°C、含0.2 mmol/L Mg2+以及菊粉浓度为8%。在该条件下,酶量为10 U时菊粉被完全水解。水解产物中单糖和二糖含量仅为9.25%,而低聚果糖(C3-C8)含量为90.75%,且C3-C5低聚果糖含量高达72.92%。【结论】克隆了K.marxianus菊粉酶基因并实现了高效表达,获得了水解菊粉制备低聚果糖的最佳工艺条件。为菊粉酶的大量生产及低聚果糖的酶法制备奠定了良好的基础。     

黑曲霉生产菊粉酶工艺条件的研究

从徐州市沛县河口镇秦庄村牛蒡种植基地取得的土壤样本中,筛选出产菊粉酶的菌株,对从土壤中分离到的40株产菊粉酶的各类微生物进行酶活的测定。通过透明圈法初筛及摇瓶复筛,获得产菊粉酶能力较高的霉菌菌株3株,为C122803、D081506和D081513,这3株菌株的酶活分别达到：C122803：1.411 U/ml;D081506 ：1.895U/ml;D081513 ：1.792U/ml。其中D081506的酶活最高,为1.895U/ml;对D081506号黑曲霉产菊粉酶的发酵条件进行了研究,确定了优化的发酵条件为：牛蒡汁2%,酵母膏1.6%,(NH4)2SO41.6%,NaCl 0.5%,K2HPO4 0.5%,pH 5.0,在27℃、140 r/min条件下,摇瓶培养24h, D081506酶活力为2.958U/ml,酶活力提高56.09%。     

内切酶法制取菊苣低聚果糖工艺条件的研究

目的:利用毕赤嗜甲醇酵母表达的重组黑曲霉菊粉内切酶,从菊苣菊粉中制取低聚果糖。方法:用重组黑曲霉菊粉内切酶酶解水提菊粉,用薄层色谱、离子色谱分析酶解产物,研究酶解条件,分析产物组成。结果与结论:菊粉浓度为20～160g/L、酶浓度为12U/g菊粉时,低聚果糖的最大产率逐渐升高然后下降;在接近最大溶解度200g/L时,菊粉寡糖最大产率为62.0%,最大产率与酶的用量无关,但提高酶的浓度可以缩短达到最大产率的时间。pH5.5、50℃、菊粉浓度为40g/L、酶用量为12U/g菊粉时,可达到最大产率64.6%;酶解的产物主要为GF2到GF10的菊粉寡糖。     

黑曲霉产菊粉酶菌株的选育及培养基优化

为获得高产菊粉酶的黑曲霉菌株,以Aspergillus niger YH-1为出发菌株,经过亚硝基胍(NTG)诱变,以高温高菊芋粉相结合的方式进行梯度驯化,选育出一株产菊粉酶菌株YH-3,并运用响应面实验方法对该菌株的培养基进行优化。确定了最佳培养基组成:菊芋粉25.2 g/L、豆饼粉40 g/L、蔗糖酯4.9 g/L、NaCl 5.5 g/L。发现内切菊粉酶活力(I)由60.9 U/mL提高到165.0 U/mL,比出发菌株提高了1.7倍。研究证明蔗糖酯对于黑曲霉YH-3发酵产菊粉酶是一种有效的促进剂。     

角毛壳菌几丁质酶基因chi58多点突变及在毕赤酵母中的高效表达

应用重叠延伸PCR技术(gene splicing by overlap extension PCR,gene SOEing),简称SOE-PCR对角毛壳菌(Chaetomium cupreum)的几丁质酶基因chi58进行多点突变.依据毕赤酵母密码子偏爱性,将毕赤酵母中编码Arg使用频率几乎为0的密码子CGC突变为使用频率高的AGA,构建了含有正确突变的酵母表达载体pPIC9K-chi58A,电转化毕赤酵母GS115,获得的重组酵母株在诱导120 h酶活力最高,平均可达101.71 U/mL±3.33 U/mL;其活力比未优化重组酵母株(31.83 U/mL±4.85 U/mL)提高了约3倍,且经10代传代培养后遗传稳定性良好.表达产物的SDS-PAGE分析表明,酶蛋白分子大小为58 kD.     

来源于软化芽孢杆菌的环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-CGT酶基因,将基因片段分别克隆到毕赤酵母表达载体pPIC9K和大肠杆菌-枯草杆菌穿梭载体pMA5中,分别转化毕赤酵母KM71和枯草杆菌WB600。结果表明,重组毕赤酵母发酵上清液中α-CGT酶活性仅0.2U/mL,重组枯草杆菌产酶达到1.9U/mL。对重组枯草杆菌发酵条件进行了优化,当以TB为出发培养基,初始pH6.5,温度为37oC时,摇瓶培养24h后α-CGT酶环化活性达到4.5U/mL(水解活性为3200IU/mL),是野生菌株软化芽孢杆菌表达量的9.8倍。     

黑曲霉(Aspergillus niger)产菊粉酶菌株的筛选及培养条件的研究

Ak3个地区7个土壤样品中筛选高产菊粉酶的黑曲霉菌株,对筛选出的黑曲霉菌株进行了形态鉴定,探讨了温度、pH值对菊粉酶活力的影响,比较了固体和液体两种扩大培养法对黑曲霉菌株产菊粉酶的影响。结果表明：经过初筛和复筛得到A4、A6、A8、A12、At4、A15和A16共7株黑曲霉菌株,菊粉酶活力大于15．0U／mL,经数码生物摄影显微镜镜检符合黑曲霉的形态特征。A8菌株的菊粉酶酶活最高,达到25．0U／mL,在60℃、pH值5．0的条件下酶活性最大。     

菊粉酶产生菌的筛选及60Co诱变选育简

目的：从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法：通过稀释平板涂布法分离微生物;利用^60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果：分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行^60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46．62U/mL,是未诱变菌株酶活的2．72倍。结论：经诱变得到1株高产菊粉酶活力的突变菌株。     

菊粉酶基因在酿酒酵母中的表达及乙醇发酵

以乙醇耐受力较强的酿酒酵母为受体菌,构建了能够分泌菊粉酶的基因工程菌并进行了菊芋粉的生料发酵。首先,以马克斯克鲁维酵母Kluyveromyces marxianus中的基因组DNA为模板,PCR扩增菊粉酶编码基因inu,分别使用菊粉酶自身启动子和酵母磷酸甘油激酶 (Phosphoglycerate kinase,pgk) 启动子,构建重组表达质粒HO/p-inu和HO/pgk-inu。经NotⅠ线性化后,采用电击法转化酿酒酵母工业菌株Saccharomyces cerevisiae 6525,分别得到含菊     

Improved ethanol production in Jerusalem artichoke tubers by overexpression of inulinase gene in Kluyveromyces marxianus

Ethanol production from Jerusalem artichoke tubers through a consolidated bioprocessing (CBP) strategy using the inulinase-producing yeast Kluyveromyces marxianus is an economical and competitive than that from a grainbased feedstock. However, poor inulinase production under ethanol fermentation conditions significantly prolongs the fermentation time and compromises ethanol productivity. Improvement of inulinase activity appears to be promising for increasing ethanol production from Jerusalem artichoke tubers by CBP. In the present study, expression of the inulinase gene INU with its own promoter in K. marxianus (K/INU2) was explored using the integrative cassette. Overexpression of INU was explored using chromosome integration via the HO locus of the yeast. Inulinase activity and ethanol were determined from inulin and Jerusalem artichoke tubers under fed-batch operation. Inulinase activity was 114.9 U/mL under aerobic conditions for K/INU2, compared with 52.3 U/mL produced by the wild type strain. Importantly, inulinase production was enhanced in K/INU2 under ethanol fermentation conditions. When using 230 g/L inulin and 220 g/L Jerusalem artichoke tubers as substrates, inulinase activities of 3.7 and 6.8 U/mL, respectively, were measured using K/INU2, comparing favorably with 2.4 and 3.1 U/mL, respectively, using the wide type strain. Ethanol concentration and productivity for inulin were improved by the recombinant yeast to 96.2 g/L and 1.34 g/L/h, respectively, vs 93.7 g/L and 1.12 g/L/h, respectively, by the wild type strain. Ethanol concentration and productivity improvements for Jerusalem artichoke tubers were 69 g/L and 1.44 g/L/h, respectively, from the recombinant strain vs 62 g/L and 1.29 g/L/h, respectively, from the wild type strain.     

Enhanced expression of the codon-optimized exo-inulinase gene from the yeast Meyerozyma guilliermondii in Saccharomyces sp. W0 and bioethanol production from inulin

In the present study, after the exo-inulinase gene INU1 from Meyerozyma guilliermondii was optimized according to the codon usage bias of Saccharomyces cerevisiae, both the optimized gene INU1Y and the native gene INU1 were ligated into the homologous integration expression vector pMIRSC11 and expressed in Saccharomyces sp. W0. It was determined that the inulinase activity of the recombinant yeast Y13 with the optimized gene INU1Y was 43.84 U/mL, which was obviously higher than that (31.39 U/mL) produced by the recombinant yeast EX3 with the native gene INU1. Moreover, it was indicated that the recombinant yeast Y13 could produce 126.30 mg/mL ethanol from 300.0 g/L inulin while the recombinant yeast EX3 and Saccharomyces sp. W0 produced 122.75 mg/mL and 114.15 mg/mL ethanol, respectively, under the same conditions. In addition, the ethanol productivity of the recombinant yeast Y13 was 2.25 mg/mL/h within 48 h of the fermentation, which was obviously higher than that of the recombinant yeast EX3 (1.97 mg/mL/h) and Saccharomyces sp. W0 (1.77 mg/mL/h) within the same period. The results demonstrated that the recombinant yeast Y13 had higher ethanol production and productivity than the recombinant yeast EX3 and Saccharomyces sp. W0. Therefore, it was concluded that the codon optimization of the exo-inulinase gene from M. guilliermondii effectively enhanced inulinase activity and improved ethanol production from inulin by Saccharomyces sp. W0 carrying the optimized inulinase gene.     

Enhanced inulinase production in solid state fermentation by a mutant of the marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> using surface response methodology and inulin hydrolysis

In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl₂. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.     

Partial purification and characterization of exoinulinase produced from Bacillus sp.

Inulinase are industrial food enzymes which have gained much attention in recent scenario. In this study, Inulinase producing eight bacterial colonies were isolated and screened from three different plant root tubers soil sample. Among 8 inulinase producing colonies, the higher yielding colony was selected with 25.10?U/mL for further studies. The best inulinase producing colony was identified by partial 16S rRNA gene sequence as Bacillus sp. The crude inulinase was purified by using ammonium sulphate precipitation, dialysis and ion exchange chromatography on DEAE – sephacel and obtained 1.9 purification fold with total activity 293 U. The purified enzyme was subjected to characterization studies and it was found to be stable at 30–60?°C and optimum temperature was at 55?°C. The enzyme was stable at pH 3.0–7.0 and optimum pH was at 6.5. The K_m and V_max value for inulinase was found to be 0.117?mg/mL and 4.45?μmol?min?mg^?1 respectively, demonstrate its greater affinity. Hence, this enzyme can be widely used for the production of fructose, and fructooligosaccharides, which are important ingredients in food and pharmaceutical industry.     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

Molecular and biochemical characterization of a new endoinulinase producing bacterial strain of Bacillus safensis AS-08

Microbial inulinases are an important class of industrial enzymes, which are used for the production of fructooligosaccharides and high-fructose syrup. Endoinulinase producing bacterial strains were isolated from soil samples taken from the vicinity of Asparagus sp. root tubers. All the bacterial strains were screened for inulinase activity. The primary screening was carried out based on hydrolytic zone on agar plates containing inulin-based medium and Lugol’s iodine solution. Thus 30 inulinase producing bacterial strains were isolated. Out of 30 strains, 5 bacterial strains were found endoinulolytic, whereas 25 were exoinulolytic on the basis of action pattern of the enzyme. In tertiary screening, the bacterial isolate AS-08 was found to be most efficient for inulinase activity. Morphological, biochemical and physiological characteristics of the bacterial isolate AS-08 confirmed it as Bacillus sp. Furthermore, species-specific identification by 16S rDNA sequencing and phylogenetic analysis revealed the isolate as Bacillus safensis. Bacillus pumilus SH-B30 was found to be the nearest homolog. The strain showed maximum inulinase activity (12.56 U/mL) after 20 h of incubation at 37°C.     

生淀粉糖化酶高产菌的选育

从土壤及霉变淀粉质等样品中分离出对生淀粉具有降解作用的菌种约 6 0株 ,其中生淀粉糖化酶最高的一株根霉OR-1 ,其酶活为 90U/mL ,通过一次紫外和亚硝基胍诱变 ,酶活分别达到 200U/mL、325U/mL ,RDA值分别为 70 %、 65%。诱变株是一株产生淀粉糖化酶较高的根霉。