一株产耐高温蛋白酶蜡样芽胞杆菌的分子鉴定和酶学性质研究

为筛选具有工业应用价值的产蛋白酶新菌种,本研究采用酪素培养基进行新菌株筛选,利用生理生化试验和16S rDNA测序进行菌种鉴定,并结合生化方法测定其蛋白酶的酶活。结果表明,本研究筛选的一株产蛋白酶菌株HSU-2 (GenBank登录号:MN315516)为蜡样芽胞杆菌(Bacillus cereus)。该菌种产蛋白酶的最适pH值和温度分别是7.0和60℃,为一种耐高温的中性蛋白酶。该蛋白酶可耐受5.0%过氧化氢、5.0%十二烷基磺酸钠和5.0%去污剂Triton X-100作用;且5.0%去污剂吐温80可显著提高该蛋白酶的酶活力。此外,Mg²⁺、Zn²⁺、K⁺、Ni²⁺和Cu²⁺ 5种金属离子可显著降低该蛋白酶酶活力,其中Zn²⁺抑制作用最强;而Ca²⁺可提高该蛋白酶酶活力。本研究为产蛋白酶菌株HSU-2的应用开发和大规模生产奠定了理论基础。     

一株产高温蛋白酶耐热菌BY25的产酶条件与酶学性质研究

对菌株BY25的生长条件、产酶条件及其产生的蛋白酶的酶学性质进行了研究。结果发现,BY25的最高生长温度为55℃,最适生长温度为50℃,最佳产酶温度为30℃,最佳培养起始pH为8.0,最佳C源为葡萄糖,高通气量明显提高菌株产酶能力。在以上条件下培养52 h,上清液蛋白酶活力达4 170 U/ml。酶学性质研究表明：该蛋白酶为高温中性金属蛋白酶,最适反应pH为7.0,最适反应温度为55℃,具有良好的pH耐受性和较好的热稳定性;EDTA能强烈抑制酶活力,而Fe2+、Cu2+对酶活力也具有一定抑制作用。     

纤维素降解真菌A25-2的分离、鉴定及其产纤维素酶的酶学特性

本研究以Avicel-刚果红选择培养基为初筛培养基,从云南哀牢山国家级自然保护区和广西猫儿山国家级自然保护区的土壤样品中分离筛选得到4200株真菌,从中筛选出透明圈与菌落直径比较大、透明程度较为清晰的12个菌株。通过液体培养发酵,测定其上清液中的羧甲基纤维素酶活力、滤纸酶活力和Avicel酶活力,最终筛选出一株产该三种酶且其活力均最高的真菌菌株A25-2。通过对菌株A25-2形态学观察和其内转录间隔区（internal transcribed spacer,ITS）序列同源性比对分析,将菌株A25-2鉴定为哈茨木霉（Hypocrea lixii）。酶活测定结果表明菌株A25-2产纤维素酶的酶活力较高,在最适作用pH4.5和最适作用温度55℃下,其羧甲基纤维素酶活力为2.26IU/mL,滤纸酶活力为0.58IU/mL,Avicel酶活力为0.39IU/mL。薄层层析实验表明A25-2具有完整的纤维素酶系统。因此,真菌A25-2可作为饲料加工等生产和纤维素酶相关研究的备选菌株。     

纤维素降解菌CMC-4的分离鉴定、诱变和酶学特性研究

从秸秆还田土壤中初筛获得一株高效纤维素降解菌CMC-4,根据菌株形态、理化性质及16S rDNA序列分析,初步鉴定为地衣芽孢杆菌(Bacillus licheniformis)。以此为出发菌株,经亚硝酸钠诱变获得一株稳定高产纤维素酶突变株CMC-4-3。对CMC-4和CMC-4-3的产酶条件和酶学性质进行比较分析,结果表明:CMC-4-3纤维素酶活力较CMC-4提高67.5%;其最适产酶条件是:37℃、pH 6.0、葡萄糖为碳源、蛋白胨为氮源、接种量2.0%、装液量60 ml/250 ml。该菌株所产纤维素酶的最适反应pH为6.0,且在4.0~7.0酶活力较稳定;最适反应温度为50℃,在20~80℃范围内均保持稳定;金属离子Fe~(2+)、Mg~(2+)、Co~(2+)、K~+对酶有激活作用,其余离子均有不同程度抑制作用,而Cu~(2+)和Ca~(2+)抑制作用最强,酶活力减弱近50%,是该酶的强效抑制因子。诱变前后菌株产酶条件和酶学性质等部分表型发生了变化,而突变菌株显示出了更宽泛的环境适应范围。据此,获得一株高效产纤维素酶、耐受范围广的具纤维素降解能力的地衣芽孢杆菌,而CMC-4-3和CMC-4的表型可作为深入探讨基因型变化的线索。     

黑胸散白蚁体内产纤维素酶细菌的筛选及其纤维素酶基因的鉴定与原核表达

白蚁(Isoptera)常以富含纤维素的木材为食,体内存在纤维素酶产生菌。从源于陕西佛坪的白蚁(经鉴定为黑胸散白蚁(Reticulitermes chinensis))体内筛选出了4株产纤维素酶菌株,采用单因素实验设计对酶活最高的菌株进行了产酶条件优化,并对其所产纤维素酶进行了酶学性质研究;同时,依据NCBI数据库设计的引物扩增出了该菌株的内切-β-1,4-葡聚糖酶基因Cbs和β-葡萄糖苷酶基因Ba G,之后在大肠杆菌(Escherichia coli)中进行了表达实验。结果表明:从黑胸散白蚁体内筛选出4株菌分别是阿氏肠杆菌(Enterobacter asburiae)、炭疽芽胞杆菌(Bacillus cereus)、枯草芽胞杆菌(Bacillus subtilis)、解淀粉芽胞杆菌(Bacillus amyloliquefaciens),其中枯草芽胞杆菌所产纤维素酶的活力最高,该菌发酵产酶的最适温度和pH分别是42℃、6.5,酶促反应的最适温度和pH分别是70℃、4.5,在此条件下该菌株酶活力为2.36 U/mL。该菌株所产的纤维素酶在50℃、pH 6.5的条件下热稳定性和pH稳定性最佳,使用该菌株所产的粗酶液发酵粗饲料,结果发现其对玉米(Zea mays)秸秆、小麦(Triticum aestivum)秸秆、苜蓿(Medicago polymorpha)干草和青贮饲料的粗纤维降解率分别为12.21%、1.3%、3.24%和17.42%。基因克隆结果表明该菌株具有Ba G、Cbs 2个纤维素酶基因,且大肠杆菌表达结果显示Ba G基因的粗酶液pro Ba G无纤维素酶活性,而Cbs基因的粗酶液pro Cbs在60℃、pH 5.0时酶活可达4.14 U/mL,将Ba G和Cbs基因进行原核融合表达,产生的蛋白约35 k D,其在最适条件70℃、pH 4.5时的酶活为4.57 U/mL,说明无纤维素酶活性的Ba G基因与Cbs基因融合后可能表达出了一个活性更高的纤维素酶蛋白。本研究对后期构建可降解木质纤维素的人工重组菌具有重要的理论价值。     

秸秆纤维素降解真菌QSH3-3的筛选及其特性研究

为了获得高效降解秸秆纤维素的微生物菌株,采用滤纸降解法和刚果红染色法从含纤维素类物质的土壤中筛选到一株产纤维素酶菌株QSH3-3,通过形态观察和ITS序列分析,鉴定为草酸青霉Penicillium oxalicum QSH3-3。摇瓶产酶试验结果表明,该菌株的最佳产酶条件为:碳源为0.5%的碱处理过的玉米秸秆粉,氮源为0.2% 硫酸铵,起始pH为7,接种量为5%,产酶温度为30℃,培养时间为4 d。最佳产酶条件下,滤纸酶（FPase）、内切酶（CMCase）和木聚糖酶（Xylanase）分别为12 U、33 U、605 U（U为酶活性单位）;在15℃,其残余酶活力可达70%～80%;在pH 4～9 范围内,其残余酶活力可达70%以上。酶学稳定性研究表明,FPase、CMCase和Xylanase在pH 4～9范围残余酶活力达85%以上,具有较强的酸碱适应能力;FPase、CMCase和Xylanase在45℃以上酶活力迅速下降,耐热性较差。该菌株具有较高的木聚糖酶活力以及较强的低温、pH的耐受力,因而该菌株在田间温差大、土壤偏碱性等复杂条件下对秸秆纤维素类物质的降解具有较高的应用潜力。     

发酵鲭鱼中一株组胺降解菌的系统发育分析及特性研究

为筛选出具有优良组胺降解能力的细菌,并探索其在发酵食品中的应用,使用乳酸细菌(MRS)、结晶紫中性红胆盐葡萄糖琼脂(VRBDA)和2216E琼脂培养基对传统发酵鲭鱼(Scomber japonicus)中的组胺降解菌进行分离纯化,测定其生长、产酶特性及对发酵鲭鱼生物胺的降解作用。结果表明,菌株V-TB-2-5具有较高的降组胺活性,通过16S rRNA基因序列系统发育学分析结合形态特征确定为产氮假单胞菌(Pseudomonas azotoformans)。在营养肉汤培养基中,该菌的生长和产酶条件范围为20~40℃、pH值5~8、NaCl浓度0~3.5%(质量分数w/v),最适生长和产酶条件为30℃、pH值6、1.5%NaCl。酶反应的最适条件为30℃、pH值7。Mn²⁺、Mg²⁺和K⁺等金属离子对酶有激活作用,Fe³⁺或EDTA则有抑制作用。在传统发酵鲭鱼中,人工强化接种菌株V-TB-2-5与对照组相比组胺含量明显下降,组胺降解率为18.2%~40.7%;同时,接种组中亚精胺含量下降极显著,而精胺则上升。本研究为菌株V-TB-2-5在水产品加工和贮藏过程中组胺生物降解的控制提供了应用依据。     

三株产ACC脱氨酶的植物根际促生菌产酶条件优化及促生作用研究

为提高植物根际促生菌（PGPR）菌株产1-氨基环丙烷-1-羧酸（ACC）脱氨酶的能力,对3株PGPR菌株进行了最佳产酶条件优化及玉米促生试验。在单因素试验的基础上,研究了p H、温度、ACC底物浓度、Na Cl浓度、摇床转速对ACC脱氨酶酶比活力的影响,以p H、温度、ACC底物浓度为主要因素,进行响应面最佳产酶工艺优化。最终得到W5菌株的产酶条件为p H 8、温度32℃、ACC脱氨酶底物浓度6.0 mmol/L;BS2-2菌株的产酶条件为p H 10、温度30℃、ACC脱氨酶底物浓度6.0 mmol/L;5#菌株产酶条件为pH 9、温度30℃、ACC脱氨酶底物浓度6.0 mmol/L;较未优化前酶比活力分别提高了6.6、2、4倍。在盆栽试验中,W5菌株促生效果最为显著。利用高产ACC脱氨酶的PGPR能够促进玉米生长,可为制作为微生物复合菌剂奠定基础。     

啤酒用β-葡聚糖酶高产菌株的选育及发酵条件优化

对产β-葡聚糖酶的地衣芽孢杆菌(Bacillus licheniformis)A302菌株进行紫外线和硫酸二乙酯(DES)复合诱变和选育，获得产β-葡聚糖酶达27．3U／mL的突变株H302。采用均匀实验设计对H302菌株产β-葡聚糖酶的液体培养基组成及发酵条件进行优化实验，所获优化配方(W／V)为麦麸1．41％，鱼粉0．80％，硝酸钾0．34％，硫酸镁0．11％，起始pH6．0；用含孢量10^8个／mL的种子菌液按体积比3．3％接种上述液体培养基，在36℃下发酵52h，菌株H302的产酶活力达到115．1U／mL，是原出发菌株及培养条件下产酶活力的9．4倍。对突变株H302所产β-葡聚糖酶性质的研究表明，该酶最适作用温度为60℃，最适作用pH为5．5，适用于啤酒生产中的麦芽糖化。     

一株高活性纤维素降解细菌的筛选鉴定及酶学特性

利用小麦/玉米秸秆还田土壤样品,通过富集培养和刚果红平板染色法筛选分离出纤维素降解细菌XWS-12;对分离的菌株进行16S rRNA基因序列系统发育分析,初步鉴定为伯克氏菌属(Burkholderia),定名为Burkholderia sp.XWS-12。以玉米秸秆和麸皮为碳源,研究了氮源、发酵时间、初始发酵温度、培养基初始pH等条件对该伯克氏菌产纤维素酶的影响。结果显示,该菌株产纤维素酶最适氮源为硝酸钠,培养时间为60 h,培养温度为37℃,培养基初始pH为4,该菌株的CMC酶活力最高,可达25 U/ml。其粗酶液的最适反应温度为50℃,最适反应pH为5,在pH 4~8的范围内酶活力较稳定。粗酶液的热稳定较差,当温度超过50℃时,该酶活力显著下降;当温度为50℃时,保温1 h,该酶活力损失53%。     

BAE_2菌株淀粉酶某些特性的研究

嗜热菌ＢＡＥ２产生热稳定的淀粉酶，研究表明它的最适温度为８５℃，在ｐＨ５～９的范围内酶活力有很高的稳定性，在无添加剂存在的情况下，酶于８０℃稳定，通过添加乙二醇、甘油、甘露醇及聚乙二醇６０００，可使酶于９０℃非常稳定。     

产淀粉酶和蛋白酶中度嗜热细菌的分离、筛选及培养

嗜热菌广泛分布于地理热环境中，从温泉中分离出了中度嗜热的细菌，从中又筛选出了产淀粉酶的优良菌株ＡＥ２和产蛋白酶优良的菌株ＰＥ１，并测定了它们的生长与温度、ＰＨ值、ＮａＣｌ浓度及碳源的关系．将嗜热菌的培养方法进行了改进。     

海洋菌W11产中温淀粉酶的酶学特性

本研究从威海文登海域筛选获得一株产淀粉酶海洋菌W11,初步鉴定为弧菌,并探讨pH、温度、无机离子对淀粉酶活性和稳定性的影响及该酶底物浓度效应和Km值。结果表明：在pH7.5左右酶活性最高,pH在4.0～7.5范围内体现较强的稳定性。最适酶解温度为55℃,酶液在60℃以下有较好的热稳定性;Ba2＋、Mn2＋对淀粉酶有激活作用,而Cu2＋、Mg2＋、Zn2＋则抑制淀粉酶活性,表观Km值为0.973mg/mL。海洋菌W11所产的中温淀粉酶保存温度范围较广、适应pH作用的范围广及稳定性较强,将有着广泛的应用潜力。     

人工老化处理对啤酒大麦籽粒淀粉酶和麦芽品质的影响

为探究老化对啤酒大麦籽粒淀粉酶和麦芽品质的影响,采用高温高湿的人工老化方法处理大麦籽粒,以未经老化处理的大麦籽粒为对照,研究经老化处理后啤酒大麦籽粒萌发早期阶段中淀粉酶活性及主要麦芽品质性状的变化。结果表明,老化使α-淀粉酶、β-淀粉酶和极限糊精酶活性均呈降低的趋势,其中老化15 d后的淀粉酶活性与对照相比差异显著。随着老化时间的延长,各品种麦芽的无水浸出物、α-氨基氮、库尔巴哈值和糖化力均呈降低的趋势,粘度均呈增加的趋势。α-淀粉酶活性、β-淀粉酶活性、极限糊精酶活性均与α-氨基氮、无水浸出物、库尔巴哈值和糖化力呈显著正相关,但与粘度呈显著负相关。综上,人工老化会引起啤酒大麦籽粒萌发早期淀粉酶活性降低,麦芽品质变劣。本研究结果为啤酒生产过程中优质原料的选择提供了理论参考。     

Molecular Size Distribution and Amylase Resistance of Maize Starch Nanoparticles Prepared by Acid Hydrolysis

The molecular size distribution of maize starch nanoparticles (SNP) prepared by acid hydrolysis (3.16M H₂SO₄) and their amylase‐resistant counterparts, before and after debranching, was investigated. The weight average molecular weight (M_w) and linear chain length distribution were determined by high‐performance size‐exclusion chromatography (HPSEC) and high‐performance anion‐exchange chromatography (HPAEC), respectively. The objective was to understand the role of amylose involvement in the formation of SNP showing different crystalline structures (A‐ and B‐types). The HPSEC profiles of SNP before debranching from waxy, normal, and high‐amylose maize starches showed broad monomodal peaks. Debranched SNP from waxy maize eluted in a single narrow peak, whereas those from nonwaxy starches showed a multimodal distribution. Similar trends were also observed for the chain length distribution patterns, for which the longest detectable chains (degree of polymerization [DP] 31) in waxy maize were significantly lower than those of nonwaxy maize starches (DP 55–59). This indicated the potential amylose involvement in the SNP structure of normal and high‐amylose starches. Further evidence of amylose involvement was ascribed to the resistance of SNP toward amylolysis (Hylon VII > Hylon V > normal > waxy). The amylase‐resistant residues of SNP from high‐amylose maize starches were composed of both low M_w linear and branched chains.     

Combining Maltogenic Amylase with CMC or Wheat Gluten to Prevent Amylopectin Recrystallization and Delay Corn Tortilla Staling

Antistaling properties of a bacterial maltogenic amylase, sodium carboxymethylcellulose (CMC), and vital wheat gluten on quality of corn tortillas were evaluated during 14 days of storage. Amylopectin recrystallization was the driving force behind the staling of corn tortillas. Increasing levels of recrystallized amylopectin measured by differential scanning calorimetry (DSC) correlated significantly with increased tortilla stiffness (r = 0.43) and reduction in tortilla pliability (r = ‐0.42) during storage. Maltogenic amylase (275–1,650 activity units) made tortillas less stiff but did not preserve pliability and extensibility as effectively as CMC (0.25–0.5%). The combination of 825 MANU of maltogenic amylase (to interfere with intragranular amylopectin recrystallization) and 0.25% CMC (to create a more flexible intergranular matrix than retrograded amylose and amylopectin) produced less stiff, equally flexible, and less chewy tortillas than did 0.5% CMC. Vital wheat gluten was not as effective as CMC in preserving tortilla flexibility or as good as the maltogenic amylase in reducing stiffness. Further research is required to optimize the addition of maltogenic amylases in continuous processing lines that use fresh masa instead of nixtamalized corn flour (NCF) and to determine how these amylases interfere with amylopectin recrystallization.     

Wet‐Milling and Dry‐Milling Properties of Dent Corn with Addition of Amylase Corn

A transgenic corn (amylase corn) has been developed that produces an endogenous α‐amylase that is activated in the presence of water and elevated temperature (>70°C). Wet‐ and dry‐milling characteristics of amylase corn were evaluated using laboratory wet‐ and dry‐milling procedures. Different amounts of amylase corn (0.1–10%) were added to dent corn (with the same genetic background as the amylase corn) as treatments. Samples were evaluated for wet‐ and dry‐milling fraction yields using 1‐kg laboratory procedures. Milling yields for all amylase corn treatments were compared with the control treatment (0% amylase corn or 100% dent corn). No significant differences were observed in wet‐ and dry‐milling yields between the control and the 0.1, 1, and 10% amylase corn treatments. Most of the amylase activity (77%) in wet‐milling fractions was detected in the protein fraction. In dry‐milling, amylase activity (68.8%) was detected in endosperm fractions (fines, small grits, and large grits).     

Determining the Role of Starch in Flour Tortilla Staling Using α‐Amylase

Effects of α‐amylase modification on dough and tortilla properties were determined to establish the role of starch in tortilla staling and elucidate the antistaling mechanism of this enzyme. Control and amylase‐treated tortillas were prepared using a standard bake test procedure, stored at 22°C, and evaluated over four weeks. Amylase improved shelf‐stability of tortillas. The enzyme also produced a significant amount of dextrins and sugars, decreased loss of amylose solubility, and weakened starch granules. Amylopectin crystallinity increased with time, but was similar for the control and treated tortillas. Staling of tortillas appears to mainly involve the starch in the amorphous phase. As such, amylase activity does not significantly interfere with amylopectin crystallization. It is proposed that amylase partially hydrolyzed the dispersed starch (i.e., mostly amylose), starch bridging the crystalline region, and protruding amylopectin branches. Starch hydrolysis decreases the rigid structure and plasticized polymers during storage. The flexibility of tortillas results from the combined functionalities of the amylose gel and amylopectin solidifying the starch granules during storage. Protein functionality may also be involved in tortilla staling, but this needs further research.     

Effects of Falling Number Sample Weight on Prediction of α‐Amylase Activity

Reports vary on the effects of falling number (FN) sample weight on test precision, reproducibility, and predictability of α‐amylase activity. Straight grade flours of 200 samples (25 cultivars × 2 locations × 2 N₂ levels × 2 repetitions) were assayed for α‐amylase activity and FN. Location significantly affected α‐amylase activity and FN values. The coefficients of variation (CV) for the FN tests were 5.75, 2.12, 1.93, 1.72, 4.27, and 14.47%, when assayed with sample weights of 7, 6, 5.5, 5, 4.5, and 4 g, respectively. The FN test with the greatest reproducibility between sample replicates (lowest LSD and highest ratio of range/LSD) was also produced using the 5‐g sample weight. By reducing FN sample weight from 7 to 5 g, FN values that averaged 350 sec, considered essentially sound, averaged 215 sec, thus shortening the FN test time by an average of 2 min and 15 sec when assaying sound wheat flour. The results suggest a review of the 7‐g stipulation of AACC Approved Method 56‐81B for FN in favor of reduced sample weight.