氧载体、表面活性剂及H2O2对L-phe发酵影响的研究

用添加氧载体(油酸、豆油)、表面活性剂(Triton-X100)及H2O2的方法,改善L-苯丙氨酸发酵体系中的氧传递速率,以提高苯丙氨酸的产量。实验结果表明,在发酵0h添加1%的豆油、3%的油酸均可使产酸提高,分别可以使L-phe产量提高21.1%和39.5%;发酵0h同时加入3%油酸和0.05%Triton-X100时,提高产量78.95%;发酵12h添加0.075%H2O2,可以提高产苯丙氨酸产量18.42%。     

制霉菌素抗性筛选高活性苎麻脱胶菌诱变菌株

利用制霉菌素抗性筛选高渗透性突变株,提高黑曲霉菌对苎麻纤维的脱胶能力,使微生物脱胶能用于工业生产实践之中.分别用紫外线、硫酸二乙酯、亚硝酸作为诱变剂对黑曲霉3.0.2菌株进行诱变处理.以制霉菌素抗性为遗传标记,从突变菌株中定向筛选得到一株高活性苎麻脱胶菌黑曲霉3.0.2-26.在以未经刮制的苎麻韧皮为主要碳源,0.7%(NH_4)_2SO_4为氮源,添加00.5%KCl;00.5%MgSO_4;0.1%K_2HPO_4; 0.1%酵母膏;Tween80 0.1%的培养液中,接入黑曲霉3.0.2-26,置30℃下,150 r/min处理30 h左右,脱胶苎麻纤维的残胶率平均为14.43%.     

表面活性剂对出芽短梗霉多糖生产影响的研究

研究了表面活性剂对出芽短梗霉细胞培养过程中多糖释放的影响。在摇瓶中,比较添加0.05%(w/v)的Tween 80、Tween 60、Tween 40,结果显示几种表面活性剂均能促进细胞释放多糖,其中以Tween 80的效果最佳。在5L发酵罐中,以100g/L玉米粉水解液做碳源的出芽短梗霉细胞培养液中分别添加了表面活性剂Tween 80 0.01%、0.05%、0.1%,其中以添加Tween 800.05%时的效果最好,与不添加表面活性剂相比多糖产量提高25%左右,发酵周期缩短了将近2d。     

表面活性剂对球孢白僵菌生物合成D-HPPA的影响

[目的]提高目的产物R-(+)-2-(4-羟基苯氧基)丙酸(D-HPPA)的产量，研究不同表面活性剂对球孢白僵菌生物合成D-HPPA的影响。[方法]在固态发酵、液态发酵和全细胞催化三种生物合成方式中，分别考察表面活性剂的种类、浓度以及添加时间对D-PPA转化率的影响。[结果]固态发酵中添加0.5%的斯盘-80,液态发酵及全细胞催化中添加2.5%的斯盘-80均取得较高的D-PPA转化率，分别为99.64%±0.47%、60.60%±0.85%和54.50%±0.69%。在固态发酵和液态发酵方式中，发酵48 h时添加表面活性剂结果均为最佳，且表面活性剂添加时机与D-PPA一致。[结论]在三种生物合成方式中添加斯盘-80均能提高D-PPA的转化率。     

磷脂酰胆碱特异性和磷脂酰肌醇特异性磷脂酶C酶学性质及其在油脂脱胶中的应用

通过基因工程方法将具有磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)特异性磷脂酶C(PC-PLC)和磷脂酰肌醇(PI)特异性磷脂酶C(PI-PLC)基因分别在枯草芽孢杆菌WB800中进行胞外表达。对2种重组蛋白粗酶的酶学性质进行研究,并探究其用于油脂脱胶中的效果。结果表明:PC-PLC和PI-PLC均在30～45℃和中性偏酸的条件下具有较好的稳定性,2 h后残余活性仍在60%以上。Zn~(2+)、Mg~(2+)对PC-PLC的活性有促进作用,而Ca~(2+)对PI-PLC的活性有促进作用。油脂脱胶研究结果表明,PC-PLC的最适脱胶条件为50℃、pH 6.0、反应2 h、酶添加量为80 mg/kg;PI-PLC的最适脱胶条件为45℃、pH 7.0、反应2 h、酶添加量为60 mg/kg。PC-PLC和PI-PLC联合脱胶实验中大豆毛油中磷含量从531 mg/kg降至4.1 mg/kg,此时甘油二酯(DAG)含量增加0.95%。     

表面活性剂对嗜热脂肪芽孢杆菌产高温蛋白酶的影响

研究了表面活性剂对嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)WF146产胞外高温蛋白酶的影响。结果表明,表面活性剂Tween80在0.05%～0.1%(体积比)浓度范围内对WF146产酶有一定的促进作用。在培养基中添加0.1%Tween80可使发酵液酶活提高12.7%,Tween20和TritonX100则抑制嗜热脂肪芽孢杆菌WF146产酶。另外,TritonX100抑制嗜热脂肪芽孢杆菌WF146生长,而Tween80和Tween20不抑制其生长。     

木瓜籽毛油精炼条件筛选及其理化指标分析

以磷脂含量为指标对木瓜〔Chaenomeles sinensis ( Thouin) Koehne〕籽毛油水化脱胶过程中脱胶剂种类、脱胶剂添加量、脱胶时间、加水量和脱胶温度进行单因素实验,并在此基础上对脱胶时间、加水量和脱胶温度进行L9(33)正交实验;以酸价为指标对碱炼脱酸过程中的碱液(NaOH溶液)浓度、碱炼温度和超碱用量进行单因素实验和L9(33)正交实验;并比较了毛油、脱胶油、脱酸油和精炼油的主要理化指标变化。单因素实验和正交实验结果表明：在木瓜籽毛油水化脱胶过程中采用不同的脱胶剂种类(包括柠檬酸、草酸和蒸馏水)、脱胶剂添加量(质量分数0.1%~0.5%)、脱胶时间(10~70 min)、加水量(质量分数1%~6%)和脱胶温度(65℃~85℃),毛油中的磷脂含量均有明显差异;而碱炼脱酸过程中采用不同的碱液浓度(质量分数6%~14%)、碱炼温度(40℃~80℃)和超碱用量(质量分数0.15%~0.40%),毛油酸价也有明显变化。总体上看,木瓜籽毛油水化脱胶的适宜条件为添加质量分数0.2%柠檬酸为脱胶剂、脱胶温度75℃、加水量为质量分数4%、脱胶时间50 min;碱炼脱酸的适宜条件为碱液浓度为质量分数12%、碱炼温度80℃、超碱用量为质量分数0.30%。理化指标的测定结果表明：与毛油相比,脱胶油、脱酸油和精炼油的碘值略升高但差异不明显、过氧化值明显升高、磷脂含量和皂化值均明显下降,而脱酸油和精炼油的酸价也明显下降。研究结果显示：经过脱胶、脱酸、水洗干燥一系列过程后获得的木瓜籽精炼油的理化指标基本符合国家食用植物油卫生标准。     

表面活性剂对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

研究了不同浓度表面活性剂Tween-80,Triton X-100,SDS对大肠杆菌生产α-环糊精葡萄糖基转移酶（α-CGT酶）的影响。结果表明：发酵初始添加Tween-80和Triton X-100的最适浓度分别为2％,0.5％,最终胞外酶活分别达2.03U/ml和4.92U/ml,相对于未添加表面活性剂时提高4.6倍和12.67倍,且改变添加时间不能提高酶的产量;发酵36 h添加0.02％SDS对α-CGT酶产量促进最大,最终胞外酶活达5.31U/ml,较对照组提高12.75倍。表面活性剂对α-CGT酶生产的促进作用可能是由大肠杆菌细胞内外膜渗透性增加所致,使细胞周质空间中α-CGT酶能更加快速地渗透到胞外。     

两株芽孢杆菌在苎麻纤维复合脱胶中的应用

【背景】苎麻纤维细长、强韧、洁白、有光泽,被誉为"天然纤维之王",应用广泛。但其被以半纤维素和果胶为主要成分的胶质所包裹,脱胶是生产精干麻工艺的核心工序。利用单一菌株脱胶,往往因其脱胶酶系不全,存在胶质去除率低的问题,导致后期仍需要大量的碱和漂白剂处理。【目的】丰富苎麻脱胶过程中关键酶系,从而提高苎麻胶质去除率,并降低脱胶后期化学试剂的用量,推进苎麻生物脱胶的工艺应用。【方法】选用2株芽孢杆菌HG-9 (高果胶酶和甘露聚糖酶)和HG-25(高木聚糖酶)建立了复合微生物脱胶技术,并对其进行了优化。【结果】当2株菌接种量均为6%,水料比16:1,初始pH值5.9,在温度37.6°C下脱胶处理14 h时脱胶效果最佳,与菌株HG-9单独脱胶相比,脱胶时间减少2 h,胶质去除率、半纤维素去除率和木质素去除率分别提高9.32%、21.24%和17.93%,次氯酸钠用量减少20%。通过电子显微镜分析其形貌特征发现,混合脱胶获得的纤维表面更加平滑,无明显扭曲和损伤且纤维分散度较高。【结论】通过复合微生物协同作用,丰富脱胶过程中关键酶系,提高了苎麻纤维胶质去除率,缩短了脱胶时间,而且减少了脱胶后期漂白剂的用量,为苎麻生物脱胶工业化应用的进一步发展提供了指导。     

闽江河口湿地土壤CH4产生与氧化速率对外源氮、硫添加的响应

通过室内培养实验,研究了外源氮、硫添加对闽江河口湿地土壤CH_4产生/氧化速率以及土壤理化性质的短期影响。NH_4Cl(N1)和NH_4NO_3(N3)处理在各培养阶段均显著促进土壤CH_4产生速率(P0.05),较对照分别提高136.70%和136.55%;NH_4Cl+K_2SO_4(NS1)和NH_4NO_3+K_2SO_4(NS3)处理在培养第3、6、12、15和18天均显著促进了CH_4产生速率(P0.05)。KNO_3(N2)、K_2SO_4(S)处理在不同培养时间对CH_4产生速率影响均不显著(P0.05);KNO_3+K_2SO_4(NS2)处理除在第21天外(P0.05),其他时间影响均不显著(P0.05)。N2、N3、NS2和NS3处理均显著促进了土壤CH_4氧化速率(P0.05),平均CH4氧化速率较CK分别提高了145.30%、142.93%、139.48%和112.68%。整体而言,不同添加处理并没有显著改变湿地土壤CH_4产生/氧化速率的时间变化规律,各处理均表现为随培养时间先增加而后逐渐降低。短期培养结束后,土壤可溶性有机碳(DOC)、电导率、p H值在不同处理间均不存在显著差异(P0.05);土壤NH+4-N含量在N1、N3、NS1和NS3处理下,NO_3~--N含量在N2、N3、NS2和NS3处理下,SO_4~(2-)含量在S、NS1、NS2和NS3处理下均显著高于对照处理(P0.05)。相关分析显示,DOC、铵态氮(NH+4-N)和硝态氮(NO_3~--N)是氮、硫添加处理下影响闽江河口湿地土壤CH_4产生/氧化速率短期变化的主要控制因素。     

Enzymatic degumming of ramie bast fibers

Bast fibers from ramie (Boehmeria nivea) were treated with cell-free culture supernatants from an Amycolata sp. and a recombinant Streptomyces lividans strain expressing the Amycolata pectate lyase to investigate the degumming effects of different extracellular polysaccharide-degrading enzymes. Culture supernatants from the Amycolata sp. with high pectate lyase activities were most effective in fiber separation and reduced the gum content of ramie fibers by 30% within 15 h. Xylanase activity produced by the Amycolata sp. contributed little to the degumming. Electron micrographs showed that the crude pectate lyase from the Amycolata sp. removed plant gum more efficiently from decorticated ramie bast fibers than the purified enzyme. Similarly, degumming with the crude enzyme of the Amycolata sp. and the recombinant S. lividans strain for 24 h resulted in fibers with a residual gum content of 14.7 and 17.3%, respectively. Degumming with the crude enzyme of the recombinant Streptomyces strain was slightly improved by the addition of a commercial pectinesterase. No significant degumming was observed with the crude enzyme from an S. lividans strain that did not produce the Amycolata pectate lyase. These results indicate that the pectinolytic activity of the Amycolata sp. plays an active role in degumming of ramie bast fibers.     

Large-scale degumming of ramie fibre using a newly isolated <Emphasis Type="Italic">Bacillus pumilus</Emphasis> DKS1 with high pectate lyase activity

A combined (enzymatic and chemical) process using a Bacillus pumilus strain (DKS1), isolated from the soil, was used to degum ramie bast fibres. After 24 h of incubation with the isolated pectinolytic strain using a low-cost medium, the weight loss of the ramie fibre was found to be 25% under small scale. High activity of pectate lyase was detected in the culture supernatants; 400 kg of ramie fibres was degummed with 24% weight loss in large-scale degumming under field conditions. No cellulase activity was found. Microbial intervention followed by mild (0.1%) alkali treatment showed high percentage of weight loss from the ramie fibre. Bacterial degumming followed by chemical treatment resulted in an increase of single fibre tenacity (cN/tex) by more than 20.81% as compared to non-degummed (decorticated) fibre samples. Scanning electron micrographs (SEM) and fluorescence microscope showed that after Bacillus pumilus DKS1 treatment the surface of the decorticated ramie fibre becomes very smooth. These results indicate the process provides an economical and eco-friendly method for the small scale as well as large-scale degumming of decorticated ramie fibre. This study has great relevance to the textile as well as paper industry.     

Degumming of ramie fibers by alkalophilic bacteria and their polysaccharide-degrading enzymes

Three strains of alkalophilic bacteria, Bacillus sp. NT-39, NT-53 and NT-76, were selected for the degumming of ramie fibers and production of polysaccharide-degrading enzymes. After 48 h of incubation with the strains, the loss of the gum might amount to 5.0% or more of the fibers and a number of polysaccharide-degrading enzymes were secreted to the culture supernatants. The residual gum of the fibers decreased to 9.4% after 5 h of enzymatic degumming. Analysis of gum contents and enzyme activities revealed that pectate lyase and xylanase played an important role in the degradation of residual gum. Enzymatic degumming resulted in an increment of 5.4 ISO units in fiber brightness, whereas the reduction in bundle breaking tenacity of the fibers was less than 5.%. The results confirmed that degumming of ramie fibers by alkalophilic bacteria and their enzymes had substantial advantages.     

Screening of pectinase producer from alkalophilic bacteria and study on its potential application in degumming of ramie

Four strains designated as NT-2, NT-6, NT-33 and NT-82 were selected from alkalophilic bacteria. They all can produce pectinase and xylanase. The polygalacturonase (PGase) and xylanase activities of strain NT-33 were 1025 U ml^-1 and 26U ml^-1 respectively. The pH and temperature optimum for the activity of PGase were 10.5 and 70°C, respectively. From batch experiments, it was found that strain NT-33 had an excellent capacity for degumming ramie fibers. After 24 h fermentation this strain can remove 70% of the gum existing in ramie fibers. The experiments showed that a series of noncellulosic polysaccharide degrading enzymes were involved in the ramie degumming process.     

宏基因组来源果胶酸裂解酶在毕赤酵母中的表达及应用

果胶酸裂解酶可用于含果胶废水的处理、纸浆漂白以及棉麻纺织品的生物精炼等。基于高通量宏基因组测序技术,从富含果胶土壤宏基因组中挖掘得到一个果胶酸裂解酶基因pela。将pela连接表达载体pPIC9转化毕赤酵母Pichia pastoris GS115。在3 L发酵罐水平,甲醇诱导10 h后,培养基中果胶酸裂解酶活力达到10.8 U/mL。重组PELA的最适温度为45℃,最适pH为9.0,在pH 7.5~11.0具有良好的稳定性。PELA的比活力为244.12 U/mg,以聚半乳糖醛酸为底物时催化反应的Km和Vmax分别为0.26 mg/mL和488.40 μmol/min·mg。EDTA及金属离子Cd2+、Zn2+、Mn2+、Cu2+、Fe3+能够高度抑制酶的活性,1 mmol/L Ca2+和2 mmol/L K+对酶活力有促进作用。将重组PELA作用于苎麻纤维4 h后,纤维质量损失率达到9.2%,苎麻纤维分散度和白度都明显提高。结果表明从宏基因组来源的果胶酸裂解酶PELA在苎麻脱胶中具有良好的应用潜力。     

Partial characterization ofAspergillus fumigatus polygalacturonases for the degumming of natural fibers

Summary Aspergillus fumigatus strain 4, cultured on citrus pectin as the sole carbon source, produced polygalacturonases whose activity was optimum at 65°C and pH 3.5–4.5. The enzymes presented a bimodal thermostability for 10 min, but not 60 min, of incubation. Polygalacturonases showed pH stability between 3.0 to 9.0. The enzymes were stable when stored at 4–6°C for 90 days, but their activity was reduced by 24% when they were stored at 26–30°C. Orange pulp was the best pectic carbon source tested for the production of pectinases capable of retting ramie fibers. The reutilization of these enzymes was possible, suggesting the viability of industrial use of pectinases for degumming ramie fibers.     

In vitro detachment of bacteria from ruminal digesta by buffered sodium oleate solutions

The effectiveness of a buffered sodium oleate solution was evaluated for detaching bacteria from ruminal digesta samples. A response surface derived from an octagonal design was used to determine the pH and concentration combination for maximum detachment of total and cellulolytic bacteria. The total number of bacteria detached increased up to 81% over control with treatment of a pH 8.8 and 1.5% sodium oleate solution. The recovery of cellulolytic bacteria was decreased to 35% of control with treatment of a pH 9.0 and 0.1% sodium oleate solution. Attempts to improve the recovery of viable bacteria exposed to sodium oleate solutions were unsuccessful. This response surface design identified an optimal pH and concentration that were consistent with existing information regarding detachment of total bacteria, and suggested that sodium oleate, at the concentrations tested, was toxic to the cellulolytic population of the rumen.     

Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from Bacillus sp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca²⁺. However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in the t₅₀ (time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in the t₅₀ value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.     

苎麻酶法脱胶的研究进展*

苎麻酶脱胶法是一种高效能、优质量、无污染的脱胶方法,它是直接利用微生物发酵后期产生的胞外酶或酶制剂降解苎麻的胶质,使得苎麻纤维释放出来。到目前为止,国内外的科研人员在这方面已经进行了大量的研究,筛选了许多不同的脱胶菌,包括需氧菌和厌氧菌等用于苎麻的脱胶。相比化学脱胶,苎麻的酶脱胶具有提高精干麻的质量、大幅度降低环境污染等显的优点,是苎麻脱胶未来的主要发展方向,有着不可估量的前途。同时,随着酶脱胶技术在工业化生产中的推广应用,酶的使用量势必大幅度增加,从而将带动酶工业的发展。