黑曲霉原生质体诱变选育果胶酶高产菌株

通过UV和NTG诱变筛选获得了2株高产果胶酶突变株。以果胶酶产生菌黑曲霉EIM6为诱变材料,采用1．5％的溶壁酶和1．5％的纤维素酶处理其对教生长期菌丝体2h获得高质量的原生质体。采用UV25S或50μg／mL NTG诱变30min,构建原生质体突变库,经刚果红果胶平板筛选获得果胶酶突变株,通过液体深层培养复筛获得高产突变株EIM6-U11、EIM6-N5,酶活力分别从46598．08、46598．08U／mL提高至68596．57、68879．56U／mL,分别提高了47．21％、47．82％。连续8次传代经发酵测酶活力表明高产突变株EIM6-U11、EIM6-N5具有较高的遗传稳定性。     

一株产纤维素酶真菌的筛选、鉴定及酶学性质初步研究

经过初筛和复筛从土样中分离出1株高产纤维素酶真菌SNB9,经形态学和ITS序列分析。鉴定为黑曲霉（Aspergu Uusniger）。生长条件的测定显示该菌生长范围偏酸。发酵后纤维素酶的最适作用pH在4．0—5．0,最适作用温度在45—55℃。滤纸酶活为9．29U／mL,C,酶活为23．69U／mL,CMCase酶活为38．23U／mL,β-葡萄糖苷酶活为65．52U／mL。发酵液中除了纤维素酶,还发现有辅助酶,包括木聚糖酶、淀粉酶、果胶酶、蛋白酶。     

碱性果胶酶高产菌株的构建和高密度发酵

碱性果胶酶可用于苎麻脱胶和棉织物前处理的精练工艺,与传统的高温碱煮相比,具有保护纤维、降低能耗和化学污染的优势,因此获得高表达的碱性果胶酶基因工程菌,低成本生产碱性果胶酶对于纺织工业节能减排具有重要的意义。前期研究工作已经将来源于枯草芽孢杆菌Bacillus subtilis 168的碱性果胶酶基因pel经过密码子优化后在毕赤酵母Pichia pastoris GS115中成功表达。本研究为了提高其表达量,首先利用启动子和信号肽都优化的载体pHBM905BDM进行表达,摇瓶酶活从68 U/mL增加到100 U/mL,qPCR检测转录水平提高了27%。再利用果胶底物平板筛选水解圈大的转化子进行摇瓶发酵获得菌株GS115-pHBM905BDM-pels4,摇瓶酶活为536 U/mL。随后构建重组质粒pPIC9K-pels,电转化菌株GS115-pHBM905BDM-pels4,利用抗生素G418平板进行筛选,在含4 mg/mL的G418抗性平板上得到菌株GS115-pHBM905BDM-pPIC9K-pels1,摇瓶酶活为770 U/mL,qPCR测定含7个拷贝目的基因。最后将该菌株在5 L的发酵罐中进行高密度发酵,果胶酶酶活提高至2 271 U/mL。该碱性果胶酶酶活已达到目前酵母表达的最高水平,说明其具有很好的应用于纺织工业的潜力。     

pH控制下绿色木霉（Trichoderma viride）流加发酵生产纤维素酶

绿色木霉（Trichodermaviride）在pH控制发酵条件下,采用流加葡萄糖发酵策略,可显著提高综合滤纸酶活力（FPA）和内切酶（endo—β—1,4-glucanase,EG）、外切酶exo—β-1,4-glucanase,CBH）、纤维二糖酶（cellobiase,CB）酶活。在5L发酵罐中采用pH控制和流加葡萄糖工艺,可提高CB酶含量,改变酶组分之间的比例,使得FPA、EG、CB和CBH酶活分别达到50．0U／mL,210．0U／mL,4．0U／mL和2．5U／mL,比摇瓶发酵分别提高了6．7．4．2、19、2．5倍。     

Sul folobus sol fataticus P2嗜热嗜酸α-淀粉酶在大肠杆菌和毕赤酵母中的表达

通过PCR方法从Sulfolobus solfataticus P2中扩增到2．6kb的α-淀粉酶基因（SS01172）,将其分别克隆到表达载体pET32a（＋）和pPICZaA,并在E．coliRosetta和Pichia pastoris GS115中进行表达。结果表明α-淀粉酶基因在Rosetta中得到了高效表达,酶活为143．466U／mL;而在GS115中表达量稍低,发酵液酶活力为98．102U／mL。     

适于罗布麻生物脱胶的果胶酶产生菌分离筛选与表征

在青海柴达木盆地沤制的罗布麻表皮中分离得到两株适于罗布麻脱胶的高产果胶酶菌株,经形态、生理生化指标鉴证和16SrDNA菌种鉴定,确定一株为新的果胶酶产生菌琼氏不动杆菌（Acinetobacter junii）,一株为枯草芽孢杆菌（Bacillus subtilis）。水解圈实验得出前者H／C值（H／C为水解圈直径H与菌落直径C之比）为5,后者为3;经酶活力测定,在37℃下,琼氏不动杆菌（Acinetobacter junii）在11h达到产酶高峰（酶活力为103．2IU／mL）,枯草芽孢杆菌（Bacillus subtilis）培养至9h达到产酶高峰（酶活力为91．6IU／mL）,但前者最高酶活力比后者高12．5％;经脱胶实验得出两菌残胶率分别为18．47％和17．31％,对罗布麻生物脱胶有较好的适用性。     

黑曲霉SL2-111复合酶固体发酵工艺研究

以酸性蛋白酶酶活为响应值,采用单因素搜索和正交试验对黑曲霉（Aspergillus niger）SL2－111固体发酵工艺进行优化,结果表明最适培养基的组成为：新鲜麸皮8．25g,米糠4．5g、豆饼粉1．5g、（NH4)2SO40.3g、K2HPO40．66g、CaCl20.075g、水8．6mL,pH5．5,变温培养,前30h28℃、后30h为23℃,培养时间为60h。采用最适培养基和优化工艺,在250mL三角瓶中进行验证实验,酸性蛋白酶酶活可达12586U/g,果胶酶和纤维素酶分别为16490U/g、9822U／g。     

一株产纤维素酶的甲醇利用细菌的鉴定及其纤维素降解条件优化

采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt-04。形态特征、生理试验及16SrDNA序列和gyrB序列分析表明,该菌株属于Bacillusmethylotrophicus。为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC—Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化。结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5．0和2％（20mg／mL）;响应面法得出的最高酶活力条件：反应温度、pH和底物浓度分别为66．1℃、4．81和19．01mg／mL。在最优条件下,酶活力达到17．85U／mL,比优化前的酶活力12．84U／mL提高了39．01％。因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景。     

利用Design-Expert软件优化丝氨酸羟甲基转移酶产酶培养基

利用Design-Expert软件中水平设计和响应面分析法对产酶基本培养基主要成分进行了优化,经过逐步回归分析建立了丝氨酸羟叫基转移酶（SHMT）活力对培养基主要成分的二次回归模型,其回归方程的决定系数达到了0．9984。得到的最佳培养琏主要组成为：葡萄糖29．5g／L、硫酸铵18．1g/L、玉米浆3．79g／L。SHMT活力最高达到113．7U／ml,比优化前（77U／mL）提高了47．7%。优化后的酶液经酶促反应50h,能催化产生10g／L的L-丝氨酸,比优化前（6g／L）提高66．7％。     

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。     

棉秸秆降解高温菌株的筛选及产酶分析

从新疆地区分离具有降解棉秸秆纤维素功能的菌株,得到4株耐高温真菌(50°C)。纤维素酶学性质分析表明,该4株菌的纤维素酶具有良好的耐酸性(最适pH为4.5)和耐高温性(最高达60°C)。以羧甲基纤维素钠(CMC-Na)、微结晶纤维素、棉花、滤纸、淀粉、果胶为底物测定酶活力,滤纸酶活力(FPA)最高达2.63 U/mL、淀粉酶活力最高达6.17 U/mL、果胶酶活力最高达5.86 U/mL。4株真菌酶学特性分析表明,该系列菌株在秸秆生物质利用方面有很大的应用潜力。     

三株散囊菌属真菌的茶叶液态发酵与其成分分析

散囊菌属真菌(Eurotium spp.)又称“金花菌”,能在六堡茶表面生长出金黄色的子囊果而得名,是一类对发酵茶叶的品质有积极影响的真菌。本研究前期从广西梧州六堡茶中筛选到的三株散囊菌属真菌Aspergillus chevalieri E2(简称E2),Aspergillus chevalieri E3(简称E3)与Aspergillus cristatus E6(简称E6),将菌株接种于自制液体茶粉培养基中培养,检测发酵液中产生的色素物质;测定发酵液中纤维素酶、果胶酶、淀粉酶的酶活力、总糖和还原糖糖含量、氨基酸含量、总抗氧化能力、自由基清除能力和茶多酚含量。结果表明:三株真菌在液体茶粉培养基中生长良好,E3生长尤其迅速。在8 d的培养期限内,E2、E3和E6的最高果胶酶活力分别为2.52、3.44和5.15 U/mL;最高纤维素酶活力分别为0.57、0.82和1.10 U/mL;最高淀粉酶活力分别为0.31、0.35和0.49 U/mL。三株真菌的发酵后培养液中还原糖含量大幅增加,增幅分别达到131.70%、627.02%和459.12%;总糖含量同样呈上升趋势,增幅分别达到6.84%、35.25%和2.07%;E2与E3的氨基酸含量分别下降了17.43%与32.68%,E6则上升了0.52%;E3的总抗氧化活性提升最大,达65.87%;DPPH·自由基清除能力提升最大的是E2,达128.33%;茶多酚含量都显著提升,增幅分别为50.85%、51.58%和53.06%。本研究对改善六堡茶的品质及茶叶的发酵的研究有一定意义和参考价值。     

片烟产果胶酶细菌的鉴定及酶活测定

以果胶为碳源, 对津巴布韦片烟烟叶表面产果胶酶细菌进行分离, 采用16S rDNA限制性酶切片段长度多态性分析(ARDRA)和测序方法, 结合形态学、生理生化实验, 对所分离产果胶酶菌株进行鉴定, 同时研究培养时间、温度、起始pH、接种量对菌株产酶的影响。结果表明, 从津巴布韦片烟烟叶表面分离得到的产果胶酶菌株主要为芽孢杆菌属的枯草芽孢杆菌Bacillus subtilis和产碱菌属的粪产碱菌Alcaligenes faecalis。在所分离的菌株中, 枯草芽孢杆菌T10酶活力最高, 以6%的接种量, 在温度为35 °C、起始pH为7.5条件下培养48?56 h, 其果胶酶酶活为571 U/mg, 聚半乳糖醛酸裂解酶酶活为297 U/mg。     

Enhanced co-production of pectinase,cellulase and xylanase enzymes from Bacillus subtilis ABDR01 upon ultrasonic irradiation

The effect of low-intensity ultrasound irradiation was studied to improve the co-production for pectinase, cellulase, and xylanase enzymes using Bacillus subtilis ABDR01. Different parameters such as ultrasonic irradiation at the different growth phases of the bacterial strain, ultrasound power, irradiation duration, and irradiation duty cycle were assessed. Sonication with 90 W ultrasound power, 25 kHz frequency with 70 % duty cycle for 5 min at 6 h of bacterial growth phase gave the maximum productions of 87.82 U/ mL pectinase 22.17 U/ mL cellulase and 137.95 U/ mL xylanase respectively. The enzyme activity of pectinase, cellulase, and xylanase was enhanced by about 38.15 %, 53.77 %, and 24.59 %, respectively, compared to non-sonicated control cultivation. This optimized low-frequency ultrasound irradiation to bacterial cells enhanced the nutrient uptake rate and increased the cell wall permeability, which results in higher enzyme productivity. Our results signify the effectiveness of low-frequency ultrasound irradiation for improved enzyme yields and hyperactivation during microbial fermentation.     

离子注入结合紫外线及~(60)Co-γ射线诱变选育碱性果胶酸裂解酶高产菌株的研究

以碱性果胶酸裂解酶产生菌芽孢杆菌WZ008为出发菌株,经形态鉴定和16S鉴定为类芽孢杆菌,命名为Paenibacillus sp.WZ008,通过N~+注入诱变、紫外线诱变、~(60)Co-γ射线诱变等多次反复诱变,选育得到一株产碱性果胶酸裂解酶性能稳定且酶活明显提高的突变株,其酶活为97.8U/mL,比出发菌株产碱性果胶酸裂解酶能力提高了1.04倍。     

Characterisation of pectin and optimization of pectinase enzyme from novel Streptomyces fumigatiscleroticus VIT-SP4 for drug delivery and concrete crack-healing applications: An eco-friendly approach

Pectinases are enzymes which are widely distributed in microbes that are present in pectin enriched sites. The agro-industrial residues can be utilized in the industrial scale for low-cost and efficient pectinase production in an eco-friendly approach. This study employs low-cost substrates (i.e. culinary fruit peels) for maximum pectinase production from novel Streptomyces fumigatiscleroticus VIT-SP4. The extraction and characterization of pectin from different fruit peels were investigated and pectinase activity was analyzed. The orange pectin gave maximum pectinase activity of about 45.93 (U/mL). Further, statistical optimization of process parameters was studied by using Taguchi method showed optimum values of pH-6, temperature −35 °C, orange pectin% − 2.5, incubation time- 48 h and RPM- 200 rpm and pectinase activity was found to be 98.65 (U/mL). The response surface methodology (RSM) was used for the optimization of media components which revealed that starch −1.17%, yeast extract-2%, and orange pectin% − 0.75% produces maximum pectinase of about 170.05 (U/mL). The drug-delivery study showed drug release was not observed at initial pH 3 after 4 h. The immediate drug release was noted at pH 6 caused due to disintegration of pectin by the pectinase activity. The self-healing of cracks by spray culture technique was investigated. The crack healing was observed up to 0.50 mm wide after 12 days. This confirms the ability of actinomycete spores to survive and they react to form calcite complex directly helps in crack healing process. This low-cost microbial pectinase can be used in drug delivery and concrete crack-healing applications sectors in future.     

Co-production of tannase and pectinase by free and immobilized cells of the yeast Rhodotorula glutinis MP-10 isolated from tannin-rich persimmon (Diospyros kaki L.) fruits

Hyper tannase and pectinase-producing yeast Rhodotorula glutinis MP-10 was isolated from persimmon (Diospyros kaki L.) fruits. The main pectinase activity of yeast was exo-polygalacturonase. No pectin methyl esterase and too low pectin lyase activities were detected for this yeast. The maximum exo-activities of tannase and polygalacturonase were determined as 15.2 and 26.9 U/mL for free cells and 19.8 and 28.6 U/mL for immobilized cells, respectively. Immobilized cells could be reused in 13 successive reaction cycles without any loss in the maximum tannase and polygalacturonase activities. Besides, too little decreases in activities of these enzymes were recorded between 14 and 18 cycles. At the end of 18 successive reaction cycles, total 503.1 U/mL of polygalacturonase and 349.6 U/mL of tannase could be produced using the same immobilized cells. This is the first report on the use of free and/or immobilized cells of a microorganism for the co-production of tannase and pectinase.     

响应面法优化黑曲霉产果胶酶培养基中无机盐成分的研究

利用SAS软件中的二水平设计和响应面分析方法较系统地研究了发酵培养基中无机盐组分对黑曲霉(Aspergillus niger)JW-1菌株产果胶酶的影响.得到了在一定条件下果胶酶随无机盐组分的变化规律,并根据分析结果优化了产酶培养基.最终确定KH_2PO_4,FeSO_4·7H_2O,CaCl_2·2H_2O的最优浓度分别为0.85mg/mL,1.86mg/mL和2.52mg/mL,此时果胶酶活力可达5054.6U·g~(-1),为该菌株今后的研究和应用奠定了基础.