枯草芽孢杆菌FM208849产果胶酶的固定化及酶学性质研究

为了提高游离果胶酶的稳定性,对罗布麻脱胶具有特异性的枯草芽孢杆菌(FM208849)进行产果胶酶发酵时,采用交联酶聚集体(CLEAs)技术制备固定化果胶酶,并对交联果胶酶聚集体的制备条件、酶学性质进行研究。结果表明,游离果胶酶经80%饱和硫酸铵沉淀后,在30℃,经4%的戊二醛溶液交联135 min,所形成的交联果胶酶聚集体的活回收率为61.5%,其最适反应温度45℃和最适pH10,在对交联果胶酶聚集体的热稳定性和有机溶剂稳定性分析中,均显示了比游离酶更高的稳定性。     

Biosynthesis of Pectinase by Fungi of the Genera Bjerkanderaand Coriolusduring Solid-Phase Fermentation

Production of an extracellular pectinase by wood-rot fungi of the genus Bjerkanderaand Corioluswas studied. The active producersB. adusta40 and C. versicolor24 were selected. The dynamics of production of pectinase and effects of temperature, initial pH, humidity of the medium, and addition of nitrogen sources on the biosynthesis of pectinase were studied.     

果胶酶的细胞化学定位:马尾松树脂道发生的细胞化学证据

运用薄切片和电镜细胞化学方法观察了马尾松(Pinus massoniana Lamb.)茎皮层树脂道的发育及发育过程中果胶酶的变化.树脂道的发育过程一般可分为4个阶段,即原始细胞阶段、胞间隙形成阶段、腔道扩大阶段和树脂道成熟阶段.在原始细胞阶段,果胶酶的反应产物首先出现在原始细胞膨胀的细胞壁角隅处,然后沿细胞壁中层分布.胞间隙形成后,果胶酶的反应产物分布在细胞壁和胞间隙的交界面.随着胞间隙的扩大,反应产物的密度逐渐降低.当树脂道成熟后,上皮细胞壁上则没有果胶酶的反应产物出现.细胞化学证据表明:在树脂道的发育过程中,果胶酶降解树脂道原始细胞细胞壁中层,支持树脂道以裂生方式形成.果胶酶的细胞化学定位技术还可用于其他植物中与果胶水解有关的发育过程.     

果胶酶的细胞化学定位：马尾松树脂道发生的细胞化学证据

运用薄切片和电镜细胞化学方法观察了马尾松(Pinus massoniana Lamb.)茎皮层树脂道的发育及发育过程中果胶酶的变化。树脂道的发育过程一般可分为4个阶段,即原始细胞阶段、胞间隙形成阶段、腔道扩大阶段和树脂道成熟阶段。在原始细胞阶段,果胶酶的反应产物首先出现在原始细胞膨胀的细胞壁角隅处,然后沿细胞壁中层分布。胞间隙形成后,果胶酶的反应产物分布在细胞壁和胞间隙的交界面。随着胞间隙的扩大,反应产物的密度逐渐降低。当树脂道成熟后,上皮细胞壁上则没有果胶酶的反应产物出现。细胞化学证据表明: 在树脂道的发育过程中,果胶酶降解树脂道原始细胞细胞壁中层,支持树脂道以裂生方式形成。果胶酶的细胞化学定位技术还可用于其他植物中与果胶水解有关的发育过程。     

果胶酶的固定化研究

本文研究了以重氮化的对—氨基苯磺酰乙基纤维素为载体制各固定化果胶酶的最适条件,并比较了固定化果胶酶与游离酶的性质。结果表明,最适的固定化果胶酶的条件是：在ｐＨ７．００．１５Ｍ的磷酸盐缓冲液中,按每克载体加入２１６３活力单位的酶的比例进行偶联反应１２小时。在以上最适条件下,固定化果胶酶的表观活力为１９８０Ｕ／ｇ,活力回收率为８７％。与游离酶相比,固定化果过酶作用的最适ｐＨ由４．６移至４．２,最适温度变宽,酶的热稳定性增强,操作稳定性良好,半衰期为３２．５天。     

固定化果胶酶澄清果汁的条件及效果

利用固定化果胶酶对四种不同果汁澄清条件及效果进行研究,结果表明,固定化果胶酶澄清四种不同果汁的效果明显,其中澄清桔汁的果胶酶重复使用20次以上,酶活力及透光率仍可维持在80%以上,其最适反应条件是:果汁浓度50%;pH 3.0~3.5;温度45~50℃;反应时间2小时;酶量每毫升果汁0.05 g固定化果胶酶;澄清时间20小时。     

草酸青霉液体发酵产果胶酶条件的优化及其产物的酶学性质

采用响应面分析法对草酸青霉（Penicillium oxalicum）L5菌株液体发酵产果胶酶条件进行了优化。结果表明：桔皮粉、米糠及硫酸铵的添加量分别为4.85%、5.89%、0.97%,摇瓶初始pH为6.0~8.0,接种量为9%时,优化后的果胶酶活达54 391.70 U,是初始酶活18 148.00 U的3倍。另外,对其果胶酶性质进行了初步探讨,结果表明：该酶较适反应温度和pH分别为50℃和5.0;30~50℃时有较好的热稳定性,pH值为5.0时稳定性最佳。     

亚麻脱胶新工艺的初步研究

研究了温水浸渍亚麻脱胶过程中的产果胶酶的微生物数量、种类和果胶酶活力变化规律,分离筛选出了产果胶酶活力较高的厌氧和兼性厌氧菌各l株,研究了这2个菌株的种子培养条件,用正交实验法优化了接入厌氧和兼性厌氧菌的亚麻脱胶工艺.实验结果表明亚麻脱胶周期缩短35%,可改善麻纤维质量.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Effect of carbon sources on the synthesis of pectinase by Aspergilli

The synthesis of pectinase is investigated using six species of Aspergillus, with five media differing either in their carbon sources or level of carbon source(s). Five of the six species used, synthesized appreciable amounts of pectinase in the media containing sugars. Pectinase synthesis was highest for A. niger, NCIM 548, with all the sugar containing media. A. foetidus, NCIM 510, was the only one among the organisms studied, that responded well to the medium containing pectin in the absence of additional sugars supplied in the medium.     

Degradation kinetics of pectins by an alkaline pectinase in bioscouring of cotton fabrics

Alkaline pectinases have been proven to be effective as bioscouring agents of cotton fabrics. In order to monitor the scouring degree of cotton fabrics quantificationally, a kinetic study of the degradation of pectins in cotton by an alkaline pectinase ‘Bioprep 3000L’ was performed and the influences of initial pectinase concentration and treatment time on bioscouring were evaluated quantitatively. The results showed that although the degradation products increased as pectinase concentration grew higher at same incubation time, the growth multiples of the maximum degradation rate which was used as the starting degradation rate were less than those of initial enzyme concentration. The degradation kinetics of pectins in cotton fibers with a pectinase could be described by modified Ghose–Walseth kinetic empirical equations which had been previously applied to the degradation reaction of cellulose.     

果胶酶高产菌株的紫外线及硫酸二乙酯的诱变育种

以HDYM-02为出发菌株,用紫外线及硫酸二乙酯进行诱变,从大量突变株中进行筛选,选育出2株产果胶酶性质稳定且酶活明显提高的突变株UV-21和DES-1,株相比其产酶期提前,前者在24h时的酶活力为出发菌株的1．6倍,后者在12h的酶活力为出发菌株的1．44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.     

Exocellular hydrolases from Penicillium ulaiense

Penicillium ulaiense exhibited carboxymethylcellulase, pectinase, protease, amylase and phenolase activities, while no xylanase, cellulase, lipase or ligninase activities were found. Pectinolytic action was studied in liquid medium, showing low levels of pectinesterase and pectinase production. No mycotoxins were detected by thin-layer-chromatography.The authors are with INIQUI, Universidad Nacional de Salta, Buenos Aires 177, 4400 Salta, Argentina     

13th Annual Biotechnology Congress (BAC Madrid 2019): abstract collection

Background

Tobacco stalk (TS), a major agricultural waste abundant in pectin, has resulted in concerns about the need for its reuse. The nicotine in TS is considered a chemical that is to\xic and hazardous to the environment.

Results

In this study, Bacillus tequilensis CAS-MEI-2-33 was isolated from cigar wrappers to produce alkaline pectinase using TS. Subsequently, the medium and fermentation conditions for the production of pectinase by B. tequilensis CAS-MEI-2-33 were optimized. The optimal fermentation period, pH of the initial fermentation medium, concentration of TS, and inoculum amount for B. tequilensis CAS-MEI-2-33 were 40 h, 40 g/L, 7.0, and 3%, respectively. Under optimal conditions, the pectinase activity was 1370 U/mL. Then, the enzymatic properties, such as the optimum pH, reaction temperature, temperature stability, and effects of metal ions, were studied. The optimal pH was determined to be 10.0, indicating that the enzyme was an alkaline pectinase. The optimal temperature was 40 °C, and pectinase activity was stable at 40 °C. The Ag⁺ metal ions were shown to remarkably promote enzyme activity. The pectinase was partly purified by ammonium sulfate precipitation, ion exchange chromatography, and Sephacryl S-100 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and LC-MS/MS analyses were utilized to analyze the pectinase.

Conclusions

This study provided a new alkaline pectinase candidate and a new strategy for the use of TS.

     

尼龙网固定化果胶酶的制备及其性质研究

用尼龙网作载体,经3-二甲氨基丙胺活化,用戊二醛将果胶酶固定化。所得固定化酶Km值与自然酶接近;对温度的稳定性有较大的提高,100℃保温30min才能使其失活。固定化酶在较宽的pH范围内能保持其正常活力,它对金属离子抑制剂的耐受性有较显著的提高,用0.5％果胶溶液作底物,重复使用10次后酶活力保留44％。固定化果胶酶与自然酶相比较,对不同果汁的澄清效果不同。固定化果胶酶在无保护剂存在的条件下,室温放置四个月活力不减少。     

Physical characteristics and antioxidant effect of polysaccharides extracted by boiling water and enzymolysis from Grifola frondosa

Grifola frondosa has been widely consumed in China and other Asian countries. Recent studies on G. frondosa have focused on the activities of polysaccharides extracted by water, and the activities of polysaccharides extracted by enzymolysis have not been studied. In this work, the relationship between the physical properties and antioxidant activity of polysaccharides extracted from G. frondosa by boiling water and enzymolysis was studied. Five polysaccharide extracts from the fruit body of G. frondosa were prepared by different extracting methods including boiling water, single enzyme enzymolysis with three different single enzymes (cellulose, pectinase, and pancreatin), and combined enzyme enzymolysis (cellulose:pectinase:pancreatin; 2:2:1). Characteristics such as the viscosity, Mw, polysaccharide content, protein content, infrared spectra, and antioxidant activities of the extracts were evaluated. The highest antioxidant activity was exhibited by the extracts prepared by combined enzyme extraction. The correlation analysis between antioxidant activity and polysaccharide content, protein content, Mw or viscosity indicated that the Mw had a more important role in antioxidant activity. Overall, the results indicate that the combined enzyme polysaccharide extracts can be developed as a new potential natural antioxidant.     

Synthesis of different pectinases by filamentous growingA. niger mutants

Mutants ofA. niger K 69/26, prepared by multistep mutagenesis (UV, MNNG, heating) have been screened for pectinase activities. Mutants with altered levels of certain pectinases, such as endo- and exopolygalacturonase (PG vis, red), pectinesterase (PE) and pectinlyase (PL), were isolated. The enzyme activities of the best mutants M 1348/126 were increased 2–3-fold compared to the parent strain after a 6-d cultivation of filamentous mycelium on a shaker. Further mutagenesis of mutants with decreased pectinase activities (e.g. Se3) produced revertants. PG (vis) synthesis of revertant Se5 was increased 1.7 times compared to the control strain K 69/26. Independent of these increased rates, the general level of pectinase activities synthesized by the filamentous mycelium ofA. niger mutants amounts to about 10–20% compared with those produced by aggregated mycelium. It appears that the enzyme synthesis related to mycelium structure is independent of the mechanism which regulates the level of pectinase synthesis within a specific morphological structure.     

Potential use of nylon scouring pad cubes attachment method for pectinase production by Aspergillus niger HFD5A-1

This study investigated the novel use of scouring pad cubes as a support matrix for immobilization of fungal cell to enhance the pectinase production. Nylon scouring pad cubes were used for immobilized Aspergillus niger HFD5A-1 cells for pectinase production in flask submerge fermentation system. The enzyme activity of immobilized cell in scouring pad cubes gave higher activity compared to free cells. Various physical parameters for culture condition were studied to evaluate its effects on pectinase production. The maximum enzyme activity obtained was 11.05 U/mL on the 6th day of cultivation after using the optimized parameters of 6 scouring pad cubes, 1 × 10⁷ spores/mL of inoculum size, agitation speed of 150 rpm and incubated at 30 °C. The use of nylon scouring pad cubes gave an increment of about 335.0% of pectinase production (11.05 U/mL) compared to free cells (2.54 U/mL). The results therefore show scouring pad cubes could be a favorable carrier to immobilize the fungal cells for higher enzyme production in submerged fermentation.