Protopectinase production by Paenibacillus polymyxa Z6 and its application in pectin extraction from apple pomace

Paenibacillus polymyxa Z6 was screened as protopectinase (PPase) producing strain and its PPase activity was 44.4 U/mL. The factors influencing PPase production were identified by a two‐level Plackett–Burman design with seven variables. The results indicated that Ca²⁺ concentration, fermentation time, and temperature were the most influential factors on the PPase production, which were applied in the Box–Behnken design. The predicted maximum PPase activity was 219 U/mL and the experimental maximum PPase activity was 221 U/mL, under the predicted optimum conditions, 170 mg/L Ca²⁺, 27 °C, and 29 hr of fermentation. The present PPase was composed of both type‐A PPase, polygalacturonase; and type‐B PPase, arabinanase, and rhamnogalacturonase. Finally, the PPase was applied for the pectin extraction from apple pomace and achieved an average yield of 11.9% with properties like 8.5% moisture content, 1.6% ash content, 3.8 mPa?S viscosity, and pH 6.1 of 1% solution.

Practical applications

Protopectinase (PPase) is a “green” way for pectin extraction with the advantages of low emission, low energy consumption, and environment‐friendly. PPase includes different types, which can work on different region of protopectin in cell wall of plants and then release highly polymerized soluble pectin applicable in food industry. Present PPase from the strain Paenibacillus polymyxa Z6, contains both A‐type PPase reacting with the smooth regions of protopectin composed of partially methoxylated galacturonic acid, polygalacturonase; and B‐type PPase reacting with the hairy regions consisting of rhamnogalacturonan and neutral side‐chains of protopectin, arabinanase, and rhamnogalacturonase. This PPase was used in pectin extraction from apple pomace and harvested pectin with lower moisture, lower ash, and higher viscosity compared with the chemically produced one, which indicates that this PPase has potential for application in food industry.     

Degradation of cell wall materials from sweetpotato, cassava, and potato by a bacterial protopectinase and terminal sugar analysis of the resulting solubilized products

Cell wall materials (CWMs) from sweetpotato, cassava, and potato starch residues were degraded using a crude enzyme solution from the culture filtrate of a Bacillus sp. isolated from soil, Bacillus sp. M4. This organism has been found to secrete polygalacturonic acid lyase (PGL) and glycan depolymerase activities, especially arabinanase, but cellulase activity was nearly absent. Sugar analysis of the solubilized product after enzyme treatment at pH 7.0 revealed that it is mainly composed of galacturonic acid, galactose, and arabinose, the sugars found commonly in the pectin fraction. This suggested the presence of a protopectinase (PPase) activity in the culture filtrate. The presence of EDTA completely inhibited PGL but PPase activity was almost retained, suggesting that the PGL is not the primary activity responsible for pectin solubilization. The mode of action of the crude enzyme was determined by terminal sugar analysis using HPAEC-PAD after hydrolysis of the reduced products. Results revealed that galactose is the main neutral sugar at the reducing terminal of the products, although rhamnose was also present in the higher molecular weight component. This suggested that at neutral pH, the primary activity in the culture filtrate of Bacillus sp. M4 is a B-type PPase, which attacked the galactan as well as rhamnogalacturonan moieties of the protopectin, resulting in the release of a soluble pectin fraction.     

微生物原果胶酶高产菌株的筛选及发酵特性的研究

从24种不同来源的样品中筛选得到一株编号为XZ3的原果胶酶高产菌株，其酶活达126．4U／ml。通过对菌落形态及孢子囊形态的观察，初步鉴定是曲霉属菌株。在不同发酵时间内测定发酵液中还原糖、pH、湿菌体重、PPase及PGase活性。结果表明：该菌株发酵过程中，发酵液中的还原糖含量在菌体生长初期升高很快，在对数生长期又迅速下降；发酵液的pH保持恒定：菌体产酶与菌体生长几乎同步且PPase与PGase活性的变化规律一致，比值在一定范围内保持恒定。     

白鲢鱼背侧肌焦磷酸酶的纯化及酶学特性

通过粗分离、50%～80%饱和度硫酸铵分级沉降、DE-52阴离子交换柱层析等步骤,从白鲢鱼背侧肌中分离纯化出焦磷酸酶。通过变性凝胶电泳分析,该酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱中仅有一个分子质量为40 kD的条带。酶学特性研究表明,白鲢鱼背侧肌焦磷酸酶可专一性地水解焦磷酸盐,反应初速率时间范围为0～15 min,最适反应温度为45 ℃,最适反应pH值为7.5。Mg2＋对该酶有明显的激活作用,在Mg2＋浓度为5 mmol/L时酶活力最高。5 mmol/L的Ca2＋、Zn2＋、乙二胺四乙酸（ethylenediaminetetraacetic acid,EDTA）-Na2、EDTA-Na4、KIO3和1 mmol/L的NaF均对该酶有强烈的抑制作用。该酶水解焦磷酸四钠的最大反应速率为0.051 U/mg,米氏常数为0.54 mmol/L。     

Purification and Characterization of β-Glucosidase from Aspergillus niger

Aspergillus niger, an isolate of soil contaminated with effluents from cotton ginning mill was grown in Czapek-Dox medium containing sawdust, Triton-X 100 and urea for production of an extracellular β-glucosidase. β-Glucosidase enzyme was purified (86-fold) from culture filtrate of A. niger by employing ammonium sulphate precipitation and gel filtration on sephadex G-75. The molecular mass of the purified enzyme was estimated to be 95 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme had an optimal activity on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5.0. The K_m and V_max of the enzyme on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5 were 8.0 mM and 166 µmol/min/mg of protein, respectively. The enzyme could hydrolyze cellobiose and lactose but not sucrose. Heavy metals like Hg²⁺, Al³⁺, and Ag⁺ inhibited the activity, whereas Zn²⁺ and detergents such as Triton-X 100 and Tween-80 increased the activity at 0.01%. The enzyme activity increased in the presence of methanol and ethanol.     

Purification and Characterization of a β-Amylase of Hendersonula toruloidea

β-Amylase produced by Hendersonula toruloidea was purified to homogeneity by salting out with ammonium sulphate, ion-exchange chromatography on DEAE-cellulose and gel-filtration on Sephadex G-75. The relative molecular mass of the enzyme was estimated to be 60,000 by gel filtration. The enzyme was optimally active at pH 6.0 and 60°C, stable between pH 6 and 8 (24 h) and retained 74% activity at 70°C (30 min). It was strongly activated by Na⁺ but inhibited by Hg²⁺, Zn²⁺ and Cu²⁺. The enzyme hydrolyzed amylopectin (Km 0.42 mg/ml) forming maltose, maltotetraose and unidentified maltooligosaccharide, and hydrolyzed soluble starch (Km 0.3 mg/ml) and glycogen (Km 0.5 mg/ml) forming maltose and unidentified maltooligosaccharide.     

猪背最长肌焦磷酸酶的分离纯化与酶学特性

通过离心、50%～70%饱和硫酸铵沉淀、DEAE-52离子交换柱层析,从猪背最长肌中分离纯化出焦磷酸酶(PPase)。变性聚丙烯酰胺凝胶电泳图谱显示,PPase分子质量约72kD。焦磷酸酶酶学特性研究表明,最适反应温度和pH值分别为50℃和7.5。Mg2+是PPase的激活剂,在浓度4.75mmol/L时,酶活力最强。但Na+和K+都能抑制酶的活力,且Na+的抑制效果强于K+。PPase水解焦磷酸钠(TSPP)的动力学参数Vmax为0.086μmol/(L.min)),Km为0.36mmol/L。     

Rapid Purfication of β -Galactosidase (Aspergillus Niger) from a Commercial Preparation

β-Galactosidase (A. niger) was purified from a commercial source in order to study the protein nature of the enzyme and some of its kinetic properties. The enzyme was rapidly purified by acetone precipitation, gel filtratior, and affinity chromatography. The specific activity of the purified enzyme was twice as high as that found in previous studies. The K_m and V_max for o-nitrophenyl β-D-galactopyranoside were 2.02 mM and 345 μmoles/min/mg protein respectively at pH 4.5 and 37°C. The procedure described yields a highly active enzyme which may be suitable for immobilization and hydrolysis of lactose. The molecular weight of the enzyme was 117,000 and the isolectric point was 4.9. The enzyme appears to be a glycoprotein and may contain multiple molecular forms.     

Isolation and Characterization of Pectin Methylesterase from Apple Fruit

Two forms of the enzyme pectin methylesterase are evidenced in the apple (Malus communis). They differ both in their charge and molecular weight. The two enzymes were separated by DEAE-cellulose chromatography. Their molecular weights, determined by gel-filtration, were 55000 and 28000 daltons. The heavier form has been purified at homogeneity and subjected to investigations regarding its activity as a function of the pH and temperature, and determination of its kinetic parameters. The enzyme has a Km value of 1.05 mg/mL for citrus pectin and an optimum activity in the pH range between 6.5-7.5. The enzyme was stable up to 40°C. Incubation for 1 min at 90°C leads to its complete inactivation.     

Characterization and Cheese-Making Properties of Rennet-Like Enzyme Produced by a Local Algerian Isolate of Aspergillus niger

An ochratoxin free extracellular acid protease was produced by solid state cultivation of Aspergillus niger FFB1. The purified enzyme (48.7 kDa) showed an optimal milk clotting activity at pH 5.5 and 45°C in the presence of 0.01 M CaCl₂. The enzyme was stable at least 24 h at 35°C in the pH range of 5.5–7.0. Thermal denaturation started above 45°C. Fresh cheese manufactured with reconstituted cow milk and the purified enzyme showed similar basic characteristics (pH 4.5, acid taste, white color) as marketed cheeses obtained with calf rennet. This emphasizes the value of exploiting local biological resources for value added food processing in developing countries.     

Esterolytic and Lipolytic Activities of Lactobacillus Casei-subsp-Casei LLG

The estcrolytic and lipolytic enzymes were produced by cell lysis of Lactobacillus casei-subsp-casei LLG during the late logarithmic growth phase. The enzyme was purified to 67 fold by ion exchange chromatography and gel filtration chromatography using the FPLC system. Polyacrylamide gel electrophoresis and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis using the “Phast” system of the purified enzyme showed a single protein band for butyrate-esterase (3.2 × 10⁵ Dalton), caproate esterase (1.1 × 10⁵ Dalton) and capryate esterase (4.0 × 10⁴ Dalton), respectively. The maximum lipolytic activity was observed at pH 7.2 and 37°C. The enzyme activity was inhibited by silver and mercury ions but magnesium and calcium stimulated lipolytic activity. The Km and Vmax values for esterase-lipase of the strain LLG were 76 μM/min/mg of protein, and 0.57 mM, respectively. This enzyme was stable at room temperature for at least 2 days.     

Isolation,purification and characterisation of polygalacturonase from ripened banana (Musa acuminata cv. Kadali)

Bananas are the most important fruit crop in the world. Short shelf life of the fruit is the major limiting factor in international trade, and this is due to the softening of the pulp during ripening. Fruit softening is an important aspect of ripening process in fleshy fruits and is caused by the cumulative action of a group of cell wall‐modifying enzymes. Polygalacturonase (PG) is the key enzyme involved in the fruit softening process in banana, and this study reports the isolation, purification and characterisation of polygalacturonase enzyme from ripened fruits of a delayed ripened banana cultivar found specifically in Kerala (Musa acuminata cv. Kadali). PG was purified by ammonium sulphate fractionation followed by DEAE cellulose ion exchange chromatography and gel filtration using Sephadex G 100. The purified protein showed two subunits on SDS‐PAGE and a single band on native PAGE. Enzyme showed maximum activity at pH 3.5 and 40 °C. Fe³⁺ enhanced the activity more, while Mg²⁺ and Ca²⁺ slightly stimulated the activity of purified enzyme. Km value for substrate polygalacturonic acid was 0.06%.     

Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus

The thermo-alkaline pectinase enzyme from Hylocereus polyrhizus was purified 232.3-fold with a 73.3 % recovery through ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. Ion exchange chromatography combined with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme was monomeric with a molecular weight of 34.2 kDa. The pectinase exhibited broad specificity towards polygalacturonic acid, arabinan, oat spelt xylan, and pNP-α-glucopyranoside. The optimum pH and temperature were 8.0 and 75 °C, respectively. This enzyme was stable over a wide pH range (3.0–11.0) and at relatively high temperature (85 °C for 1 h). The K_m and V_max values of pectinase towards polygalacturonic acid were 2.7 mg/ml and 34.30 U/mg proteins, respectively. In addition, the enzyme activity was inhibited by Ni²⁺, Al³⁺, and Fe²⁺ and was increased in the presence of Ca²⁺ and Mg²⁺ by 120 and 112 %, respectively. The purified pectinase demonstrated robust stability in response to surfactants and oxidising agents. EDTA, which is a powerful chelating agent, did not exert any significant effect on the enzyme stability. Thus, enzymes with these unique properties may be widely used in different types of industries and biotechnological applications.     

硝基还原假单胞菌谷氨酰胺酶的分离纯化及酶学性质

采用离子交换层析和凝胶过滤层析等方法,分离纯化硝基还原假单胞菌(Pseudomonas nitroreducens)SK16.004所产的谷氨酰胺酶,并进一步研究该酶的酶学性质及反应动力学参数。结果表明,该酶的最适反应温度为55℃,最适pH为9.0,温度稳定范围为37～60℃,pH稳定范围为5.0～11.0;Cu2+对促进酶活提高作用最大,而Fe3+会抑制该酶的转移活力。谷氨酰胺酶对底物谷氨酰胺的亲和力最强,其Km值为0.72 mmol/L,Vmax为0.55μmol/(min.mL)。     

Bacillus sp.CBB272脱枝酶的酶学性质

脱枝酶是淀粉加工过程中水解其1,6-糖苷键的水解酶,为淀粉彻底糖化所必需。作者从高温菌Bacillus sp.CBB272的培养液中,经过盐析、凝胶过滤层析、弱阴离子交换层析以及强阴离子交换层析联用的方法,纯化出一种新型脱枝酶。该酶在SDS-PAGE上的相对分子质量约为70 000,最适作用温度为70℃,最适作用pH为6.0。该酶在30~70℃、pH 4.5~9.0之间具有优良的稳定性。该酶在50℃和pH 6.0下水解支链淀粉的Km、Vmax分别为4.0324 mg/mL和0.1841 mg/(min.mL)。研究了不同金属离子对该酶活性和热稳定性的影响,发现Ca2+、Mg2+、Mn2+等金属离子对于该酶具有显著的激活效果,并且Ca2+对该酶的热稳定性具有很好的提升作用。     

Polygalacturonase activity in starfruit

Polygalacturonase (PGase) was extracted and partially purified from ripe starfruit (Averrhoa carambola, L.). 2·3- and 7·8-fold purifications were obtained following purification by Amicon ultrafiltration and Sephadex G-100 filtration, respectively. The apparent pH optimum of PGase in the Amicon concentrate and gel filtrate (combined fractions 6–8) was 5·2. Viscometric and reductometric analyses, on PGase in the gel filtrate and paper chromatographic analysis on the enzyme reaction mixture, were carried out to ascertain the mode of action. The results obtained indicated that exoPGase was the only PGase enzyme present in ripe starfruit.     

Purification and Properties of Beta-galactosidase from Streptococcus thermophilus

A β-galactosidase from Streptococcus thermophilus was purified to homogeneity by ammonium sulfate and acetone fractionation, gel filtration on Sephadex G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme preparation exhibited an optimum pH at 6.6–7.0 and an optimum temperature of 57°C. The enzyme was stable at pH 6.8–7.0. Km and V_max for the enzyme, using ortho-nitrophenyl β-D-galactopyranoside as the substrate, were 0.25 mM and 83 μmoles/mg protein/min, respectively. It was strongly inhibited by Hg⁺⁺, Ag⁺, and Cu⁺⁺ as well as pchloro-mercuri benzoate. The enzyme had a molecular weight of about 6 × 10⁵ and was highly specific for β-galactoside bonds.     

Sweetpotato α- and β-Amylases: Characterization and Kinetic Studies with Endogenous Inhibitors

Sweetpotato α- and β-amylases were characterized to assist optimization of direct hydrolysis of starch by endogenous amylases. In kinetic studies purified starch was substrate, and ascorbate, oxalate, phenolics, phytate and sweetpotato extracts were assayed for inhibitory activity. α-Amylase had optimum pH between 5.8 and 6.4 and was stable from pH 5.0 to 9.0. Optimum activity occurred at 71.5°, but it was inactivated by heat in the absence of Ca²⁺ at > 63°. The Km for soluble starch was 2.08 mg/mL. The molecular weight was 45000 daltons. α-Amylase activity was reduced up to 70% by 0.2 mM K-ascorbate and moderately by Na-oxalate and Na-phytate. β-Amylase had optimum pH between 5.3 and 5.8, and was stable from pH 4.0 to 8.0. Its maximum activity was at 53° and it was inactivated at 60°. Km for soluble starch was 3.71 mg/mL. At 0.08 mM, K-ascorbate strongly inhibited β-amylase activity.     

Aspergillus kawachii produces an acidic pectin releasing enzyme activity

A pectin-releasing (protopectinase, PPase) activity was found in a culture filtrate of Aspergillus kawachii IFO 4308. PPase activity was highest in the pH range of 2.0-2.5 and it was highly stable at 50 degrees C (85% of residual activity was found after a 10-h incubation in citrate-phosphate buffer, pH 3.0). Among other different enzyme activities, which are usually involved in plant cell-wall degradation, only polygalacturonase activity was detected. This result suggests that the PPase activity could correspond to a particular kind of polygalacturonase. Pectin extraction from lemon peels carried out at 50 degrees C for 2 h (pH 3.5) gave yields of ethanol-precipitated pectin equivalent to 17.4% of the initial total solids contained in the peels. Thus, this enzyme activity would allow carrying out a pectin extraction process at lower reaction pHs and higher temperatures in comparison with similar reports using other PPases. These properties seem to be very interesting from the practical point of view.     

牛肾溶菌酶的分离纯化及部分酶学性质

将新鲜牛肾匀浆后,经由缓冲液提取,正丁醇除脂,硫酸铵分级沉淀,CM-Sepharose阳离子交换层析,Superdex-200凝胶过滤层析后得到电泳纯的牛肾溶菌酶。结果表明：该酶比活力为15 145.63 U/mg,回收率为29.13%,纯化倍数为231.05;分子质量约12.66 kD,为单亚基酶。最适反应温度为65 ℃,在65 ℃以下较稳定;最适pH 9.0,pH值在3.0～9.0范围内稳定性较好;测得最适条件下牛肾溶菌酶对溶壁微球菌的Km值为0.399 μg/mL;甲醇、乙醇、异丙醇和硫氰化钾（KSCN）对该酶有激活作用;十二烷基硫酸钠、Pb2＋、Ag＋对该酶有明显抑制作用。