Enzyme Yield Research Articles

Scale-up studies for production of α-amylase by A. Tamarii MTCC5152 and development of enzyme product

α-amylases are one of the most vital commercially produced starch hydrolyzing enzymes. Increasing enzyme production is becoming one of the most important goals in biotechnological processes. In this study, the production of α-amylase by A.tamarii MTCC5152 in flask level studies is experimentally validated by scale-up studies in Koji room under controlled conditions using wheat bran, a low cost agro-industrial waste as substrate. The production of amylase was increased by optimising various culture conditions such as inoculum size, incubation period and moisture content. It was found that A.tamarii MTCC5152 produced highest titre of α-amylase activity (668.01 u/g), after 4 days of incubation period at 5% inoculum concentration and 80% moisture content. The results of the comparative analysis of protease and amylase production demonstrated 15% increase in α- amylase and 20% increase in protease production compared to flask level studies. Studies conducted for the development of enzyme products as liquid enzyme concentrate and dry enzyme powder showed that addition of hydrophilic agents such as poly ethylene glycol (PEG- 6000) was found to be an effective way to increase the activity and enzyme yield in the preparation of a dry enzyme powder using A.tamarii MTCC5152. The ammonium sulfate precipitated enzyme at saturation, lyophilized by freeze drying gave the best recovery (96%) of the enzyme activity.

Challenges and prospects of microbial α-amylases for industrial application: a review.

α-Amylases are essential biocatalysts representing a billion-dollar market with significant long-term global demand. They have varied applications ranging from detergent, textile, and food sectors such as bakery to, more recently, biofuel industries. Microbial α-amylases have distinct advantages over their plant and animal counterparts owing to generally good activities and better stability at temperature and pH extremes. With the scope of applications expanding, the need for new and improved α-amylases is ever-growing. However, scaling up microbial α-amylase technology from the laboratory to industry for practical applications is impeded by several issues, ranging from mass transfer limitations, low enzyme yields, and energy-intensive product recovery that adds to high production costs. This review highlights the major challenges and prospects for the production of microbial α-amylases, considering the various avenues of industrial bioprocessing such as culture-independent approaches, nutrient optimization, bioreactor operations with design improvements, and product down-streaming approaches towards developing efficient α-amylases with high activity and recyclability. Since the sequence and structure of the enzyme play a crucial role in modulating its functional properties, we have also tried to analyze the structural composition of microbial α-amylase as a guide to its thermodynamic properties to identify the areas that can be targeted for enhancing the catalytic activity and thermostability of the enzyme through varied immobilization or selective enzyme engineering approaches. Also, the utilization of inexpensive and renewable substrates for enzyme production to isolate α-amylases with non-conventional applications has been briefly discussed.

Increasing planting density can improve the yield of Tartary buckwheat.

Planting densities and nitrogen fertilizer application rates determine the yield of crops. Tartary buckwheat is a pseudocereal crop with great health care and development values. However, little is known about application of nitrogen fertilizer and planting density on the physiological characteristics of Tartary buckwheat. This study aims to clarify the effect of planting density on the senescence and yield of Tartary buckwheat under low nitrogen conditions. A 2-year field experiment was conducted on Tartary buckwheat (Jinqiao 2) to study the effects of different planting densities (8 × 105, 10 × 105, 12 × 105, 14 × 105, and 16 × 105 plants·ha-1) on the root morphology and activity, chlorophyll and malondialdehyde (MDA) contents, antioxidant enzyme activity, photosynthetic characteristics, agronomic traits, and yield of Tartary buckwheat in the absence of nitrogen fertilizer treatment. With the increase in planting density, the root morphological indices and activities; chlorophyll a, chlorophyll b, and carotenoid contents; superoxide dismutase and peroxidase activities; net photosynthetic rate; transpiration rate; intercellular CO2 concentration and transpiration rate; main stem node, branch, and leaf numbers; grain number and weight per plant; and 1000-grain weight of Jinqiao 2 decreased continuously, whereas plant height and leaf MDA content increased continuously. The yield of Tartary buckwheat first increased and then decreased with the increase in planting density. The yield under 14 × 105 plants·ha-1 treatment increased by 68.61%, 44.82%, 11.00%, and 22.36%, respectively, relative to that under 8 × 105, 10 × 105, 12 × 105, and 16 × 105 plants·ha-1treatments. In summary, planting at an appropriately high density (14 × 105 plants·ha-1) can promote the increase in the yield of Tartary buckwheat populations under low nitrogen conditions and is recommended for use in production to achieve the high-yielding and nitrogen saving cultivation of Tartary buckwheat. This research can serve as a theoretical basis to jointly achieve the high yield and nitrogen saving of Tartary buckwheat.

Purification and characterization of L-asparaginase produced from Bacillus sp.

The objective of this study was to isolate and identify the asparaginase-producing bacteria, then purify and characterize the enzyme in order to investigate their properties in the future. Fifteen local bacterial isolates were isolated from various sites in the city of Baghdad, identified by conventional morphological and biochemical procedures, and confirmed using vitek 2 methods, and submitted to primary screening processes for asparaginase production. For secondary screening, eight isolates with the greatest yellow zone ability on a specific solid medium were chosen. Bacillus sp. was reported to have the highest enzyme production (7.5 U/mg proteins). After 24 hours of incubation, submerged fermentation yielded optimal conditions for the production of L-asparaginase (L-ASNase) by the chosen isolate, with medium (2) serving as the optimal medium for production and fructose serving as the optimal source of carbon. In pH 6 at 40°C, Sephadex G-150 gel filtration chromatography was used to purify the enzyme. The final purification folds were increased by 2.5 times, resulting in an enzyme yield of 93.7%. It also showed the highest purified enzyme activity and stability was at 37°C. Also it revealed the highest activity and stability at pH 7.0 and pH 8.0 respectively. Enzyme lost activity when exposed to several metallic ions at concentrations of 1, 5, and 10 mM.

Enhancing biocatalytical N[sbnd]N bond formation with the actinobacterial piperazate synthase KtzT

Natural compounds with nitrogen-nitrogen bonds are diverse and have applications in medicine and agriculture. l-Piperazic acid (Piz), an α-hydrazino acid, is one of few naturally occurring compounds of its kind. Yet, Piz and its derivatives are valuable building blocks for bioactive compounds. Few NNzymes, enzymes capable of forming NN bonds, have been identified thus far. The hemoenzyme KtzT from Kutzneria sp. 744 catalyzes the intramolecular NN bond formation of N5‑hydroxy-l-ornithine (OH-Orn) to form Piz, a natural building block of kutznerides. The latter has antifungal and antibiotic properties. In our study, we established an improved expression method, with significantly improved yields (ca. 35-fold) of heme-loaded enzyme, making the enzyme much more accessible for laboratory studies. In vitro biochemical characterization under conditions for NN bond formation indicated a considerable thermo- and pH-flexibility, with optimal reaction conditions at 30 °C and 10 mM Tris buffer at pH 9 together with low salinity, paving the way for more complex applications involving KtzT. We have also identified two homologous enzymes from extremophilic organisms to exhibit piperazate-forming activity. In silico structural studies, combined with phylogenetic analysis, resulted in a heme- and substrate-binding model, suggesting target enzyme residues that we propose are critical for the structural integrity and catalytic activity of KtzT. Following this approach, we investigated the potential role of a cysteine residue in a dimer-stabilizing disulfide bridge. The interplay of in vitro and in silico data therefore provides crucial functional information on this enzyme class.

A scalable overexpression of a thermostable recombinant poly-histidine tag carboxyl esterase under lambda promoter: purification, characterization, and protein modelling

BackgroundAs a white biotechnological trend, esterases are thought to be among the most active enzymes’ classes in biocatalysis and synthesis of industrially importance organic compounds. Esterases are used in many applications such as the manufacture of pharmaceuticals, cosmetics, leather, textile, paper, food, dairy products, detergents, and treatment of some environmental pollutants. ResultsA poly-histidine moiety was added to the C-terminal end of the Geobacillus sp. gene encoding carboxyl esterase (EstB, ac: KJ735452) to facilitate one-step purification. This recombinant protein was successfully expressed in Escherichia coli (E. coli) under control of Lambda promoter (λ). An open reading frame (ORF) of 1500 bps encoding a polypeptide of 499 amino acid residues and a calculated molecular weight (54.7 kD) was identified as carboxyl-esterase B due to its conserved glycine-X-serine-X-glycine motif (G-X-S-X-G) and its high similarity toward other carboxyl esterases, where the 3-D tertiary structure of EstB was determined based on high homology % (94.8) to Est55. The expression was scaled up using 7-L stirred tank bioreactor, where a maximum yield of enzyme was obtained after 3.5 h with SEA 51.76 U/mg protein. The expressed protein was purified until unity using immobilized metal affinity chromatography (IMAC) charged with cobalt and then characterized. The purified enzyme was most active at pH 8.0 and remarkably stable at pH (8–10). Temperature optimum was recorded at 65 °C, and it kept 70% of its activity after 1-h exposure to 60 °C. The active half-live of enzyme was 25 min at 70 °C and a calculated T melting (Tm) at 70 °C. The determined reaction kinetics Michaelis–Menten constant (Km), maximum velocity rate (Vmax), the turnover number (Kcat), and catalytic efficiency (Kcat/Km) of the pure enzyme were found 22.756 mM, 164.47 U/ml (59.6 min−1), and (2.619 mol/ min), respectively. ConclusionCreation of a recombinant 6 × -His estB derived from a thermophile Geobacillus sp. was performed successfully and then overexpressed under λ-promoter. In a bench scale bioreactor, the overexpression was grown up, followed by one-step purification and biochemical characterization. The recorded promising pH and temperature stability properties suggest that this expressed carboxyl esterase could be used in many industrial sectors.

Application of statistical methodology for the optimization of l-glutaminase enzyme production from Streptomyces pseudogriseolus ZHG20 under solid-state fermentation

BackgroundActinomycetes are excellent microbial sources for various chemical structures like enzymes, most of which are used in pharmaceutical and industrial products. Actinomycetes are preferred sources of enzymes due to their high ability to produce extracellular enzymes. l-glutaminase has proven its essential role as a pharmaceutical agent in cancer therapy and an economic agent in the food industry. The current study aimed to screen the potent l-glutaminase producer and optimize the production media for maximum enzyme yield using one factor at a time (OFAT) approach and statistical approaches under solid-state fermentation (SSF). ResultsOut of 20 actinomycetes strains isolated from rhizosphere soil, 5 isolates produced extracellular l-glutaminase. One isolate was chosen as the most potent strain, and identified as Streptomyces pseudogriseolus ZHG20 based on 16S rRNA. The production and optimization process were carried out under SSF, after optimization using OFAT method, the enzyme production increased up to 884.61 U/gds. Further, statistical strategy, response surface methodology (RSM), and central composite design (CCD) were employed for the level optimization of significant media component (p < 0.05), i.e., wheat bran, sesame oil cake, and corn steep liquor which are leading to increase 3.21-fold l-glutaminase production as compared to unoptimized media. ConclusionsThe presented investigation reveals the optimization of various physicochemical parameters using OFAT and RSM-CCD. Statistical approaches proved to be an effective method for increasing the yield of extracellular l-glutaminase from S. pseudogriseolus ZHG20 where l-glutaminase activity increased up to 1297.87 U/gds which is 3.21-fold higher than the unoptimized medium using a mixture of two solid substrates (wheat bran and sesame oil cake) incubated at pH 7.0 for 6 days at 33 °C.

Exploring the potential of immobilized phytase to enhance phosphorus uptake by Catharanthus roseus

The current study was designed to establish stable phytases, using poly-(acrylamide-co-acrylic acid) hydrogels as an entrapment matrix, for their application in the promotion of plant growth. Different production parameters were optimized to gain the maximum enzyme yield of 14.3 ± 0.41 U/g and a protein content of 0.7 ± 0.03 mg/ml from Rhizopus oligosporus under solid-state fermentation. For plants, phytase is a source of phosphorus, an essential nutrient for their growth. The study outlined the standardized conditions for the physical entrapment of phytase within polyhydrogel as; 1.5 ml APS, 0.25 ml enzyme, and a 12-minute procurement period. Moreover, the optimal temperature and pH for the partially purified immobilized enzyme were recorded as 50 ºC and 7.5, respectively, under the optimized conditions. The immobilized enzyme has shown enhanced stability over the free enzyme at 70 °C and a wide pH range. During the study, the maximum enzyme activity achieved by partially purified free and immobilized phytase was 7.65 ± 0.43 and 9.12 ± 0.28 U/g. Furthermore, the characterization of the poly-hydrogel revealed that it contains a rough and porous 3-D structure, with abundant cross-linking channels. In a pot experiment, phytase-entrapped poly-(acrylamide-co-acrylic acid) hydrogel was applied to the soil and displayed a positive impact upon different growth parameters as well as phosphorus content of the C. roseus than free enzyme and control groups. The phosphorus-status of the soil having entrapped phytase, increased up to 1.69 and 2.17-fold compared to the plants of free enzyme and control. The study has shown that immobilized phytase was effective to deliver phytate-P to Catharanthus roseus as it significantly increased its rate of photosynthesis (25.9 ± 1.4 µ mol m−2s−1), shoot length (36.1 ± 1.3 cm), and leaf chlorophyll content (56.7 ± 2.8) as compared to the other experimental groups. Overall, the work declares the considerable potential of phytases in phosphorus uptake and plant growth-promoting activities.

PURIFICATION AND CHARACTERIZATION OF AMYLASE EXTRACTED FROM LOCAL WHEAT

This study was aimed to test four types of different plants for the purpose of selecting the optimal plant as a source of the amylase enzyme, including wheat, barley, rice, and potatoes. Among these plants, wheat was selected as the best plant source for enzyme extraction because it possessed the highest effectiveness of the enzyme specific activity (3.0 U/mg protein). Furthermore, sodium acetate buffer (0.2 M, pH 6.0) was determined to be the optimal extraction buffer with a 1:5 (w:v) ratio after 75 min, and it provided 68 U/mg protein. The enzyme was concentrated with sucrose before being purified by gel filtration chromatography using Sephadex G-150. The results demonstrated a 1.8-fold increases in final purification folds with a 332% yield of enzymes. At pH 6.0, the purified enzyme showed its highest activity and stability. The activity of the purified enzyme was most effective at 45 °C and remained stable up to 37 °C. At concentrations of 5 and 10 M, various ionic and chemical substances had an impact on amylase.

Enhancing bacterial fitness and recombinant enzyme yield by engineering the quality control protease HtrA of Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is well-known for its high secretory capacity. Although secretion of homologous proteins is extremely efficient, secretion of heterologous proteins imposes what is known as "secretion stress" on the cells. This is not desirable in an industrial setting, because the bacterial secretion stress responses can adversely affect product yields. So far, the main approach to reduce such counterproductive secretion stress responses has been to remove components of the CssR-CssS two-component regulatory system, including the CssR-CssS-regulated quality control proteases HtrA and/or HtrB. However, there are several examples indicating that a complete loss of these proteases does not correlate with improved protein secretion or a reduced secretion stress response. In this study, an alternative approach to limit the effects of secretion stress by modulating the protease activity of HtrA rather than completely removing this protease was investigated. Indeed, expression of the proteolytically inactive HtrA led to a generally improved fitness of the bacteria when producing the heterologous α-amylase AmyQ. Moreover, compared to control strains, the strain expressing the mutant HtrA provided higher AmyQ yields, which correlated with superior α-amylase enzymatic activity in shake flasks and fermenter-scale cultivations. Proteome analysis of the engineered strains revealed critical differences in stress responses and metabolism. Altogether, our present findings show how proteolytically inactive HtrA can be employed to achieve improved bacterial fitness and higher enzyme yields. IMPORTANCE In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.

Bioethanol production by enzymatic hydrolysis from Aspergillus calidoustus employing different lignocellulosic wastes

Cellulolytic enzymes were necessary for the decomposing of forest waste cellulose present in the environment. Based on the molecular characterization, the N6.2 potent cellulolytic fungal isolate was identified as determined Aspergillus calidoustus. The physical parameters of 5 days of incubation at 30 °C with seven pH resulted in high yield of cellulase enzymes. The endoglucanase activity was determined 87.36 U/ml, followed by β-glucosidase activity of 43.11 U/ml and exoglucanase, 21.03 U/ml under optimized conditions. Treatment of cellulosic substrate Pongamia and wood wastes by Aspergillus calidoustus resulted in breakdown of ligocellulosic waste with exoglucanase activity 234.50 ± 2.13 & 125.00 ± 2.50 U/ml, followed by endoglucanase activity 208.55 ± 4.03 & 112.08 ± 1.51 U/ml and β-glucosidase of around 90.57 ± 0.72 & 54.71 ± 2.50 U/ml. Further, the fermentation of pre-treated cellulosic polymer broth resulted in the production of bioethanol production of around 4.4 g/l/g of Pongamia leaves waste and 2.2 g/l/g of wood waste by the action of Saccaharomycetes spp. Hence, the current work outlines the cellulosic substrates utilization for sustainable two-step bioethanol synthesis, and potential implications for extensive research.

Long-term bioorganic and organic fertilization improved soil quality and multifunctionality under continuous cropping in watermelon

Continuous cropping has can majorly damage the soil ecosystem, leading to an increased prevalence of pathogenic microorganisms and severe yield losses for watermelons. Multiple prevention strategies have been adopted to overcome this obstacle, among which the application of bioorganic fertilizer can improve microbiome deterioration. However, the response of soil ecosystem multifunctionality and soil quality to fertilizer regimes under continuous cropping remains poorly understood. In this study, based on an 8-year field experiment, we evaluated soil health and ecosystem multifunctionality under different fertilization treatments. Long-term bio-organic fertilization preserved soil electrical conductivity and increased total Cu, Zn, Fe, Mn, Ca, and Mg, total and available nutrients, and enzyme activities compared with those in unfertilized control (CK) and chemical fertilizer treatments. Enzyme activities and yield rapidly changed, indicating their sensitivity to these management practices. Conversely, soil pH, biodiversity, medium/trace elements, and total NPK content were more resistant to change than enzyme activities, suggesting higher stability over time. The area of soil quality index under bio-organic fertilization was 1.2–2.9 times larger than that under CK treatment. Applying biological organic fertilizer and a mixture of chemical and organic fertilizers improved the multifunctionality decline caused by continuous cropping by 1.1–1.4 times, whereas the effect of the chemical fertilizer was weak. Soil health was positively correlated with ecosystem multifunctionality. These findings suggest that long-term bioorganic and organic fertilization is an effective strategy for improving soil quality and multifunctionality in continuous watermelon crops while also contributing toward a more sustainable agricultural system.

Asparagine and nitric oxide jointly enhance antioxidant capacity and nitrogen metabolism to improve drought resistance in cotton: Evidence from long‐term field trials

AbstractAsparagine (Asn) and nitric oxide (NO) can enhance plant tolerance to abiotic stress, but their interaction is not well understood. Cotton is a vital resource for the textile industry, but its yield is reduced by drought stress, which could threaten its global supply in a warming and stressful world. Improving cotton's drought tolerance is crucial for supporting the textile industry. Two simultaneous field experiments were conducted to study the effect of Asn and sodium nitroprusside (SNP), a NO donor, on cotton's drought tolerance. Two irrigation treatments were applied: control (C: 100% A pan) and drought stress (50% A pan). The plants were also sprayed with two plant stimulants before imposing drought stress: Mock control, 20 mM Asn, and 0.2 mM SNP, either alone or together. Drought stress impaired plant growth, photosynthesis, yield, nitrogen metabolism, and antioxidant defense, while increasing oxidative stress and free amino acid levels. However, Asn and SNP treatments alleviated these negative effects and improved antioxidant enzyme activity, plant growth, yield, and nitrogen content. The Asn + SNP treatment also increased irrigation water productivity under water‐limited conditions, suggesting its potential for enhancing water use efficiency in cotton production. The combined treatment was more effective than the single treatments, indicating a synergistic effect of Asn and SNP in enhancing drought tolerance in cotton. These results imply that Asn and SNP could be useful tools for sustaining cotton production under drought conditions by boosting nitrogen metabolism and antioxidant defense, thereby contributing to the global supply of cotton and supporting the textile industry.

Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its Asp8Asn variant

Homo sapiens adenosine deaminase isoform 1 (HsADA1) hydrolyzes adenosine and 2-deoxyadenosine as a key step in the purine nucleoside salvage pathway. Some HsADA1 mutations have severe deleterious effects, as is the case in a severe combined immunodeficiency resulting from loss of enzyme activity (ADA-SCID). Other mutations that reduce enzyme activity, for instance the Asp8Asn (D8N) variant, do not cause ADA-SCID but are correlated with other consequences to health. To ease further study of HsADA1 and its variants, we optimized an inexpensive, recombinant expression process in an Escherichia coli host through multiplexed parameter testing enabled by a lysate-based microtiter plate assay. We demonstrate the importance of gene codon usage, induction time and temperature, and alcohol supplementation towards improving enzyme yield to a final titer of 5 mg per liter of culture. We further show that use of a double-histidine-tag (his-tag) system greatly improves purity. We then utilize our expression and purification framework to produce the HsADA1 D8N variant, which had previously not been purified to homogeneity. We confirm that the D8N variant is ∼30% less active than the wildtype HsADA1 and show that it better retains its activity in human serum. Additionally, we show that both HsADA1 and the D8N variant have heightened activity in serum, driven in part by a previously undescribed phenomenon involving albumin. Therefore, this work presents a valuable process to produce HsADA1 that allows for insights into it and its variants’ behavior. We also confirm the utility of lysate-based activity assays towards finding optimal E. coli expression conditions for enzymes and show how fusing his-tags in tandem can enhance product purity.

Cyclodextrin Glucanotransferase of Alkalophilic Strain &lt;i&gt;Caldalkalibacillus mannanilyticus&lt;/i&gt; IB-OR17-B1

Extracellular cyclodextrin glucanotransferase (CGTase, K.F.2.1.19) was characterized for the first time in a strain of bacteria of the species Caldalkalibacillus mannanilyticus IB-OR17-B1. The enzyme was isolated from the culture supernatant using ultrafiltration and affinity adsorption on corn starch. The specific activity of the CGTase was increased in 18-fold as a result of purification with the enzyme yield 56%. The molecular mass of the purified enzyme was 70 kDa according to the denaturing electrophoresis in polyacrylamide gel. The CGTase of C. mannanilyticus IB-OR17-B1 demonstrated a maximal cyclizing activity under pH 8 and temperature 60°C, respectively, and it was stable in the pH range 7–10 and temperatures ≤70°C. The thermal stability of the enzyme under 70°C increased by 10–15% in the presence 5–10 mM of calcium and magnesium salts. The cations of Ag+, Cu2+, Zn2+, Fe2+ and Fe3+ in concentration 5 mM inhibited a CGTase activity by 90, 26, 23, 18 and 11%, respectively. The purified CGTase under optimal conditions and enzyme-substrate ratio 1 U/g converted a potato starch during 24 h to mixture of α-, β- and γ-cyclodextrins with mass ratio 38.8 : 52.6 : 8.6 and yield 42%.

EVALUATION THE PROPERTIES OF PURIFIED LACCASE EXTRACTED FROM SOME LOCAL PLANTS UNDER THE OPTIMUM CONDITIONS

This study set out to screen 36 common plants which have the greatest level of laccase enzyme activity. It revealed that the enzymatic activity of fenugreek seeds was the highest in comparison with other plants. The optimal enzyme-specific activity was 5340.38 units per milligram protein which were obtained by extracting the enzyme with a sodium phosphate buffer at a concentration of 0.02 M and pH 8.0, at a ratio of 1:40 (weight to volume), and extracting time of 210 minutes. The enzyme yield was 27.6% after extraction and purification by gel filtration using Sephacryl S-3 after 1.01 purification fold. The optimum circumstances for enzymatic activity and stability were found by using 0.1 M sodium acetate as a buffer at pH 5. Also, the maximal activity and stability of purified laccase was obtained at 20 oC for 15 min by using o-tolidine as a substrate. This research sheds light on how to isolate and characterize the laccase enzyme, an important biochemical with numerous biotechnological and technological uses through fenugreek seeds as a source of the laccase.

Effects of drip irrigation on yield, soil fertility and soil enzyme activity of different potato varieties in Northwest China.

The effects of different irrigation and fertilization on potato yield, soil fertility and soil enzyme activity of different varieties under drip irrigation fertilization mode were studied, which could provide support for selecting the best varieties in Northwest China. Three factors and three levels orthogonal experimental design method, a total of nine treatments. The three irrigation levels were W1 (100% crop evapotranspiration (ETC)), W2 (80% ETC) and W3 (60% ETC). The three fertilization levels were F1 (N-P2O5-K2O, 240-120-300 kg ha-1), F2 (180-90-225 kg ha-1) and F3 (120-60-150 kg ha-1). The three varieties were V1 (Feiuritar), V2 (Longshu7) and V3 (Qingshu 9). The results showed that different irrigation and fertilization had significant effects on potato yield, soil fertility and soil enzyme activity in root zone. The highest yield of T5 (80%ETC, 180-90-225 kg ha-1, Qingshu 9) was 49,222.3 kg ha-1. With the increase of fertilizer application rate, potato yield and soil enzyme activity in root zone increased first and then decreased, but soil electrical conductivity (SEC), soil nitrate-N content (SNNC), soil alkali-hydrolyzable nitrogen content (SAHC), soil available potassium (AK), soil available phosphorus (AP), soil ammonium-N content (SANC) and soil organic matter (SOM) in root zone increased continuously. The yield, soil catalase activity, soil urease activity and soil sucrase activity at W2 were 2.81% and 22.2%, 1.84% and 7.04%, 8.26% and 9.62%, 5.34% and 13.36% higher than those at W1 and W3, respectively. The overall trend of soil water content, soil nutrient content and enzyme activity in root zone was 0-20 cm >20-40 cm >40-60 cm soil layer. There were many soil factors affecting tuber yield, among which soil enzyme activity, pH value and root zone conductivity were the key factors. The results showed that T5 (80%ETC, 180-90-225 kg ha-1, Qingshu 9) was the best treatment to improve soil enzyme activity and yield.

Obtaining Efficient Mutant from the Wild Type Bacillus subtilis E6-5 by Physical and Chemical Mutagenesis for High Efficiency Protease Production, Optimizing the Mutant's Culture Medium

In this study, to enhance protease production, the wild type of Bacillus subtilis E6-5 was mutagenized by random mutagenesis using ultraviolet radiation and ethidium bromide. After combined treatment, several mutants were obtained. Among these mutants, the mutant strain with the largest proteolytic zone diameter (25 mm) was selected and named Bacillus subtilis ATA38. The enzyme production capacity of the obtained mutant was tested and the mutant strain (404 IU/mL at 24 hours) produced 6.7 times more enzyme than the parental strain (60 IU/mL at 32 hours). The effects of some important parameters in the growth medium on enzyme production were examined. The best carbon, organic nitrogen and metal ion were obtained with wheat starch (525 IU/mL), meat extract (850 IU/mL) and KCl+CaCl2 (548 IU/mL), respectively. pH 6.0, 37°C, 200 rpm, inoculum age 18 hours and inoculation amount 1% were obtained as the best physical factors.To further increase the yield, the best nutritional and physical parameters were combined to create a new modified medium. It was determined that the enzyme yield with mutant strain increased 2.7 times in the modified medium (1096 IU/mL) compared to the control (404 U/mL). The mutant strain (1096 IU/mL) showed an 18.2-fold increase in production compared to the wild type (60 IU/mL) in the modified medium. Protease enzyme obtained from ATA38 mutant strain may have great potential in industry for different purposes.

Soil enzyme activities, physiological indicators, agronomic traits and yield of common buckwheat under herbicide combined with safeners

In the pursuit of green agricultural development, alleviating the harmful effects of herbicides is critical. Herbicide safeners have been identified as an effective solution to safeguard crops without compromising the herbicidal efficacy. However, the impact of combined applications of herbicide and safeners on the physiological characteristics, growth, yield of common buckwheat, and soil enzyme activities remains unclear. Therefore, a two-year (2021 and 2022) field experiment was conducted in the Loess Plateau region of Northwest China under seven treatments: herbicide metolachlor application alone (H1); herbicide metolachlor combined with gibberellin (H1S1); herbicide metolachlor combined with brassinolide (H1S2); herbicide metolachlor combined with naian (H1S3); herbicide metolachlor combined with jiecaotong (H1S4); manual weeding (CK1) and spraying the same volume of water (CK2). The results indicated that H1S3 minimized herbicide toxicity while sustaining the herbicide control efficacy. H1S2 treatment significantly increased the chlorophyll content (SPAD value), superoxide dismutase (SOD), peroxidase (POD) activities, and decreased the malondialdehyde (MDA) content of the leaves compared to H1 treatment. Additionally, the safeners helped restore the biochemical homeostasis of the soil by preventing the inhibition of invertase and urease activities and increasing soil catalase activity. Furthermore, H1S2 promotion of dry matter accumulation, alleviation of herbicide inhibition on plant height, stem diameter, grainnumber per plant and thousand-grain weight resulted in a significant increase in grain yield (14.36 % in 2021 and 27.78 % in 2022) compared to other safener treatments. Overall, this study demonstrates that brassinolide as a safener can effectively mitigate the negative effects of herbicide on the growth and development of common buckwheat while also improving grain yield. These findings provide valuable technical guidance for sustainable and intensive production of common buckwheat in the Loess Plateau of Northwest China.

Screening and genome-wide analysis of lignocellulose-degrading bacteria from humic soil.

Crop straw contains huge amounts of exploitable energy, and efficient biomass degradation measures have attracted worldwide attention. Mining strains with high yields of cellulose-degrading enzymes is of great significance for developing clean energy and industrial production of related enzymes. In this study, we reported a high-quality genome sequence of Bacillus velezensis SSF6 strain using high-throughput sequencing technology (Illumina PE150 and PacBio) and assessed its lignocellulose degradation potential. The results demonstrated that the genome of B. velezensis SSF6 was 3.89 Mb and contained 4,015 genes, of which 2,972, 3,831 and 158 genes were annotated in the COGs (Clusters of Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genomes) and CAZyme (Carbohydrate-Active enZymes) databases, respectively, and contained a large number of genes related to carbohydrate metabolism. Furthermore, B. velezensis SSF6 has a high cellulose degradation capacity, with a filter paper assay (FPA) and an exoglucanase activity of 64.48 ± 0.28 and 78.59 ± 0.42 U/mL, respectively. Comparative genomic analysis depicted that B. velezensis SSF6 was richer in carbohydrate hydrolase gene. In conclusion, the cellulose-degrading ability of B. velezensis SSF6 was revealed by genome sequencing and the determination of cellulase activity, which laid a foundation for further cellulose degradation and bioconversion.