Biochemical Characterization of Cellulase From Bacillus subtilis Strain and its Effect on Digestibility and Structural Modifications of Lignocellulose Rich Biomass

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. The present study reports the partial purification and characterization of cellulase from Bacillus subtilis CD001 and its application in biomass saccharification. Out of four different substrates, carboxymethyl cellulose, when amended as fermentation substrate, induced the highest cellulase production from B. subtilis CD001. The optimum activity of CMCase, FPase, and amylase was 2.4 U/ml, 1.5 U/ml, and 1.45 U/ml, respectively. The enzyme was partially purified by (NH4)2SO4 precipitation and sequenced through LC-MS/MS. The cellulase was found to be approximately 55 kDa by SDS-PAGE and capable of hydrolyzing cellulose, as confirmed by zymogram analysis. The enzyme was assigned an accession number AOR98335.1 and displayed 46% sequence homology with 14 peptide-spectrum matches having 12 unique peptide sequences. Characterization of the enzyme revealed it to be an acidothermophilic cellulase, having an optimum activity at pH 5 and a temperature of 60°C. Kinetic analysis of partially purified enzyme showed the Km and Vmax values of 0.996 mM and 1.647 U/ml, respectively. The enzyme activity was accelerated by ZnSO4, MnSO4, and MgSO4, whereas inhibited significantly by EDTA and moderately by β-mercaptoethanol and urea. Further, characterization of the enzyme saccharified sugarcane bagasse, wheat straw, and filter paper by SEM, ATR-FTIR, and XRD revealed efficient hydrolysis and structural modifications of cellulosic materials, indicating the potential industrial application of the B. subtilis CD001 cellulase. The findings demonstrated the potential suitability of cellulase from B. subtilis CD001 for use in current mainstream biomass conversion into fuels and other industrial processes.

Purification and Characterization of an Extracellular Lipase Produced by Aspergillus oryzae ST11 as a Potential Catalyst for an Organic Synthesis

The lipase producing Aspergillus sp. ST11 was identified by molecular and morphological methods. The primers ITS1/ITS4 were used for amplifying the ITS region. It showed that the strain was grouped with Aspergillus oryzae and Aspergillus flavus (98 % bootstrap value). The colony morphology of Aspergillus sp. ST11 on malt extract agar and Czapek yeast agar showed a characteristic of A. oryzae. Therefore, it was identified as Aspergillus oryzae ST11. The lipase produced by the strain was purified and characterized. The purification steps involved precipitation with chilled acetone and separation by column chromatography, with HiTrap® Q HP and Toyopearl Butyl-650M, respectively. After purification, the lipase activity was increased 13 fold and with 7.9 % yield. Its molecular mass was 25 kDa. The purified lipase was stable at a pH between 5.0 - 8.0 and had optimum activity at pH 7.5. It was stable at 30 °C and had optimum activity at 37 °C. Its activity was promoted in the presence of Mg2+ but it was greatly decreased in the presence of Co2+, Cu2+, Hg2+ and Zn2+. Surfactants (Triton X-100, Tween-80, Tween-20, arabic gum, and sodium dodecyl sulfate) showed negative effects on lipase activity, while inhibitors (PMSF, EDTA, and β-mercaptoethanol) did not reduce the activity significantly. Polar solvents, such as methanol and ethanol, had much negative effect on lipase activity compared to non-polar solvents, such as hexane and isooctane. The concentrated lipase from A. oryzae ST11 was used to catalyze the transesterification and gave the highest bioconversion (90 %) after 24 h. HIGHLIGHTS Extracellular lipase produced by Aspergillus oryzae ST11 could be applied in many applications which is more flexible compared with the use of whole-cell biocatalysts High stability toward a wide range of pH and temperature obtained from this extracellular lipase The concentrated lipase shows the capability to produce the high biodiesel from natural oil GRAPHICAL ABSTRACT

Metaproteomic Discovery and Characterization of a Novel Lipolytic Enzyme From an Indian Hot Spring

Lipolytic enzymes are produced by animals, plants and microorganisms. With their chemo-, regio-, and enantio-specific characteristics, lipolytic enzymes are important biocatalysts useful in several industrial applications. They are widely used in the processing of fats and oils, detergents, food processing, paper and cosmetics production. In this work, we used a new functional metaproteomics approach to screen sediment samples of the Indian Bakreshwar hot spring for novel thermo- and solvent-stable lipolytic enzymes. We were able to identify an enzyme showing favorable characteristics. DS-007 showed high hydrolytic activity with substrates with shorter chain length (<C8) with the maximum activity observed against p-nitrophenyl butyrate (C4). For substrates with a chain length >C10, significantly less hydrolytic activity was observed. A preference for short chain acyl groups is characteristic for esterases, suggesting that DS-007 is an esterase. Consistent with the high temperature at its site of isolation, DS-007 showed a temperature optimum at 55°C and retained 80% activity even after prolonged exposure to temperatures as high as 60°C. The enzyme showed optimum activity at pH 9.5, with more than 50% of its optimum activity between pH 8.0 and pH 9.5. DS-007 also exhibited tolerance toward organic solvents at a concentration of 1% (v/v). One percent of methanol increased the activity of DS-007 by 40% in comparison to the optimum conditions without solvent. In the presence of 10% methanol, DMSO or isopropanol DS-007 still showed around 50% activity. This data indicates that DS-007 is a temperature- and solvent-stable thermophilic enzyme with reasonable activity even at lower temperatures as well as a catalyst that can be used at a broad range of pH values with an optimum in the alkaline range, showing the adaptation to the habitat’s temperature and alkaline pH.

Optimization of Pectinase and Protease Produced from Bacillus subtilis Isolated from Market Waste

Aims: The objective of the study was to produce and optimize protease and pectinase from Bacillus subtilis isolated from market waste. Place and Duration of Study: Department of Microbiology (laboratory unit), Michael Okpara University of Agriculture Umudike, Abia State Nigeria. Methodology: The production and optimization of protease and pectinase from bacteria isolated from solid market waste was investigated. Isolated bacteria from the waste were screened for protease and pectinase production using skim milk agar and pectin agar respectively. Using morphological, biochemical and molecular technique the enzymes producing isolate was confirmed as Bacillus subtilis. Protease and Pectinase were produced by Bacillus subtilis using submerged fermentation in gelatin broth and pectin broth respectively. The enzymes were purified using ammonium sulphate precipitation, dialysis and ion-exchange chromatography. Optimization using different temperatures, pH and nutrient sources was done. Enzyme activity was measured. Results: Purified protease exhibited maximum activity of 8.72U/ml at 40oC while pectinase exhibited maximum activity of 8.98U/ml at 50oC. Glucose as a carbon source and peptone as a nitrogen source gave optimum activity for both enzymes. Both pectinase and protease exhibited optimum activity at pH 9. There was significant difference (P=.05) in enzyme activity at different temperatures, pH and nitrogen sources for both protease and pectinase. There was no significant difference in pectinase activity at P=.05 for the different carbon sources while there was significant difference for protease activity for the different carbon sources at P=.05. Conclusion: Production of microbial enzymes such as protease and pectinase from waste material is an eco-friendly process and cheaper option for large scale use of enzymes in industry.

A novel hyperthermophilic methylglyoxal synthase: molecular dynamic analysis on the regional fluctuations

Two putative methylglyoxal synthases, which catalyze the conversion of dihydroxyacetone phosphate to methylglyoxal, from Oceanithermus profundus DSM 14,977 and Clostridium difficile 630 have been characterized for activity and thermal stability. The enzyme from O. profundus was found to be hyperthermophilic, with the optimum activity at 80 °C and the residual activity up to 59% after incubation of 15 min at 95 °C, whereas the enzyme from C. difficile was mesophilic with the optimum activity at 40 °C and the residual activity less than 50% after the incubation at 55 °C or higher temperatures for 15 min. The structural analysis of the enzymes with molecular dynamics simulation indicated that the hyperthermophilic methylglyoxal synthase has a rigid protein structure with a lower overall root-mean-square-deviation value compared with the mesophilic or thermophilic counterparts. In addition, the simulation results identified distinct regions with high fluctuations throughout those of the mesophilic or thermophilic counterparts via root-mean-square-fluctuation analysis. Specific molecular interactions focusing on the hydrogen bonds and salt bridges in the distinct regions were analyzed in terms of interatomic distances and positions of the individual residues with respect to the secondary structures of the enzyme. Key interactions including specific salt bridges and hydrogen bonds between a rigid beta-sheet core and surrounding alpha helices were found to contribute to the stabilisation of the hyperthermophilic enzyme by reducing the regional fluctuations in the protein structure. The structural information and analysis approach in this study can be further exploited for the engineering and industrial application of the enzyme.

Partial Purification of a Catalase from an Improved Nigerian Sorghum Grain Variety

Catalases are key components of cellular detoxification pathways that prevent the formation of highly reactive hydroxyl radicals through catalyzing the decomposition of hydrogen peroxide into water and molecular oxygen. Their presence in brewery grains prevent the inactivation of important brewery enzymes and also stop lipid peroxidation. To determine their occurrence and establish some of its properties in sorghum, which has become as an important brewery grain similar to barley, crude catalase was obtained from a sorghum grain variety. Preliminary purification of catalase from the sorghum grain variety used, NRL-3, showed that the enzyme was purified 3.2-fold from the crude protein to give a 49% yield of the partially purified enzyme, with a final specific activity of 32 Umg-1 proteins. There was also a positive indication of sorghum catalase presence on SDS PAGE with positive bands occurring between the range of 48-62 kDa. Therefore, the molecular weight of sorghum catalase most likely falls within the two bands. The enzyme showed a narrow pH range with optimum activity occurring at pH 7. Similarly, its optimum activity temperature occurred at 40°C. This work is the first reported attempt at purifying catalase from sorghum.

Effect of Cross-Linked Enzyme Aggregates Strategy on Characterization of sn-1,3 Extracellular Lipase from Aspergillus Niger GZUF36

Background: A novel Aspergillus. niger strain GZUF36 with two high sn-1,3 position selectivity of lipases, including an intracellular lipase and an extracellular one was selected from oil-rich soil in our previous work. The sn-1,3 extracellular lipase from Aspergillus niger GZUF36 (EXANL1) has important potential applications. However, the structure and properties of this lipase need further study for its better application, the immobilization of enzyme is an effective method to study its structural properties and obtain the best catalytic properties. Cross-linked enzyme aggregates (CLEAs) have been widely used in carrier-free immobilization technologies because of their low cost and fast preparation. To this end, it is necessary to investigate the effect of CLEAs strategy on the characteristics, secondary structure, and positional selectivity of EXANL1 before and after immobilization.Results: The CLEAs of purified EXANL1 (CLEA-EXANL1) was achieved optimum activity recovery (100.3±1.1%) with 80% tert-butanol as the precipitant, a glutaraldehyde (GA) concentration of 30 mM, a GA treatment time of 1.5 h, and a centrifugal speed of 6000 g. CLEA-EXANL1 exhibited a broader optimum pH range (4–6) compared with free EXANL1 (6.5). CLEA-EXANL1 presented optimum activity at 40 °C, which was 5 °C higher than that of free EXANL1. CLEAs strategy decreased the maximum reaction rate and increased the Michaelis–Menten constant of EXANL1 when olive oil emulsion was used as a substrate. Moreover, after 30 days, free EXANL1 lost more than 80.0% of its activity, whereas CLEA-EXANL1 retained more than 90.0% of its activity. CLEAs strategy improved the tolerance of EXANL1 in polar organic solvents. Fourier transform infrared spectroscopy results showed that the CLEAs technique increased the content of the β-sheets and β-turns of EXANL1 and reduced α-helixes and irregular crimp contents. CLEAs strategy did not change the sn-1,3 selectivity of EXANL1.Conclusion: The effect of CLEAs technology on catalytic properties, structure, selectivity and other characteristics of the EXANL1 was comprehensively explored, which laid a foundation for its subsequent rational transformation and industrial application.

Inulinase Activity of Extracellular Protein of Lactobacillus casei AP in Different Growth Conditions

Inulinase is an enzyme that catalyzes the reaction involving the hydrolysis of inulin into fructose and/or small fructooligosaccharides by cutting the β-2,1 terminal. The present study aimed to determine the inulolytic activity of extracellular inulinase, extracted from L. casei AP, when grown under different conditions. Extracellular inulinase was extracted from the growth supernatant of L. casei AP, following which the extract was subjected to the qualitative inulinase test; inulinase activity was determined under growth conditions involving different combinations of pH and temperature. The results showed that L. casei AP had inulinolytic ability; therefore, it degraded inulin both in inulin media and inulin extract. The optimum activity of inulinase occurred at the combination of a temperature of 41 °C and pH of 5, with the activity of 20.53 in inulin media and 17.73 in inulin extract.