Background The extracellular lignin peroxidase (LiP) secreted by bacterial isolates is the key enzyme in lignin degradation in several species of Streptomyces (actinomycetes). Random mutations were induced for bacterial strains using ultraviolet (UV) and ethyl methanesulfonate (EMS). Moreover, protoplast fusion is an important tool in strain improvement to achieve genetic recombination and developing hybrid bacterial strains. The molecular analysis of mutants and fusants by random amplification of polymorphic DNA (RAPD-PCR) was done. Objective Streptomyces lavendulae R-St strain, which produces the highest LiP, was discovered and investigated in a previous study by the authors. It has been deposited in NCBI under the accession number ‘OL697233.1.’ S. lavendulae was used in the present study to produce novel, higher LiP-producing mutants using EMS-mutagenesis and UV light. Most mutant strains that produce LiP fuse their protoplasts. To assess the genetic diversity of isolated S. lavendulae R-St-1 with its mutants and fusants, RAPD-PCR was used. Materials and methods Lignin was extracted and purified from black wood liquor. UV and EMS were used for creating super LiP-producing mutants of S. lavendulae R-St. Protoplast fusion between EMS and UV-treated mutants was performed for isolating LiP-productive fusants (s) from S. lavendulae R-St-1 as the original isolate. Fermentation medium (FM) (g/l) was used for lignin-degrading bacterial screening after dilution of the soil samples: K2HPO4, 4.55, KH2PO4, 0.53, MgSO4,0.5, NH4NO3, 0.1, yeast extract, 0.1, lignin (0.1% v/v), agar 15, and the pH should be 7.0. The aforementioned FM medium was supplemented with 50 mg/l of azure B and toluidine dyes and 100 mg/l of tannic acid. FM was used without any supplements and agar for the isolation of lignin-degrading bacteria using lignin (0.1% v/v). The molecular analysis of mutants by RAPD-PCR was applied using different primers, and different separate bands were determined. Results and discussion S. lavendulae R-St-1 strain was mutagenized with alkylating EMS (200 mm) and UV. Results showed that from the S. lavendulae R-St-1 (W.T) isolate, two EMS-treated mutants (Rst/60/7E and Rst/40/8E), which showed activities of 8.5 and 7.3 U/ml, respectively, and two UV-treated mutants (Rst/9/2U and Rst/9/6U), which showed activities of 9.4 and 7.8 U/ml, respectively, were the most efficient ligninolytic mutants. Protoplast fusion between two higher LiP-producing mutants (cross 1 and 2) proved to be the most effective, and the two isolated fusants C1/St/5 and C1/St/6 showed activity of 12.8 and 11.8 U/ml, respectively, after protoplast fusion between Rst/9/6U and Rst/60/7E mutants of S. lavendulae R-St-1 (W.T). To determine molecular variability of two EMS mutants, and their recombinant fusants as well as S. lavendulae (W.T) (parental), three random primers were used. RAPD primer (P1) was employed. Fusant C1/St/5 shared the parental isolate with the bands 850 and 300 bp, whereas fusant C1/St/6 had five new unique bands (1470, 750, 650, 520, and 250 bp). The DNA loci of the obtained banding profiles using P1, P2, and P3 primers were 12, 17, and three loci after RAPD assay. A total of 14 unique loci were obtained using the primers P1 and P2.

Background Lipase is a type of hydrolytic enzyme that has several applications and industrial efforts. Lipases are used as biological catalysts to manufacture products such as food ingredients and applied in making fine chemicals. The type of lipase produced from microbes, mainly from bacteria and fungi, represents the most widely used class of enzymes in biotechnological applications and organic chemistry. Microbial enzymes are also more stable than their corresponding plant and animal enzymes, and their production is more convenient and safer, which makes them more important in commercial uses. The oily environment of vegetable oil-processing factories, industrial wastes, soil contaminated with oil, and diesel fuel-polluted soil provides a suitable habitat for lipase-producing microorganisms. Objective This study aims to detect new strains of lipase-producing bacteria from diverse sources and different areas in Jeddah, Saudi Arabia. Furthermore, the detected bacterial strains have been identified based on morphological, biochemical, and molecular characterization. The plasmid profile of some isolated bacterial strains has been detected. Materials and methods A total of 36 soil samples contaminated with fuel and engine oil were collected from different areas in Jeddah, Saudi Arabia. Tween 20 medium was used to detect the lipolytic activity of the bacterial strains. The isolated bacteria in this study were identified by morphological and biochemical tests and 16SrRNA. Results and discussion Results showed that 53 isolates were positive and able to produce lipase, and 15 isolates have been selected as strong lipase-producing bacteria. The sequences were submitted to the NCBI GenBank under accession numbers, accession numbers, ON360988.1 for Acinetobacter sp. (FS5), ON360990.1 for Alcaligenes faecalis (FS8), ON360991.1 for Acinetobacter baumannii (FS9), ON360992.1 for Bacillus tropicus (FS10), ON360993.1 for A. baumannii (FS11), ON360994.1 for Sphingomonas aeria (FS15), and ON360996.1 for A. baumannii (FS17). Plasmids were isolated from selected strains that showed lipase production using a plasmid-isolation miniprep. Results indicated that isolates FS6 and FS15 have no plasmids, whereas FS8 has one plasmid (≈1295.5 bp). Furthermore, isolates FS10 and FS11 have two plasmids (≈1539.3 and 1295.5 bp). In addition, isolate FS9 has three plasmids (≈1539.3, 1295.5, and 417.7 bp). The isolates showed strong lipase activity and could be good sources for the production of lipase.

Background The identification of naturally occurring bacteria with lignin-oxidizing enzymes would be significant. Several species of filamentous bacteria belonging to the genus Streptomyces (Actinomycetes) have been identified as degraders of lignin. Such species play the most important role in biodegradation of lignin. Objective This study aimed to isolate and discover promising isolates and ideal conditions for lignin peroxidase (LiP) production as well as 16S-rRNA identification of the ligninolytic bacterial strains. Materials and methods Lignin was isolated and purified from black wood liquor. The ligninolytic bacterial colonies were isolated from three types of soil farms (F1, F2, and F3) from Jeddah, KSA. Fermentation medium (FM) was used for screening of lignin-degrading bacteria after dilution of the soil sample using lignin (0.1% v/v). The FM medium was supplemented with 50 mg/l of Azure B and toluidine dyes and 100 mg/l of tannic acid. FM was used without any supplements and agar for isolation of lignin-degrading bacteria after dilution of the soil samples. Different concentrations of lignin (0.1–0.9%) were applied to optimize LiP production by the selected strains under different temperatures (30, 35, 40, and 45°C); different pH values (7, 7.5, 8.0, and 8.5); eight different carbon sources (0.1%, w/v), such as glucose, fructose, xylose, lactose, sucrose, carboxymethyl cellulose, and xylan; and four organic sources (0.1%, w/v), such as peptone, meat extract, sodium nitrate, and potassium nitrate. The enzyme productivity was evaluated in the culture supernatant. The bacterial strain genomic DNA was extracted from pure culture isolated from soil and subjected to amplification and sequencing of 16 S ribosomal RNA gene. Results and discussion Nine ligninolytic bacterial colonies that excrete peroxidases based on the use of lignin (as sole carbon source) were isolated from three types of soil farms (F1, F2, and F3) from Jeddah, KSA, and the promising isolates and the optimum conditions for LiP production using FM under three incubation periods were evaluated. Two most active isolates for production of LiP belonging to Actinomycetes and Bacilli designated (R-St-1 and R-B-1) were identified using 16S-rRNA. Results showed that the highest LiP producer was Streptomyces R-St-1 isolate (3.8 U/ml) followed by Bacilli R-B-1 isolate (2.4 U/ml) after 3 days of fermentation. Different concentrations of lignin (0.1–0.9%) were tested for their effect on LiP production by Streptomyces R-St-1 . As lignin concentration increased, LiP production increased, and the maximum productivity of 4.9 U.mL−1 was observed at 0.5% lignin after which the LiP production was decreased. At the ideal temperature recorded of 35°C and at the optimum pH of 7.5, the production of LiP rose significantly (4.6 U.mL-1 and 4.0 U.mL-1). Various carbon sources were examined for LiP production, and glucose was shown to be the best option for producing a high yield of LiP by Streptomyces R-St-1 , followed by lactose (4.6 and 4.0 U/ml, respectively). However, neither organic nor inorganic nitrogen sources were shown to be suited for high LiP output. As lignin concentration increased, LiP production increased, and the maximum productivity of 4.4 U/ml from Bacilli R-B-1 isolate was observed at 0.7% lignin, after which LiP production was decreased. The optimum temperature was 40°C, where LiP production showed a significant increase (4.5 U/ml), whereas the optimum pH was 8.0, and sucrose was found to be more suitable for high yield of LiP production followed by glucose (4.3 and 3.3 U/ml, respectively). The identified bacterial DNA sequences were conserved in the GenBank under two accession numbers OL697233.1 (Streptomyces lavendulae R-St-1) and Priestia aryabhattai R-B-1 (OL697234.1) (formerly known as Bacillus aryabhattai).