Production purification and characterization of psychrotolerant alkaline lipase and protease and their application in detergent formulation

Abstract

Proteases are important enzymes for various applications of industrial importance. Microbial proteases show an imperative task in different biotechnological processes. The purpose of the present study was to screen, isolate and production optimization of cold-active alkaline protease producing psychrotrophic bacteria from soil and water samples collected from Juglot, Jutial and Rakaposhi glacier Gilgit, Northern Areas of Pakistan. Soil samples were serially diluted and 0.1ml of sample was spread on skim milk agar plates, at 15 to 25oC for 48 hrs. Total four of the bacterial colonies from glacier soil and water showed clear zone around the colony indicating protease activity. Among these, Stenotrophomonas sp. strain PAK-01 produced highest protease activity (5559 unit/ml) and the isolated thermolabile alkaline protease producing bacterium was identified as Stenotrophomonas sp. (NCBI GenBank Accession no. MG662181) on the basis of 16S ribosomal RNA. Factors influencing the maximum hydrolytic catalysis of extracellular thermolabile alkaline protease by Stenotrophomonas sp. PAK-01 were optimized through the procedure of the one-factor at a time manner. Highest catalytic activity was observed toward temperature was 25oC (4469 unit/ml) at pH 9 (4516 unit/ml) in fermentation production medium with a 24hour old inoculum (5765 U/ml) alongside 5 % size of the inoculum (5754 U/ml) and constant agitation at 150 rpm for 96 hours incubation (6886 unit/ml). Stenotrophomonas sp. PAK-01 exploited multiple sources of carbon for its highest proteolysis and glucose (5833 unit/ml) was observed the best inducer for the highest catalytic activity followed by sucrose and galactose. Amongst the numerous organic and inorganic nitrogen nutrients experienced highest proteolytic activity value (6965 unit/ml) was examined by the supplementation of yeast extract in the basal fermentation production medium. Overall activity of thermolabile protease secretion from Stenotrophomonas sp. strain PAK01 in the basal production medium observed was induced in the existence of Ca2+ (5231 unit/ml) and Mg2+(3984 unit/ml). These metal ions enhanced the enzymatic activity and had stimulatory effect on enzyme production. Amongst the ions examined, HgCl2, CuSO4 and ZnSO4 constrained the enzymatic activity, while FeCl3 had no observable effect on enzymatic activity. The above results indicate that this bacterial isolate can be use as biotechnological tool for industrial purpose. Pry gene extracted from Stenotrophomonas sp. strains PAK-01 translating alkaline protease was amplified from genomic DNA correspondingly, cloned in pET28a plasmid and afterward nucleotide genomic DNA was sequenced. Sequence analysis of the pET28a /protease gene revealed an open reading frame of 1740 base pair and exhibited a 96 % resemblance to the Pr2 nucleotide sequence encoding trypsin of S. maltophilia strain and accessible in GenBank database (JF317278). Interpreting the analyzed 1740 bp nucleotide sequence of the serine protease gene was translated into amino acid sequence and was observed a polypeptide chain of 578 monomers of amino acid with molecular weight of 58 KDa. Protein comprise numerous domains discovered in serine peptidase belonging to family of peptidase protein S8 domain in the position of 172 to 448 and proprotein-processing peptidases C-terminal domain to combined basic monomers of amino acids and was examined between the protein sequence at position of 501 and 567 aa. PrY gene cloned in pET28a plasmid multiple cloning region of T7 promoter with N-terminal His6 residues and subsequently expressed in Escherichia coli BL21 (DE3) in the existence of 1 mM IPTG. Recombinant PrY gene protease protein was expressed as a soluble fraction in E. coli BL21 (DE3) and purified by nickel chelate chromatography followed another technique of Sepharose column chromatography. Purified monomer proteins were observed from the protein analysis. Specific activity of 245 U/mg with yield recovery of 48% was observed from the overall purification procedure. Our study was designing to isolate a novel psychrotrophic bacteria that has the ability to produce alkaline stable protease from temperate region of Gilgit-Baltistan. Universal primers of 16s RNA was used for the identification of the selected strain PAK-03. Enzyme purification was obtained using distinctive techniques of purification such as (NH4)2SO4 precipitation, dialysis, ion exchange and gel permeation chromatography and then characterized by assessing its stability and activity on the basis of different parameters and its formulation in washing detergent was performed by washing analysis using proteinaceous pigments (Blood and Yolk). Strain PAK01 was identified as Stenotrophomonas sp. with a gene bank accession no MG662181. SDS PAGE analysis of PAK-01 supernatant showed that the purified thermolabile protease has a molecular size of about 58 kDa. The highest catalytic efficiency of enzyme was achieved at cold environment in comparison with other commercial proteases and does not exposed the characteristics of thermostability. To check the catalytic activity of enzyme, the maximum hydrolysis for protease enzyme was observed at 9 pH and showed resistance against wide- ranging pH distending from 6 to 10. Enhanced catalytic activity was experimented when bivalent cations such as magnesium chloride MgCl2 and calcium chloride CaCl2 are supplemented in the reaction mixture and hydrolytic activity was inhibited by the supplementation of iron sulfate and copper sulfate (Fe2+ and Cu2+). The thermolabile alkaline protease was delicate to denaturing agents such as EDTA and PMSF and was resilient regarding to H2O2, SDS, DTT and mercaptoethanol. Thermolabile molecules enzyme from Stenotrophomonas sp. PAK-01 with optimum hydrolysis at 25 to 30°C and buffer of Tris-HCl was discovered active in detergents at concentration of 5 % of 10 mg/ml (purex and xtra) and also functional in 500 mg/l of sodium hypochlorite at low environment such as 25oC. It preserved 51 % and 41 % hydrolysis even subsequently after an incubation with (5 % of 15 mg/ml) (purex and xtra) at 24 hours at 25°C. Blood plasma pigments from test fabric were removed within 15 min in 100 U/ml molecule at Tris-HCl buffer with 1 % of detergent (purex) and it acquired 100 U/ml molecules of enzymes to eradicate egg yolk from test fabric (cotton) within 15 min only. It is concluded that protease isolated from this psychrotrophic bacterium can be a better option in detergent industry. An extracellular cold active lipase producing psychrotrophic pseudomonas peli (PAK-03) isolated from the soil samples collected from Rakaposhi glacier, in northmost territory of Gilgit Baltistan, Pakistan and was identification as pseudomonas peli by 16S rRNA nucleotide sequencing. Psychrotrophic pseudomonas peli produced maximum thermolabile alkaline lipase at 25°C after 96 h in alkaline conditions by using yeast extract (3923 unit/ml), tributyrin (3557 unit/ml) as a substrate. CaCl2 stimulated activity by 3766 U respectively. whereas ZnSO4 and FeCl3 strongly inhibited the activity. A novel organic solvent stable lipase gene yLip (GenBank ID MH338242), was amplified from Pseudomonas peli (MH338242). Cloned yLip was expressed in BL21 (λDE3) gave protein of molecular mass 32-kDa soluble protein after induction with 1 mM IPTG and was purified by standard procedure. It maintained thermal stability from 4 to 50oC and half of the catalytic activity was inhibited subsequently 60 min of incubation at 60oC. The activity in organic solvents was increased in the existence of DMSO and ethanol. Present results suggest YLip as thermolabile lipase with comparatively maximum thermal stability and remarkable resistance toward organic solvents. Purified thermolabile lipase enzyme from Pseudomonas peli PAK-03 was obtained by distinctive techniques of purification such as (NH4)2SO4 precipitation, dialysis, ion exchange and gel permeation chromatography. Alkaline lipase secreted by Pseudomonas peli PAK-03 was purified 68.42-fold with an overall yield of 20.52 %, and a specific activity of 1381 U/mg beside molecular mass of purified protein was estimated to be approximately 32 kDa by SDS-PAGE analysis. While the lipase was active at a temperature range of 15–40°C, it exhibited maximum catalytic activity at 30°C, at pH 9. Thermostability analysis of the enzyme was experimented at different temperature from 4 to 75oC. Cold adapted enzyme demonstrated stability at low temperature 4°C and after the incubation of reaction mixture at 240 minutes observed retained 38 % of the residual activity. Half of its activity was inhibited at 55oC and preserved 50 % of its residual preliminary activity by incubation of the reaction mixture at 90 min and the thermolabile enzymes was supposed to have stability toward high temperature. Highest residual activity was observed at pH 9 and by increasing the alkaline condition of the reaction mixture the enzyme stability observed was progressively deteriorated. CaCl2 was found to have stimulated effect on catalysis of enzyme. Whereas the enzyme preserved its catalysis levels toward the existence of a diversity of organic solvents, DMSO boosted this. Extraordinary stability against diverse ions, solvents, high alkalinity and activity at low temperatures make the alkaline thermolabile lipase of Pseudomonas peli PAK-03 a contender for industrial applications. Lipases as an additive to the washing detergent formulation are the enzymes of best choice for laundry cleaner industries due to outstanding to their triacylglyceride eradicating potential from dirtied cloth which ultimately decreases the practice of peroxide-based bleaches cleansers in the detergent formulation. In this research, pseudomonas peli PAK-03 secreted lipase was purified and evaluated for its triacylglyceride eradicating potential by formulating a presoak mixture so as to usage of lipase as an improved in washing detergent formulations. Alkaline lipase stability toward the effect of different marketable detergents, oxidizing compounds and surfactants were investigated in a preliminary assessment to aimed at its further potential in the industrial environment. It was observed that Purified lipase exhibited decent stability in the existence of varied chemical compounds. Laundry competence was observed to be boosted while exploiting alkaline lipase with 0.6 % nonionic detergent as compared towards anionic detergent. Washing performance exploiting against 0.6 % wheel alongside 60 U lipase at 25oC in 40 min consequences in maximum removal of oil was 71 % from dirtied cloth. Therefore, present-day XX analysis begins the innovative objective in bio detergent sector for making of chemicalfree detergent formulation by means of alkaline bacterial lipase.