Industrial applications of microbial lipases

https://www.massey.ac.nz/%7Eychisti/Lipases.pdf

Production, purification, characterization, and applications of lipases.

Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.

Bacterial lipases: an overview of production, purification and biochemical properties.

Developments in industrially important thermostable enzymes: a review.

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.

Microbial keratinases and their prospective applications: an overview.

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65°C with its specific growth rate (2.50 h−1) and its lipase activity reached the maximum value of 520 U l−1 during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70–75°C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60°C and 30 min at 70°C and its catalytic function was activated in the presence of Ca2+ or Zn2+.

https://repository.ipb.ac.id/bitstream/handle/123456789/27838/b3.pdf;sequence=1

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID‐1

A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 °C and pH 7 with a maximum specific growth rate (0.14 h–1) in Thermotoga-Fervidobacterium (TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 °C and pH 9, and had a half-life of 90 min at 100 °C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe

Characteristics of thermostable chitinase enzymes from the indonesian Bacillus sp.13.26

A chitinase produced by Bacillus licheniformis MB-2 isolated from Tompaso geothermal springs, Indonesia, was purified and characterized. The extracellular enzyme was isolated by successive hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme was a monomer with an apparent molecular weight of 67 kDa. The optimal temperature and pH of the enzyme were 70 °C and 6.0, respectively. It was stable below 60 °C for 2 h and over a broad pH range of 4.0–11.0 for 4 h. The enzyme was resistant to denaturation by urea (1 M), Tween-20 (1%) and Triton-X (1%), but unstable toward organic solvents such as dimethyl sulphoxide, DMSO, (5%) and polyethylene glycol, PEG, (5%) for 30 min. The enzyme hydrolysed colloidal chitin, glycol chitin, chitosan, and glycol chitosan. The first 13 N-terminal amino acids of the enzyme were determined as SGKNYKIIGYYPS, which is identical to those in chitinases from B. licheniformis and B. circulans.

Purification and characterization of a thermostable chitinase from Bacillus licheniformis Mb-2

Bacillus sp. MTS isolated from Tangkuban Perahu crater Indonesia was found capable of degrading whole chicken feather effectively. The bacteria produced extracellular alkaline keratinase and disulfide reductase. When grown in feather media, Bacillus sp. MTS produced multi-fractions of both enzymes. The purified enzymes worked optimally at alkaline pHs, for keratinase at pH 8–12, and for disulfide reductase at pH 8–10. Optimum temperature for the extracellular keratinase was within 40–70 °C, while that for disulfide reductase was 35 °C. When the purified keratinase was mixed with purified disulfide reductase, enzyme activities on the natural keratin substrates (feather and wool) were greatly increased compared to activity of each enzyme alone, activity of proteinase K or activity of purified keratinase in the presence of reducing agents. The mutual action of the two enzymes on feather was examined by Scanning Electron Microscope.

Maggy Thenawidjaja Suhartono

Papers

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID‐1

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe

Characteristics of thermostable chitinase enzymes from the indonesian Bacillus sp.13.26

Purification and characterization of a thermostable chitinase from Bacillus licheniformis Mb-2

Degradation of keratin by keratinase and disulfide reductase from Bacillus sp. MTS of Indonesian origin